Articles de revues : « PCR-based diagnostic »

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Mirmajlessi, Seyed Mahyar, Maria Jennifer Sjölund, Marika Mänd, Marianne Loiseau, Vincenza Ilardi, Geert Haesaert, Reet Karise, Richard Alexander Gottsberger, Jason Sumner-Kalkun et Assunta Bertaccini. « PCR-based diagnostic methods for ‘Candidatus Liberibacter solanacearum’ – Review ». Plant Protection Science 55, No. 4 (13 septembre 2019) : 228–41. http://dx.doi.org/10.17221/145/2018-pps.

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‘Candidatus Liberibacter solanacearum’ is an economically important pathogen in the Americas, New Zealand and Europe. The primary objective of this review is to systematically investigate the polymerase chain reaction (PCR)-based methods used for its detection in plant samples. Several databases were searched from the inception of the relevant literature up to August 2018. This review identified 53 studies that met all the inclusion criteria. The performance of the different methods was also compared, however due to data heterogeneity and insufficient evidence on the sensitivity of all assays used, a meta-analysis of the data was not possible. Nonetheless, the review indicates that the rtPCR designed to the 16S ribosomal RNA gene can be routinely employed as a fast, cost-effective, and reliable detection technique in diagnostic laboratories.

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Mangayarkarasi, V., Sneka P, Sujith R et Jayaprakash Jayaprakash. « Ergonomic Diagnostic Tool based on Chip Mini RT-PCR for Diagnosis of Pulmonary and Extra Pulmonary Tuberculosis ». Journal of Pure and Applied Microbiology 13, n^o 2 (30 juin 2019) : 1185–90. http://dx.doi.org/10.22207/jpam.13.2.58.

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Volkov, A. N., L. V. Nacheva et Yu V. Zakharova. « Molecular genetic techniques in current biomedical research. Part II : PCR applications in diagnostics of human infectious diseases ». Fundamental and Clinical Medicine 6, n^o 1 (29 mars 2021) : 77–85. http://dx.doi.org/10.23946/2500-0764-2021-6-1-77-85.

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Polymerase chain reaction (PCR)-based diagnostics is currently established as a gold standard for the detection of microorganisms. The features of PCR include rapid amplification of DNA and RNA as well as high sensitivity and specificity. In contrast to diagnostic microbiology, PCR diagnostics does not require preliminary culture of the microorganisms for their identification, reducing both time and costs of the diagnostic procedure. The lecture discusses the molecular basis behind the modern technical solutions for the PCR diagnostics of human infectious diseases including multiplex and reverse transcription PCR. We describe the principles of qualitative and quantitative PCR-based detection of pathogens in biological samples and provide the examples of PCR application for solving specific diagnostic scenarios. The lecture is primarily designed for students of biomedical specialties and healthcare professionals using molecular genetic techniques in their practice.

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Reinitz, David M., Timothy P. Yoshino et Rebecca A. Cole. « A Ribeiroia Spp. (Class : Trematoda)–-Specific PCR-Based Diagnostic ». Journal of Parasitology 93, n^o 5 (octobre 2007) : 1234–38. http://dx.doi.org/10.1645/ge-3584rn.1.

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Kumar, Vinay, Nitish Bansal, Trilok Nanda, Aman Kumar, Rajni Kumari et Sushila Maan. « PCR Based Molecular Diagnostic Assays for Brucellosis : A Review ». International Journal of Current Microbiology and Applied Sciences 8, n^o 02 (10 février 2019) : 2666–81. http://dx.doi.org/10.20546/ijcmas.2019.802.312.

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Kugeler, Kiersten J., Nikos Gurfield, Jean G. Creek, Kerry S. Mahoney, Jessica L. Versage et Jeannine M. Petersen. « Discrimination between Francisella tularensis and Francisella-Like Endosymbionts when Screening Ticks by PCR ». Applied and Environmental Microbiology 71, n^o 11 (novembre 2005) : 7594–97. http://dx.doi.org/10.1128/aem.71.11.7594-7597.2005.

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ABSTRACT The presence of Francisella-like endosymbionts in tick species known to transmit tularemia poses a potential diagnostic problem for laboratories that screen tick samples by PCR for Francisella tularensis. Tick samples initially considered positive for F. tularensis based on standard 16S rRNA gene PCR were found to be positive only for Francisella-like endosymbionts using a multitarget F. tularensis TaqMan assay (ISFtu2, tul4, and iglC) and 16S rRNA gene sequencing. Specificity of PCR-based diagnostics for F. tularensis should be carefully evaluated with appropriate specimen types prior to diagnostic use.

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Agrawal, Megha. « On-Chip PCR Based Plasmonic Microfluidic Platform : Ultrafast Point-of-Care Diagnostics of SARS-CoV-2 ». Biotechnology Kiosk 3, n^o 1 (7 janvier 2021) : 5–11. http://dx.doi.org/10.37756/bk.21.3.1.1.

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It is critically important to have rapid screening and identification of contagious viral diseases such as the current COVID-19 pandemic that is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Rapid and accurate diagnostic is essential for preventing worldwide spread of virus and ensuring in-time care for patients during the fast spread of pandemic diseases. Nanobiotechnology enabled tools have allowed to develop advanced polymerase chain reaction (PCR) based diagnostics of contagious viral diseases. To this end, microfluidic on-chip PCR platforms have shown huge promise for highly efficient, rapid and small-volume bioassay for point-of-care (POC) diagnostic applications in mitigating the challenges of SARS-CoV-2. Here, we discuss latest advances in ultrafast, real-time, and on-chip nanoplasmonic PCR for rapid and quantitative molecular diagnostics at POC level.

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Li, Xiaolei, Weiping Zeng, Jing Liao, Zhenbiao Liang, Shuhua Huang et Zhi Chao. « DNA Barcode-Based PCR-RFLP and Diagnostic PCR for Authentication of Jinqian Baihua She (Bungarus Parvus) ». Evidence-Based Complementary and Alternative Medicine 2015 (2015) : 1–7. http://dx.doi.org/10.1155/2015/402820.

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We established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and diagnostic PCR based on cytochrome C oxidase subunit I (COI) barcodes ofBungarus multicinctus, genuine Jinqian Baihua She (JBS), and adulterant snake species. The PCR-RFLP system utilizes the specific restriction sites ofSpeI andBstEII in the COI sequence ofB. multicinctusto allow its cleavage into 3 fragments (120 bp, 230 bp, and 340 bp); the COI sequences of the adulterants do not contain these restriction sites and therefore remained intact after digestion withSpeI andBstEII (except for that ofZaocys dhumnades, which could be cleaved into a 120 bp and a 570 bp fragment). For diagnostic PCR, a pair of species-specific primers (COI37 and COI337) was designed to amplify a specific 300 bp amplicon from the genomic DNA ofB. multicinctus; no such amplicons were found in other allied species. We tested the two methods using 11 commercial JBS samples, and the results demonstrated that barcode-based PCR-RFLP and diagnostic PCR both allowed effective and accurate authentication of JBS.

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Malorny, B. « Interlaboratory diagnostic accuracy of a Salmonella specific PCR-based method ». International Journal of Food Microbiology 89, n^o 2-3 (31 décembre 2003) : 241–49. http://dx.doi.org/10.1016/s0168-1605(03)00154-5.

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Clancy, Cornelius J., et M. Hong Nguyen. « Polymerase Chain Reaction (PCR)–Based Diagnostic Assays for Invasive Candidiasis ». Current Fungal Infection Reports 5, n^o 3 (4 août 2011) : 135–40. http://dx.doi.org/10.1007/s12281-011-0062-x.

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Reid, Scott M., et Chris C. Wilson. « PCR-RFLP based diagnostic tests for Moxostoma Species in Ontario ». Conservation Genetics 7, n^o 6 (21 février 2006) : 997–1000. http://dx.doi.org/10.1007/s10592-006-9113-1.

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Dahiya, Bhawna, Tulika Prasad, Vishwajeet Singh, Anish Khan, Ekta Kamra, Preeti Mor, Aparna Yadav, Krishna B. Gupta et Promod K. Mehta. « Diagnosis of tuberculosis by nanoparticle-based immuno-PCR assay based on mycobacterial MPT64 and CFP-10 detection ». Nanomedicine 15, n^o 26 (novembre 2020) : 2609–24. http://dx.doi.org/10.2217/nnm-2020-0258.

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Aim: To improve the diagnostic accuracy of immuno-PCR (I-PCR) in tuberculosis (TB) patients by using functionalized gold nanoparticles (AuNPs) coupled with detection antibodies and oligonucleotides, and magnetic beads (MBs) conjugated with capture antibodies in the liquid phase. Materials & methods: MB-coupled AuNP-based I-PCR (MB-AuNP-I-PCR) assay was designed to detect a cocktail of Mycobacterium tuberculosis MPT64 and CFP-10 proteins in bodily fluids of TB patients. Results: The sensitivities of 89.3 (n = 94) and 78.1% (n = 73) were observed in pulmonary TB and extrapulmonary TB patients, respectively, with specificities of 97.9–98.3%. Notably, the sensitivities attained by MB-AuNP-I-PCR in smear-negative pulmonary TB and extrapulmonary TB patients were significantly higher (p < 0.05–0.001) than Magneto-ELISA and GeneXpert assay. Conclusion: The improved technology, as well as enhanced diagnostic accuracy of MB-AuNP-I-PCR, may lead to development of an attractive diagnostic kit.

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OLCHAWA, ANNA, BEATA KRAWCZYK et ANNA BRILLOWSKA-DĄBROWSKA. « New PCR Test for Detection of Candida glabrata Based on the Molecular Target Chosen by the RAPD Technique ». Polish Journal of Microbiology 62, n^o 1 (2013) : 81–84. http://dx.doi.org/10.33073/pjm-2013-011.

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Rapid, reliable diagnosis is a necessary condition for the successful treatment of infections. Such diagnostic assays are continually being developed. The paper presents a method for selecting the molecular target for PCR-based diagnostics based on the comparison of RAPD patterns. A sequence encoding Candida glabrata CBS138 hypothetical protein was selected. The limit of detection for PCR and real-time PCR reactions with DNA extracted from blood samples spiked with Candida glabrata was estimated at 1 CFU/ml. The application of the assays developed in this study would thus seem to be promising as a complementary method in the diagnostics of C. glabrata infections.

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de Godoy, NataliaSouza, ManoelSebastião da Costa Lima-Junior, JoséAngelo Lauletta Lindoso, VeraLucia Pereira-Chioccola, ThelmaSuely Okay et LuciaMaria Almeida Braz. « A PCR and RFLP-based molecular diagnostic algorithm for visceral leishmaniasis ». Asian Pacific Journal of Tropical Medicine 13, n^o 2 (2020) : 62. http://dx.doi.org/10.4103/1995-7645.275414.

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Зернов, Н. В., А. А. Гуськова et М. Ю. Скоблов. « New approach for diagnostic of Facioscapulohumeral muscular dystrophy based on PCR ». Nauchno-prakticheskii zhurnal «Medicinskaia genetika», n^o 7() (30 juillet 2019) : 3–9. http://dx.doi.org/10.25557/2073-7998.2019.07.3-9.

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Актуальность. Миодистрофия Ландузи-Дежерина (МЛД) является одной из наиболее часто встречающихся мышечных дистрофий. В 95% случаев заболевание связано с частичной делецией массива повторов D4Z4 на одном из аллелей 4-й хромосомы. Существующие диагностические методики гибридизации по Саузерну и молекулярного комбинга являются ресурсо- и времязатратными. В настоящее время в Российской Федерации молекулярно-генетическая диагностика МЛД не проводится. Цель. Поиск новых подходов к диагностике МЛД для использования в молекулярно-генетических лабораториях. Методы. ДНК выделялась в агарозных блоках и подвергалась обработке эндонуклеазой EcoRI. Полученные фрагменты ДНК разделялись методом пульс-электрофореза в агарозном геле, после этого агарозный гель фрагментировался согласно маркеру молекулярного веса и использовался в качестве матрицы для полимеразной цепной реакции (ПЦР). Принадлежность полученных ПЦР-продуктов к последовательностям повторов D4Z4 4-й хромосомы подтверждалась секвенированием по Сэнгеру. Результаты. Протокол пульс-электрофореза оптимизирован таким образом, что после всех этапов ДНК в агарозном геле пригодна для использования в качестве матрицы для ПЦР. Разработана ПЦР-система специфичной амплификации контрольных ДНК-матриц 4-й хромосомы и подтверждена секвенированием принадлежность получаемых ПЦР-продуктов к последовательности повторов D4Z4 4-й хромосомы. Выводы. Показана возможность использования ДНК в агарозном геле после пульс-электрофореза в качестве матрицы для детекции повторов D4Z4 методом ПЦР. Представленная ПЦР-система специфично амплифицирует последовательности D4Z4 4-й хромосомы. Используя данную ПЦР-систему и геномную ДНК пациента с известной длиной массива повторов D4Z4 проведена успешная диагностика МЛД. Таким образом разработан новый подход к диагностике МЛД для использования в молекулярно-генетических лабораториях. Relevance. Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common muscular dystrophies. In 95% of cases, the disease is associated with partial deletion of the array of the D4Z4 repeats on one of the alleles of the 4th chromosome. The existing diagnostic methods of Southern blotting and molecular combing are quite resource-and time-consuming. At the moment, molecular genetic diagnostic of FSHD is not provided on the territory of the Russian Federation. Aim: to find new approaches for molecular genetic diagnostic of FSHD acceptable for use in standard molecular genetic laboratories Materials and methods: DNA isolated in agarose plugs and treated by the EcoRI restriction enzyme. DNA fragments then were separated by pulse field gel electrophoresis (PFGE) in agarose gel. After PFGE, the agarose gel was fragmented and used as a matrix for PCR. The identity of the obtained PCR products to the sequence of the D4Z4 repeats of the 4th chromosome was confirmed by sequencing by Sanger. Results. The PFGE protocol is optimized in such a way that, after all stages, DNA in agarose gel is suitable for use as a matrix for PCR. We achieve a specific amplification of the control DNA matrices of the 4th chromosome and confirm belonging of the PCR products to the sequence of D4Z4 repeats of the 4th chromosome by the Senger sequencing. Conclusions. This paper shows the possibility of using DNA in agarose gel after PFGE as a matrix for detection of D4Z4 repeats by PCR. The presented PCR system specifically amplify sequence of the 4th chromosome D4Z4 repeats. Using this PCR system and genomic DNA of a patient with a known length of the D4Z4 repeats array, a successful diagnosis of FSHD was performed. Thus, we propose a new approach for FSHD diagnostic, acceptable for use in standard molecular genetic laboratories.

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Egorova, M. S., A. N. Ignatov et E. S. Mazurin. « Development diagnostic methods of bacterial blight of rice on based PCR ». RUDN Journal of Agronomy and Animal Industries, n^o 2 (2014) : 22–27. http://dx.doi.org/10.22363/2312-797x-2014-2-22-27.

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Giannetto, Sabrina, Riccardo Velasco, Michela Troggio, Giulia Malacarne, Paolo Storchi, Severina Cancellier, Barbara De Nardi et Manna Crespan. « A PCR-based diagnostic tool for distinguishing grape skin color mutants ». Plant Science 175, n^o 3 (septembre 2008) : 402–9. http://dx.doi.org/10.1016/j.plantsci.2008.05.010.

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Holmes, N. G., T. Acheson, E. J. Ryder et M. M. Binns. « A PCR based diagnostic test for fucosidosis in English Springer Spaniels ». Veterinary Journal 155, n^o 2 (mars 1998) : 113–14. http://dx.doi.org/10.1016/s1090-0233(98)80001-4.

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Li, Z., et A. Yu. « Diagnostic Value of a PCR-Based Technique for Prosthetic Joint Infection ». Journal of Clinical Microbiology 52, n^o 6 (15 mai 2014) : 2281–82. http://dx.doi.org/10.1128/jcm.00840-14.

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Singh, R., D. Datta, Priyamvada, S. Singh et R. Tiwari. « A diagnostic PCR based assay for stripe rust resistance geneYr10in wheat ». Acta Phytopathologica et Entomologica Hungarica 44, n^o 1 (juin 2009) : 11–18. http://dx.doi.org/10.1556/aphyt.44.2009.1.2.

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Moabelo, Koena L., Darius R. Martin, Adewale O. Fadaka, Nicole R. S. Sibuyi, Mervin Meyer et Abram M. Madiehe. « Nanotechnology-Based Strategies for Effective and Rapid Detection of SARS-CoV-2 ». Materials 14, n^o 24 (18 décembre 2021) : 7851. http://dx.doi.org/10.3390/ma14247851.

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The coronavirus disease 2019 (COVID-19) pandemic has gained worldwide attention and has prompted the development of innovative diagnostics, therapeutics, and vaccines to mitigate the pandemic. Diagnostic methods based on reverse transcriptase-polymerase chain reaction (RT-PCR) technology are the gold standard in the fight against COVID-19. However, this test might not be easily accessible in low-resource settings for the early detection and diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The lack of access to well-equipped clinical laboratories, requirement for the high level of technical competence, and the cost of the RT-PCR test are the major limitations. Moreover, RT-PCR is unsuitable for application at the point-of-care testing (PoCT) as it is time-consuming and lab-based. Due to emerging mutations of the virus and the burden it has placed on the health care systems, there is a growing urgency to develop sensitive, selective, and rapid diagnostic devices for COVID-19. Nanotechnology has emerged as a versatile technology in the production of reliable diagnostic tools for various diseases and offers new opportunities for the development of COVID-19 diagnostic systems. This review summarizes some of the nano-enabled diagnostic systems that were explored for the detection of SARS-CoV-2. It highlights how the unique physicochemical properties of nanoparticles were exploited in the development of novel colorimetric assays and biosensors for COVID-19 at the PoCT. The potential to improve the efficiency of the current assays, as well as the challenges associated with the development of these innovative diagnostic tools, are also discussed.

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Fomina, E. G., E. E. Grigorieva et A. S. Vladyko. « Recombinant Retroviral Particles : Technology of Poduction and Application as Positive Controls for PCR Diagnostics of Dangerous Viral Infections ». Problems of Particularly Dangerous Infections, n^o 2 (12 juillet 2020) : 115–21. http://dx.doi.org/10.21055/0370-1069-2020-2-115-121.

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Objective. Construction of positive control samples based on recombinant retroviral particles and their application in RT-PCR diagnostic assays for RNA detection of agents of dangerous and particularly dangerous viral infections.Materials and methods. Molecular biological, genetic engineering, and immunological methods were used: polymerase chain reaction, restriction, ligation, cloning, transformation, transfection, flow cytometry.Results and discussion. Technology of positive control samples producing based on recombinant virions has been developed and tested. It includes construction of retroviral vector with cloned diagnostic sequence of the viral genome; obtaining a packaging cell line producing chimeric retroviral particles; determination of recombinant virions titer by flow cytometry and polymerase chain reaction; application of the obtained preparation as a control sample for PCR diagnostics of infectious agents. Positive controls based on retroviral vectors as carriers of genomic RNA fragments of pathogenic viruses were used in the development of PCR diagnostic kits for dangerous and particularly dangerous viral infections. Their application increased the kits quality and made it possible to exclude the work with concentrated hazardous infectious agents (Lassa virus, tick-borne encephalitis virus, lymphocytic choriomeningitis virus, Puumala virus).

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Luigi, Marta, Ariana Manglli, Antonio Tiberini, Sabrina Bertin, Luca Ferretti, Anna Taglienti, Francesco Faggioli et Laura Tomassoli. « Inter-Laboratory Comparison of RT-PCR-Based Methods for the Detection of Tomato Brown Rugose Fruit Virus on Tomato ». Pathogens 11, n^o 2 (3 février 2022) : 207. http://dx.doi.org/10.3390/pathogens11020207.

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In 2020, a test performance study (TPS) for the specific detection of tomato brown rugose fruit virus (ToBRFV) was organized in the frame of the H2020 Valitest project. Since no validated tests were available, all the protocols reported in the literature were at first screened, performing preliminary studies in accordance with the EPPO standard PM 7/98 (4). Five molecular tests, two conventional RT-PCR and three real-time RT-PCR were found to be suitable and were included in the TPS. Thirty-four laboratories from 18 countries worldwide took part in TPS, receiving a panel of 22 blind samples. The panel consisted of sap belonging to symptomatic or asymptomatic leaves of Solanum lycopersicum and Capsicum annuum. The results returned by each laboratory were analyzed and diagnostic parameters were assessed for each test: reproducibility, repeatability, analytical sensitivity, diagnostic sensitivity and diagnostic specificity. All the evaluated tests resulted in being reliable in detecting ToBRFV and were included in an EPPO Standard PM 7/146—Diagnostics.

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Sisay, Abay, Sonja Hartnack, Abebaw Tiruneh, Yasin Desalegn, Abraham Tesfaye et Adey Feleke Desta. « Evaluating diagnostic accuracies of Panbio™ test and RT-PCR for the detection of SARS-CoV-2 in Addis Ababa, Ethiopia using Bayesian Latent-Class Models (BLCM) ». PLOS ONE 17, n^o 10 (19 octobre 2022) : e0268160. http://dx.doi.org/10.1371/journal.pone.0268160.

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Background Rapid diagnostics are vital for curving the transmission and control of the COVID-19 pandemic. Although many commercially available antigen-based rapid diagnostic tests (Ag-RDTs) for the detection of SARS-CoV-2 are recommended by the WHO, their diagnostic performance has not yet been assessed in Ethiopia. So far, the vast majority of studies assessing diagnostic accuracies of rapid antigen tests considered RT-PCR as a reference standard, which inevitably leads to bias when RT-PCR is not 100% sensitive and specific. Thus, this study aimed to evaluate the diagnostic performance of Panbio™ jointly with the RT-PCR for the detection of SARS-CoV-2. Methods A prospective cross-sectional study was done from July to September 2021 in Addis Ababa, Ethiopia, during the third wave of the pandemic involving two health centers and two hospitals. Diagnostic sensitivity and specificity of Panbio™ and RT-PCR were obtained using Bayesian Latent-Class Models (BLCM). Results 438 COVID-19 presumptive clients were enrolled, 239 (54.6%) were females, of whom 196 (44.7%) had a positive RT-PCR and 158 (36.1%) were Panbio™ positive. The Panbio™ and RT-PCR had a sensitivity (95% CrI) of 99.6 (98.4–100) %, 89.3 (83.2–97.6) % and specificity (95% CrI) of 93.4 (82.3–100) %, and 99.1 (97.5–100) %, respectively. Most of the study participants, 318 (72.6%) exhibited COVID-19 symptoms; the most reported was cough 191 (43.6%). Conclusion As expected the RT-PCR performed very well with a near-perfect specificity and a high, but not perfect sensitivity. The diagnostic performance of Panbio™ is coherent with the WHO established criteria of having a sensitivity ≥80% for Ag-RDTs. Both tests displayed high diagnostic accuracies in patients with and without symptoms. Hence, we recommend the use of the Panbio™ for both symptomatic and asymptomatic individuals in clinical settings for screening purposes.

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Wang, Chong-Zhi, Ping Li, Jia-Yi Ding, Guo-Qian Jin et Chun-Su Yuan. « Identification ofFritillaria pallidifloraUsing Diagnostic PCR and PCR-RFLP Based on Nuclear Ribosomal DNA Internal Transcribed Spacer Sequences ». Planta Medica 71, n^o 4 (avril 2005) : 384–86. http://dx.doi.org/10.1055/s-2005-864112.

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Lemasova, L. V., G. A. Tkachenko, E. V. Prokhvatilova, L. I. Belitskaya, D. V. Viktorov et A. V. Toporkov. « ASSESSMENT OF THE POSSIBILITY OF APPLICATION IN LABORATORY PRACTICE OF REAGENT KIT FOR DIAGNOSIS OF GLANDERS AND MELIOIDOSIS BY REAL-TIME POLYMERASE CHAIN REACTION ». Russian Clinical Laboratory Diagnostics 64, n^o 11 (15 novembre 2019) : 700–704. http://dx.doi.org/10.18821/0869-2084-2019-64-11-700-704.

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The reagent kit AmpligenBurk-mallei/pseudomallei-RT PCR is designed for detecting in vitro diagnostics and differentiate the DNA of glanders and melioidosis pathogens by real-time multiplex PCR in biological (clinical) material and cultures of microorganisms, as well as environmental objects and solid food products (rice). During clinical testing diagnostic value of reagent kit AmpligenBurk-mallei/pseudomallei-RT PCR has been studied. Based on the results obtained, a high analytical sensitivity (1×103 microbe cells/ml) and specificity (100%) of PCR-RT with the developed reagent kit were established, regardless of the type of material being studied. The diagnostic sensitivity of PCR-RT using a set of reagents was at least 98.0% and specificity at least 99%. The stages of state examination have been completed, a registration certificate has been obtained at Roszdravnadzor, production, sale and use of reagent kit in medical laboratory practice have been permitted.

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Shipitsyna, Е. V., О. V. Budilovskaya et А. М. Savitcheva. « Nucleic acid sequence—based amplification (nasba) and its application in obstetrical and gynecological practice ». Journal of obstetrics and women's diseases 54, n^o 2 (1 octobre 2005) : 83–89. http://dx.doi.org/10.17816/jowd82490.

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Method of isothermal amplification of nucleic acids NASBA {Nucleic Acid SequenceBased Amplification) is becoming widely used in diagnostic molecular microbiology including diagnostics of infections in pregnant women and newborn infants. NASBA method possesses the unique ability to amplify RNA target selectively in the presence of DNA target of identical sequence, which determines its main application areas: diagnosis of RNA viruses, investigation of bacterial and viral gene expression, diagnosis of bacterial infections based on 16S rRNA detection. In the article the principle and the main steps of the method as well as some applications in diagnostic microbiology are reviewed. In addition, a comparative evaluation of NASBA and other amplification techniques such as polymerase chain reaction (PCR) and reversetranscriptase PCR (RTPCR) is presented.

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REBELO-de-ANDRADE, H., et M. C. ZAMBON. « Different diagnostic methods for detection of influenza epidemics ». Epidemiology and Infection 124, n^o 3 (juin 2000) : 515–22. http://dx.doi.org/10.1017/s0950268899003751.

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Linking continuous community-based morbidity recording of influenza-like illness (ILI) with virological sampling has consistently proved its value as one of the earliest indicators of circulating influenza activity. The clinical morbidity recording in the Portuguese national surveillance network, during a 7-year period, and the contribution of different diagnostic techniques, including virus isolation, multiplex RT–PCR, immunocapture enzyme linked immunoassay (EIA) and complement fixation tests (CFTs) for the detection of influenza in such a community-based setting is described and evaluated in this study. There was good correlation between the increase of morbidity, total samples taken and the detection of influenza virus by all the methods although this was less evident for virus isolation and EIA than for RT–PCR or serology. From a total of 1685 throat swabs collected from cases of ILI, 43·6% were RT–PCR positive, 17·5% were positive by capture EIA and in 5% virus isolates were made. The detection of influenza by RT–PCR occurred earlier than by any other method and showed the best correlation with epidemic patterns of morbidity registration. We conclude that in surveillance systems where virus culture is sub-optimal, RT–PCR provides a rapid, sensitive, specific method for detecting influenza viruses from community-based sampling.

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Yoo, Hee-Min, Il-Hwan Kim et Seil Kim. « Nucleic Acid Testing of SARS-CoV-2 ». International Journal of Molecular Sciences 22, n^o 11 (7 juin 2021) : 6150. http://dx.doi.org/10.3390/ijms22116150.

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The coronavirus disease 2019 (COVID-19) has caused a large global outbreak. It is accordingly important to develop accurate and rapid diagnostic methods. The polymerase chain reaction (PCR)-based method including reverse transcription-polymerase chain reaction (RT-PCR) is the most widely used assay for the detection of SARS-CoV-2 RNA. Along with the RT-PCR method, digital PCR has emerged as a powerful tool to quantify nucleic acid of the virus with high accuracy and sensitivity. Non-PCR based techniques such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) are considered to be rapid and simple nucleic acid detection methods and were reviewed in this paper. Non-conventional molecular diagnostic methods including next-generation sequencing (NGS), CRISPR-based assays and nanotechnology are improving the accuracy and sensitivity of COVID-19 diagnosis. In this review, we also focus on standardization of SARS-CoV-2 nucleic acid testing and the activity of the National Metrology Institutes (NMIs) and highlight resources such as reference materials (RM) that provide the values of specified properties. Finally, we summarize the useful resources for convenient COVID-19 molecular diagnostics.

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Dahiya, Bhawna, Suman Sharma, Anish Khan, Ekta Kamra, Preeti Mor, Abhishek Sheoran, Vishnubhatla Sreenivas et al. « Detection of mycobacterial CFP-10 (Rv3874) protein in tuberculosis patients by gold nanoparticle-based real-time immuno-PCR ». Future Microbiology 15, n^o 8 (mai 2020) : 601–12. http://dx.doi.org/10.2217/fmb-2019-0347.

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Aim: Timely and reliable diagnostic test for tuberculosis (TB) is immediately required. Attempts were made to improve the technology and diagnostic potential of real-time immuno-PCR (RT-I-PCR). Methods: We designed gold nanoparticle (GNP)-based RT-I-PCR (GNP-RT-I-PCR) assay for the detection of Mycobacterium tuberculosis CFP-10 (Rv3874) protein in clinical samples of TB patients. Results: A wide quantitative detection range of CFP-10 was found to be 0.5–5 × 104 pg/ml in bodily fluids of TB patients, which can evaluate the progression of disease. Moreover, sensitivities of 83.7 and 76.2% were observed in pulmonary (n = 49) and extrapulmonary TB (n = 42) patients, respectively, with specificities of 93.5–93.8% (n = 63). Conclusion: Conjugation of detection antibodies and oligonucleotides to functionalized GNPs of GNP-RT-I-PCR is relatively easier, compared with streptavidin-biotin/succinimidyl-4-( N-maleimidomethyl) cyclohexane-1-carboxylate system employed in RT-I-PCR. Our assay also showed better diagnostic performance than RT-I-PCR, which may provide a viable platform for the development of an efficient TB diagnostic test.

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Kil, Byeong-Heon, Ji-Seong Park, Chan-Young Park, Yu-Seop Kim et Jong-Dae Kim. « System Architecture for IIoT-Based POC Molecular Diagnostic Device ». Engineering Proceedings 6, n^o 1 (17 mai 2021) : 60. http://dx.doi.org/10.3390/i3s2021dresden-10147.

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In this paper, we investigate an efficient structure for a point-of-care (POC) molecular diagnostic system based on the industrial Internet of things (IIoT). The target system can perform automated molecular diagnosis including DNA extraction, PCR amplification, and fluorescence detection. Samples and reagents are placed in a multi-room cartridge and loaded into the system. A rotating motor and a syringe motor control the cartridge to extract DNA from the sample. The extracted DNA is transferred to a polymerase chain reaction (PCR) chamber for DNA amplification and detection. The proposed system provides multiplexing of up to four colors. For POC molecular diagnostics, the World Health Organization demands features such as low volume, low cost, fast results, and a user-friendly interface. In this paper, we propose a system structure that can satisfy these requirements by using a PCR chip and open platform. A distributed structure is adopted for the convenience of maintenance, and a web-based GUI is adopted for the user’s convenience. We also investigated communication problems that may occur between system components. Using the proposed structure, the user can conveniently control from standard computing devices including a smartphone.

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Das, Debayan, Manaswini Masetty et Aashish Priye. « Paper-Based Loop Mediated Isothermal Amplification (LAMP) Platforms : Integrating the Versatility of Paper Microfluidics with Accuracy of Nucleic Acid Amplification Tests ». Chemosensors 11, n^o 3 (28 février 2023) : 163. http://dx.doi.org/10.3390/chemosensors11030163.

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Paper-based diagnostics offer a promising alternative to traditional diagnostic methods for point-of-care use due to their low cost, ease of use, portability, rapid results, versatility, and low environmental impact. While paper-based serology tests in the form of lateral flow assays can provide rapid test results for past pathogen exposure, they currently lack the accuracy and sensitivity offered by molecular diagnostic tests such as the polymerase chain reaction (PCR). Loop-mediated isothermal amplification (LAMP)—an isothermal nucleic acid amplification test (NAAT)—provides PCR-like performance while simultaneously reducing the instrumentation and assay complexity associated with PCR. In this review, we discuss a newly emerging class of paper-based LAMP platforms that integrates the versatility of paper microfluidics with the accuracy of NAATs. Since its first adoption in 2015, we have discussed all paper-based LAMP platforms in terms of the paper substrates, reagent incorporation techniques, paper platform design, heating hardware, detection methods, and sensitivity and specificity of paper-based LAMP assays. We conclude by identifying the current challenges and future prospects of paper-based NAATs.

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Degefu, Yeshitila. « Molecular diagnostic technologies and end user applications : potentials and challenges ». Suomen Maataloustieteellisen Seuran Tiedote, n^o 23 (31 janvier 2008) : 1–6. http://dx.doi.org/10.33354/smst.75922.

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Conventional methods of pathogen identification have often depended on identification of disease symptoms, isolation and culturing of the organisms, and identification by morphology and biochemical tests. The major limitations of these culture based morphological approaches, however, are the reliance on the ability of the organism to be cultured, the time consuming nature and requirement of extensive taxonomic expertise. Furthermore, diagnosis of plant diseases can be even more difficult with asymptomatically infected propagative materials such as tree grafting stocks or potato tubers. The use of molecular methods can circumvent many of these shortcomings. Accordingly, there have been significant developments in the area of molecular detection of plant pathogens in the last three decades. The advent of antibody based detection, the monoclonal antibodies and the enzyme linked Immunosorbent assay (ELISA), was an important turning point in virology and bacteriology. Then came the DNA based technologies, such as the polymerase chain reaction (PCR) which revolutionised molecular diagnostics and biological sciences. In the last decade the range of targets that can be diagnosed using diagnostic PCR have grown tremendously. The very flexibility and application specific variations in the basic theme of the system have allowed the development of many PCR variants adapted to wide range of applications. Furthermore, diagnostic PCR has been greatly improved by the introduction of the second generation PCR, known as the Real time PCR where closed-tube fluorescence detection and quantification during PCR amplification (in real time) is possible eliminating the need for laborious post-PCR sample processing steps which greatly reduces the risk of carryover contamination. Using Real Time PCR, it is possible not only to detect the presence or absence of the target pathogen, but it is also possible to quantify the amount present in the sample allowing the quantitative assessment of the number of the pathogen in the sample. Enumerating the pathogen upon detection is crucial to estimate the potential risks with respect to diseases development and provides a useful basis for diseases management decisions. Crops can be attacked by many pathogens which, in addition, often occur in complexes. Therefore, many disease diagnostic applications require simultaneous detection and quantification of several targets. Methodological limitations, however, are in many cases the reason for developing simplex or assays designed for only few targets. The DNA Microarray technology, originally designed to study gene expression and generate single nucleotide polymorphism (SNP) profiles, is currently a new and emerging pathogen diagnostic technology, which in theory, offers a platform for unlimited multiplexing capability. It is viewed as a technology that fundamentally alter molecular diagnostics. The fast growing databases generated by genomics and biosystematics research provides unique opportunity for the design of more versatile, high-throughput, sensitive and specific molecular assays which will address the major limitations of the current technologies and benefit plant pathology.

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Kritsiriwuthinan, Kanyanan, Kanthima Choosang et Sakone Sunantaraporn. « EVALUATION OF TWO NESTED PCR-BASED DIAGNOSTIC ASSAYS FOR PLASMODIUM FALCIPARUM INFECTION ». Asian Journal of Pharmaceutical and Clinical Research 10, n^o 10 (1 septembre 2017) : 200. http://dx.doi.org/10.22159/ajpcr.2017.v10i10.20156.

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Objective: The majority of malaria cases and deaths are caused by Plasmodium falciparum. The rapid and accurate diagnosis is very important for malaria treatment and control. The aim of this study was to evaluate two nested polymerase chain reaction (PCR)-based methods (protocol A and protocol B) for P. falciparum infection, diagnosis in Thailand.Methods: A total of 90 dried blood spot samples were investigated. The samples composed of P. falciparum-, Plasmodium vivax-infected blood and normal human blood samples. The microscopic examination was used as gold standard.Results: The results showed the sensitivity of 100/83.33%, specificity of 100/100%, and accuracy of 100/94.44% for protocol A and protocol B, respectively. The analytical sensitivity of protocol A and protocol B was 0.625 and 6.25 parasites/μl, respectively. The comparison among microscopic examination, protocol A and protocol B by statistical analysis, found that they were not a significant difference. The agreements between each method were good. The kappa value between protocol A and protocol B was 0.87, protocol A and microscopy was 1.00, and protocol B and microscopy was 0.87.Conclusion: The results demonstrated that protocol A should be used for further development of P. falciparum diagnosis in Thailand, especially in case of low parasitemia such as asymptomatic infection and for screening blood donors.

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Noh, Pureum, Wook Jin Kim, Sungyu Yang, Goya Choi et Byeong Cheol Moon. « PCR-based rapid diagnostic tools for the authentication of medicinal mistletoe species ». Phytomedicine 91 (octobre 2021) : 153667. http://dx.doi.org/10.1016/j.phymed.2021.153667.

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Krokene, Paal, Irene Barnes, Brenda D. Wingfield et Michael J. Wingfield. « A PCR-RFLP based diagnostic technique to rapidly identifySeiridiumspecies causing cypress canker ». Mycologia 96, n^o 6 (novembre 2004) : 1352–54. http://dx.doi.org/10.1080/15572536.2005.11832884.

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Khadangi, Fatemeh, Maryam Yassi et Mohammad Amin Kerachian. « Review : Diagnostic accuracy of PCR-based detection tests forHelicobacter Pyloriin stool samples ». Helicobacter 22, n^o 6 (29 septembre 2017) : e12444. http://dx.doi.org/10.1111/hel.12444.

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Moore, Sinead, Michael Gunn et Dermot Walls. « A rapid and sensitive PCR-based diagnostic assay to detect bovine herpesvirus 1 in routine diagnostic submissions ». Veterinary Microbiology 75, n^o 2 (juillet 2000) : 145–53. http://dx.doi.org/10.1016/s0378-1135(00)00210-8.

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Abdulmawjood, Amir, Michael Bülte, Stefanie Roth, Hahn Schönenbrücher, Nigel Cook, Martin D’Agostino, Burkhard Malorny, Kieran Jordan, Sinikka Pelkonen et Jeffrey Hoorfar. « Toward an International Standard for PCR-Based Detection of Foodborne Escherichia coli O157 : Validation of the PCR-Based Method in a Multicenter Interlaboratory Trial ». Journal of AOAC INTERNATIONAL 87, n^o 4 (1 juillet 2004) : 856–60. http://dx.doi.org/10.1093/jaoac/87.4.856.

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Abstract The performance of a polymerase chain reaction (PCR) method for detection of Escherichia coli O157, previously validated on DNA extracted from pure cultures, was evaluated on spiked cattle swabs through an interlaboratory trial, including 12 participating laboratories from 11 European countries. Twelve cattle swab samples, spiked at 4 levels (0, 1–10, 10–100, and 100–1000 colony-forming units, in triplicate) with E. coli O157 were prepared centrally in the originating laboratory; the receiving laboratories performed pre-PCR treatment followed by PCR. The results were reported as positive when the correct amplicons were present after gel electrophoresis. The statistical analysis, performed on 10 sets of reported results, determined the diagnostic sensitivity to be 92.2%. The diagnostic specificity was 100%. The accordance (repeatability) was 90.0%, calculated from all positive inoculation levels. The concordance (reproducibility) was 85.0%, calculated from all positive inoculation levels. The concordance odds ratio (degree of interlaboratory variation calculated from all positive inoculation levels) was 1.58, indicating the robustness of the PCR method. Thus, the interlaboratory variation due to personnel, reagents, minor temperature or pH fluctuations and, not least, thermal cyclers, did not affect the performance of the method, which is currently being considered as part of an international PCR standard.

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Pokharel, Sunil, Lisa J. White, Jilian A. Sacks, Camille Escadafal, Amy Toporowski, Sahra Isse Mohammed, Solomon Chane Abera, Kekeletso Kao, Marcela De Melo Freitas et Sabine Dittrich. « Two-test algorithms for infectious disease diagnosis : Implications for COVID-19 ». PLOS Global Public Health 2, n^o 3 (31 mars 2022) : e0000293. http://dx.doi.org/10.1371/journal.pgph.0000293.

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Diagnostic assays for various infectious diseases, including COVID-19, have been challenged for their utility as standalone point-of-care diagnostic tests due to suboptimal accuracy, complexity, high cost or long turnaround times for results. It is therefore critical to optimise their use to meet the needs of users. We used a simulation approach to estimate diagnostic outcomes, number of tests required and average turnaround time of using two-test algorithms compared with singular testing; the two tests were reverse transcription polymerase chain reaction (RT-PCR) and an antigen-based rapid diagnostic test (Ag-RDT). A web-based application of the model was developed to visualise and compare diagnostic outcomes for different disease prevalence and test performance characteristics (sensitivity and specificity). We tested the model using hypothetical prevalence data for COVID-19, representing low- and high-prevalence contexts and performance characteristics of RT-PCR and Ag-RDTs. The two-test algorithm when RT-PCR was applied to samples negative by Ag-RDT predicted gains in sensitivity of 27% and 7%, respectively, compared with Ag-RDT and RT-PCR alone. Similarly, when RT-PCR was applied to samples positive by Ag-RDT, specificity gains of 2.9% and 1.9%, respectively, were predicted. The algorithm using Ag-RDT followed by RT-PCR as a confirmatory test for positive patients limited the requirement of RT-PCR testing resources to 16,400 and 3,034 tests when testing a population of 100,000 with an infection prevalence of 20% and 0.05%, respectively. A two-test algorithm comprising a rapid screening test followed by confirmatory laboratory testing can reduce false positive rate, produce rapid results and conserve laboratory resources, but can lead to large number of missed cases in high prevalence setting. The web application of the model can identify the best testing strategies, tailored to specific use cases and we also present some examples how it was used as part of the Access to Covid-19 Tools (ACT) Accelerator Diagnostics Pillar.

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Petrucelli, Monise Fazolin, Mariana Heinzen de Abreu, Bruna Aline Michelotto Cantelli, Gabriela Gonzalez Segura, Felipe Garcia Nishimura, Tamires Aparecida Bitencourt, Mozart Marins et Ana Lúcia Fachin. « Epidemiology and Diagnostic Perspectives of Dermatophytoses ». Journal of Fungi 6, n^o 4 (23 novembre 2020) : 310. http://dx.doi.org/10.3390/jof6040310.

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Dermatophytoses affect about 25% of the world population, and the filamentous fungus Trichophyton rubrum is the main causative agent of this group of diseases. Dermatomycoses are caused by pathogenic fungi that generally trigger superficial infections and that feed on keratinized substrates such as skin, hair, and nails. However, there are an increasing number of reports describing dermatophytes that invade deep layers such as the dermis and hypodermis and that can cause deep infections in diabetic and immunocompromised patients, as well as in individuals with immunodeficiency. Despite the high incidence and importance of dermatophytes in clinical mycology, the diagnosis of this type of infection is not always accurate. The conventional methods most commonly used for mycological diagnosis are based on the identification of microbiological and biochemical features. However, in view of the limitations of these conventional methods, molecular diagnostic techniques are increasingly being used because of their higher sensitivity, specificity and rapidity and have become more accessible. The most widely used molecular techniques are conventional PCR, quantitative PCR, multiplex PCR, nested, PCR, PCR-RFLP, and PCR-ELISA. Another promising technique for the identification of microorganisms is the analysis of protein profiles by MALDI-TOF MS. Molecular techniques are promising but it is necessary to improve the quality and availability of the information in genomic and proteomic databases in order to streamline the use of bioinformatics in the identification of dermatophytes of clinical interest.

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Aharonov, Ranit, Gila Lithwick Yanai, Hila Benjamin, Mats Olot Sanden, Marluce Bibbo, Craig Thurm, Laurie Horowitz et al. « New microRNA-based diagnostic test for lung cancer classification. » Journal of Clinical Oncology 30, n^o 15_suppl (20 mai 2012) : 10528. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.10528.

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10528 Background: Lung cancer is the leading cause of cancer deaths in the US. Treatment options are determined by tumor subtyping, for which there is lack of standardized, objective, and highly accurate techniques. In 20%-30% of cases significant limitations of tumor quantity and quality prevent full classification of the tumor using traditional diagnostic methods. Using microRNA microarray data generated from over a hundred formalin-fixed, paraffin-embedded (FFPE) primary lung cancer samples, we have identified microRNA expression profiles that differ significantly for the main lung cancer types. Based on these findings, we have developed and validated a microRNA-based qRT-PCR assay that differentiates primary lung cancers into four types: squamous cell carcinoma, non-squamous non-small cell lung cancer, carcinoid and small cell carcinoma. Methods: Over 700 primary tumor samples from different histological types of lung cancer were collected. Samples included FFPE blocks from resection or biopsies and cell blocks from cytology specimens including fine needle aspiration, bronchial brushing and bronchial washing. High-quality RNA was extracted from the samples using proprietary protocols. Expression levels of potential microRNA biomarkers were profiled using microarrays followed by a sensitive and specific qRT-PCR platform. An assay for lung tumors classification using 8 microRNAs on qRT-PCR was developed based on data from 261 samples. This assay was validated on an independent blinded set of 451 cytological and pathological samples. Results: Using the expression levels of 8 microRNAs measured in qRT-PCR, accurate classification of the primary lung tumors into the four main cancer types is obtained. The microRNA-based assay reached an accuracy of 94%. Moreover, cytological samples composed over 50% of the validation set and reached an accuracy of 95%. Conclusions: We present here a new microRNA-based assay for the classification of the four main types of lung cancer based only on the expression of 8 microRNAs. This assay displays very high levels of accuracy for both pathological and cytological samples. The assay comprises a standardized, well-tested and objective tool which can assist physicians in the diagnosis of lung cancer.

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Kristensen, Lasse Sommer, et Lise Lotte Hansen. « PCR-Based Methods for Detecting Single-Locus DNA Methylation Biomarkers in Cancer Diagnostics, Prognostics, and Response to Treatment ». Clinical Chemistry 55, n^o 8 (1 août 2009) : 1471–83. http://dx.doi.org/10.1373/clinchem.2008.121962.

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Abstract Background: DNA methylation is a highly characterized epigenetic modification of the human genome that is implicated in cancer. The altered DNA methylation patterns found in cancer cells include not only global hypomethylation but also discrete hypermethylation of specific genes. In particular, numerous tumor suppressor genes undergo epigenetic silencing because of hypermethylated promoter regions. Some of these genes are considered promising DNA methylation biomarkers for early cancer diagnostics, and some have been shown to be valuable for predicting prognosis or the response to therapy. Content: PCR-based methods that use sodium bisulfite–treated DNA as a template are generally accepted as the most analytically sensitive and specific techniques for analyzing DNA methylation at single loci. A number of new methods, such as methylation-specific fluorescent amplicon generation (MS-FLAG), methylation-sensitive high-resolution melting (MS-HRM), and sensitive melting analysis after real-time methylation-specific PCR (SMART-MSP), now complement the traditional PCR-based methods and promise to be valuable diagnostic tools. In particular, the HRM technique shows great potential as a diagnostic tool because of its closed-tube format and cost-effectiveness. Summary: Numerous traditional and new PCR-based methods have been developed for detecting DNA methylation at single loci. All have characteristic advantages and disadvantages, particularly with regard to use in clinical settings.

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Dalla Valle, L., L. Zanella, P. Patarnello, L. Paolucci, P. Belvedere et L. Colombo. « Development of a sensitive diagnostic assay for fish nervous necrosis virus based on RT-PCR plus nested PCR ». Journal of Fish Diseases 23, n^o 5 (septembre 2000) : 321–27. http://dx.doi.org/10.1046/j.1365-2761.2000.00255.x.

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Peng, Xiaoxiao, Weidong Li, Wenquan Wang et Genben Bai. « Identification ofLonicera japonicaby PCR-RFLP and Allele-Specific Diagnostic PCR Based on Sequences of Internal Transcribed Spacer Regions ». Planta Medica 76, n^o 05 (20 octobre 2009) : 497–99. http://dx.doi.org/10.1055/s-0029-1186235.

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Staudacher, K., P. Pitterl, L. Furlan, P. C. Cate et M. Traugott. « PCR-based species identification of Agriotes larvae ». Bulletin of Entomological Research 101, n^o 2 (1 novembre 2010) : 201–10. http://dx.doi.org/10.1017/s0007485310000337.

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AbstractClick beetle larvae within the genus Agriotes (Coleoptera: Elateridae), commonly known as wireworms, are abundant ground-dwelling herbivores which can inflict considerable damage to field crops. In Central Europe up to 20 species, which differ in their distribution, ecology and pest status, occur in arable land. However, the identification of these larvae based on morphological characters is difficult or impossible. This hampers progress towards controlling these pests. Here, we present a polymerase chain reaction (PCR)-based approach to identify, for the first time, 17 Agriotes species typically found in Central Europe. Diagnostic sequence information was generated and submitted to GenBank, allowing the identification of these species via DNA barcoding. Moreover, multiplex PCR assays were developed to identify the nine most abundant species rapidly within a single-step reaction: Agriotes brevis, A. litigiosus, A. obscurus, A. rufipalpis, A. sordidus, A. sputator, A. ustulatus, A. lineatus and A. proximus. The latter two species remain molecularly indistinguishable, questioning their species status. The multiplex PCR assays proved to be highly specific against non-agrioted elaterid beetles and other non-target soil invertebrates. By testing the molecular identification system with over 900 field-collected larvae, our protocol proved to be a reliable, cheap and quick method to routinely identify Central European Agriotes species.

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Nisavic, Jakov, Nenad Milic, Andrea Zoric, Jovan Bojkovski et Aleksandar Stanojkovic. « The application of PCR based methods in diagnostics of some viral infections of swine ». Biotehnologija u stocarstvu 32, n^o 4 (2016) : 321–29. http://dx.doi.org/10.2298/bah1604321n.

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Viral infections of swine cause significant economic losses in swine husbandry. They manifest in death of infected animals of different ages or in decreased productivity during the manufacturing process. Having that in mind, rapid and reliable diagnostics of viral infections is crucial in the prevention of disease transmission in herds of swine. Today, virological laboratories all over the world use different diagnostic methods such as isolation of virus in cell lines, ELISA, virus neutralization test, direct and indirect immunofluorescence and hemagglutination and hemagglutination inhibition tests. Virus isolation, virus neutralization test and some other standard virological methods are time consuming and rather expensive, therefore, molecular methods such as conventional PCR, RT - PCR, real-time PCR and direct sequencing methods are applied worldwide as fast and reliable. Their application is especially necessary for the detection of viruses which cannot be identified by using standard virological methods.

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Kulichenko, A. N., et N. S. Sarkisyan. « To the question regarding accuracy of COVID-2019 laboratory diagnostics ». Russian Journal of Infection and Immunity 11, n^o 1 (28 février 2021) : 9–16. http://dx.doi.org/10.15789/2220-7619-ttq-1622.

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Issues of accuracy (sensitivity and specificity) of PCR-analysis depending on features of performing preanalytical and analytical stages of laboratory diagnostics of COVID-19, as well as comparing PCR and lung computed tomography (CT) results have been analyzed in the study. Currently, a molecular genetic test based on polymerase chain reaction (PCR) is used for diagnostics of a new coronavirus infection (COVID-19). As of November 1, 2020, more than 750 million PCR tests have been conducted globally. Evidence accumulated by now allows to estimate diagnostic sensitivity and specificity of the SARS-CoV-2-specific PCR as high as 82—91% and 99—100%, respectively. In addition, increased PCR sensitivity may be noted upon performing repeated testing of the upper respiratory tract samples comprising 82.2% during the primary analysis that was further elevated up to 90.6% after two consecutive tests. A whole set of factors affect the PCR accuracy. In particular, false negative data might result from insufficient amount of virus-coupled genetic material in the sample, timeframe and mistakes made upon selecting biological samples. It was found that SARS-CoV-2 virus RNA was detected at the maximum diagnostic sensitivity in the upper respiratory tract 1—3 days before the onset of symptoms and sustained within the 5—6 days after disease onset. Such period of time is associated with the peak risk of SARS-CoV-2 transmission. On week 2 after disease onset, there have been noted elevated rate of detecting viral RNA in bronchopulmonary samples. The duration of detecting virus-related markers (including those found in the absence of viable virus forms) correlates with disease severity and may last for as long as 1—2 months. Another real-world issue related to PCR analysis is posed by an opportunity of obtaining false positive data, which solution requires high level organized laboratory research, especially in case large-scale studies. Upon that, it is worth noting that positive PCR results may account for detecting solely certain RNA-related fragments present in any sample, rather than a viable virus. It was noted that PCR in comparison to CT analysis demonstrates higher specificity, but does not allow to distinguish pneumonia caused by SARS-CoV-2 from pneumonia caused by other etiological agents (up to 25% false positive results). However, the diagnostic CT sensitivity was 97.2% that exceeds such parameter for PCR by 10—15%. It was concluded that the approach combining use of both PCR and CT by taking into account their own features as well as factors affecting the accuracy of the data obtained, allows us to correctly interpret the diagnostical results.

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Price, Jessica S., Melissa Fallon, Raquel Posso, Matthijs Backx et P. Lewis White. « An Evaluation of the OLM CandID Real-Time PCR to Aid in the Diagnosis of Invasive Candidiasis When Testing Serum Samples ». Journal of Fungi 8, n^o 9 (3 septembre 2022) : 935. http://dx.doi.org/10.3390/jof8090935.

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Background: Treatment for invasive candidiasis (IC) is time-critical, and culture-based tests can limit clinical utility. Nonculture-based methods such as Candida PCR represent a promising approach to improving patient management but require further evaluation to understand their optimal role and incorporation into clinical algorithms. This study determined the performance of the commercially available OLM CandID real-time PCR when testing serum and developed a diagnostic algorithm for IC. Methods: The study comprised a retrospective performance evaluation of the CandID real-time PCR assay when testing surplus serum (n = 83 patients, 38 with IC), followed by a prospective consecutive cohort evaluation (n = 103 patients, 24 with IC) post incorporation into routine service. A combined diagnostic algorithm, also including (1-3)-β-D-Glucan testing, was generated. Results: Prospective CandID testing generated a sensitivity/specificity of 88%/82%, respectively. Specificity was improved (>95%) when both PCR replicates were positive and/or the patient had multiple positive samples. When combining CandID with (1-3)-β-D-Glucan testing, the probability of IC when both were positive or negative was >69% or <1%, respectively. Conclusions: The CandID provides excellent performance and a rapid time-to-result using methods widely available in generic molecular diagnostic laboratories. By combining nonculture diagnostics, it may be possible to accurately confirm or exclude IC.

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Ferreira, Beatriz Iandra da Silva, Natália Lins da Silva-Gomes, Wagner Luis da Costa Nunes Pimentel Coelho, Vanessa Duarte da Costa, Vanessa Cristine de Souza Carneiro, Rafael Lopes Kader, Marisa Pimentel Amaro et al. « Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting ». PLOS ONE 16, n^o 11 (22 novembre 2021) : e0260087. http://dx.doi.org/10.1371/journal.pone.0260087.

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The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.

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