SARS-CoV-2 direct real-time polymerase chain reaction testing in laboratories with shortage challenges

Aim: In our study, we propose the use of direct real-time polymerase chain reaction (RT-PCR), this test does not require extraction or a preheating step, which saves a lot of cost, labor, processing time and provides a solution for supply shortage. Materials & methods: We assayed 185 nasopharyngeal samples stored in viral transport media. The indirect method was done with RNA extraction step, and the direct RT-PCR was done without an extraction step, both assays were evaluated on a commercially validated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) kit targeting E gene. Results: Our assay showed a sensitivity of 79%, a specificity of 100% and the agreement between methods was 72%. Conclusion: Overall, this simple direct RT-PCR approach can be utilized as a qualitative diagnostic tool for emergency SARS-CoV-2 surveillance in countries with limited resources and may help laboratories to continue testing and at greater frequency despite supply shortages, although with delay in cycle threshold value in comparison with indirect RT-PCR.

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1504: Molecular Diagnosis and Staging of Prostate Cancer Using Clusterin in Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

The Journal of Urology ◽

10.1016/s0022-5347(18)33708-x ◽

2006 ◽

Vol 175 (4S) ◽

pp. 485-486

Author(s):

Sabarinath B. Nair ◽

Christodoulos Pipinikas ◽

Roger Kirby ◽

Nick Carter ◽

Christiane Fenske

Keyword(s):

Prostate Cancer ◽

Polymerase Chain Reaction ◽

Real Time ◽

Molecular Diagnosis ◽

Reverse Transcription ◽

Rt Pcr ◽

Chain Reaction ◽

Polymerase Chain

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Investigation of Different Commercially Available Real Time-Polymerase Chain Reaction Kits for SARS-CoV-2 Diagnosis

JOURNAL FOR CLINICAL AND DIAGNOSTIC RESEARCH ◽

10.7860/jcdr/2021/48726.14977 ◽

2021 ◽

Author(s):

Rajeev Kumar Jain ◽

Nagaraj Perumal ◽

Rakesh Shrivastava ◽

Kamlesh Kumar Ahirwar ◽

Jaya Lalwani ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Internal Control ◽

Housekeeping Genes ◽

Housekeeping Gene ◽

Specific Gene ◽

Rt Pcr ◽

Chain Reaction ◽

Gene Target ◽

Polymerase Chain

Introduction: The whole world is facing an ongoing global health emergency of COVID-19 disease caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is a gold standard in the detection of SARS-CoV-2 infection. Presently, many single tube multiple gene target RT-PCR kits have been developed and are commercially available for Coronavirus Disease 2019 (COVID-19) diagnosis. Aim: To evaluate the performance of seven COVID-19 RT-PCR kits (DiagSure, Meril, VIRALDTECT II, TruPCR, Q-line, Allplex and TaqPath) which are commercially available for COVID-19 RT-PCR diagnosis. Materials and Methods: This observational study was conductedat the State Virology Laboratory (SVL), Gandhi Medical College, Bhopal, Madhya Pradesh, India. Seven commercially available kits have been evaluated on the basis of: (i) number of SARS-CoV-2 specific gene target; (ii) human housekeeping genes as internal control; (iii) RT-PCR run time; and (iv) kit performances to correctly detect SARS-CoV-2 positive and negative RNA samples. A total of 50 RNA samples (left over RNA) were included, master mix preparation, template addition and RT-PCR test has been performed according to kits literature. At the end of PCR run, mean and standard deviation of obtained cut-off of all kits were calculated using Microsoft Excel. Results: All seven RT-PCR kits performed satisfactory regarding the reproducibility and they could correctly identify 30 positive and 20 negative RNA samples. RNA samples (group C) having low viral loads with a high Cycle threshold (Ct) value (>30) were also detected by all these seven kits. Obtained Ct values of each group was in parallel range in comparison with the initial testing Ct values. Kits were found to be superior which contains primers and probes for three SARS-CoV-2 specific gene targets, have human housekeeping gene as internal control and taking less time to complete RT-PCR. Conclusion: All seven COVID-19 RT-PCR kits included in this study demonstrated satisfactory performance and can be used for the routine molecular diagnosis of COVID-19 disease.

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Sensitive SYBR Green—Real Time PCR for the Detection and Quantitation of Avian Rotavirus A

Veterinary Sciences ◽

10.3390/vetsci6010002 ◽

2018 ◽

Vol 6 (1) ◽

pp. 2 ◽

Cited By ~ 2

Author(s):

David De la Torre ◽

Claudete Astolfi-Ferreira ◽

Ruy Chacon ◽

Antonio Piantino Ferreira

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Sybr Green ◽

Molecular Techniques ◽

Economic Losses ◽

Rt Pcr ◽

Chain Reaction ◽

Rotavirus A ◽

Avian Nephritis Virus ◽

Polymerase Chain

Avian rotavirus A (ARtV-A) is a virus that affects young birds, causing acute diarrhea and economic losses in the poultry industry worldwide. The techniques used for the diagnosis of ARtV-A include electron microscopy, isolation in cell culture, and serology, as well as molecular techniques, such as the reverse transcription-polymerase chain reaction (RT-PCR). The objective of this work was to standardize a real-time RT-polymerase chain reaction (RT-qPCR) using SYBR Green chemistry for the rapid detection and quantification of ARtV-A from bird tissues and materials fixed on FTA cards on the basis of the nucleotide sequence of segment 6 (S6), which codes for the structural VP6 protein of ARtV-A. The results show the efficient amplification of the proposed target, with a limit of detection (LoD) of one copy gene (CG) per microliter of cDNA and a limit of quantification (LoQ) of 10 CGs per microliter. The efficiency of the primers was determined to be 95.66% using a standard curve, with an R2 value of 0.999 and a slope of −3.43. The specificity was determined using samples coinfected with ARtV-A, the chicken parvovirus, the chicken astrovirus, and the avian nephritis virus as positive controls and commercially available vaccines of the infectious bronchitis virus, infectious bursa disease virus, avian reovirus and healthy organs as negative controls. This technique, which lacks nonspecific PCR products and dimers, demonstrated greater sensitivity and specificity than conventional RT-PCR, and it reduced the analysis time by more than 50%.

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Accuracy of real-time polymerase chain reaction to detect Schistosoma mansoni – infected individuals from an endemic area with low parasite loads

Parasitology ◽

10.1017/s003118202000089x ◽

2020 ◽

Vol 147 (10) ◽

pp. 1140-1148

Author(s):

Fernanda do Carmo Magalhães ◽

Samira Diniz Resende ◽

Carolina Senra ◽

Carlos Graeff-Teixeira ◽

Martin Johannes Enk ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Schistosoma Mansoni ◽

Real Time ◽

Diagnostic Methods ◽

Control Measures ◽

Rt Pcr ◽

Chain Reaction ◽

Cross Sectional ◽

Intestinal Schistosomiasis ◽

Polymerase Chain

AbstractDue to the efforts to control schistosomiasis transmission in tropical countries, a large proportion of individuals from endemic areas present low parasite loads, which hinders diagnosis of intestinal schistosomiasis by the Kato-Katz (KK) method. Therefore, the development of more sensitive diagnostic methods is essential for efficient control measures. The aim was to evaluate the accuracy of a real-time polymerase chain reaction (RT-PCR) to detect Schistosoma mansoni DNA in fecal samples of individuals with low parasite loads. A cross-sectional population-based study was conducted in a rural community (n = 257) in Brazil. POC-CCA® was performed in urine and feces were used for RT-PCR. In addition, fecal exams were completed by 18 KK slides, saline gradient and Helmintex techniques. The combined results of the three parasitological tests detected schistosome eggs in 118 participants (45.9%) and composed the consolidated reference standard (CRS). By RT-PCR, 117 out of 215 tested samples were positive, showing 91.4% sensitivity, 80.2% specificity and good concordance with the CRS (kappa = 0.71). RT-PCR identified 86.9% of the individuals eliminating less than 12 eggs/g of feces, demonstrating much better performance than POC-CCA® (50.8%). Our results showed that RT-PCR is a valuable alternative for the diagnosis of intestinal schistosomiasis in individuals with very low parasite loads.

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Evaluation of a 384–Well Format for High-Throughput Real-Time Reverse Transcription Polymerase Chain Reaction for Avian Influenza Testing

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870902100513 ◽

2009 ◽

Vol 21 (5) ◽

pp. 679-683 ◽

Cited By ~ 4

Author(s):

Pamela J. Ferro ◽

Jason Osterstock ◽

Bo Norby ◽

Geoffrey T. Fosgate ◽

Blanca Lupiani

Keyword(s):

Polymerase Chain Reaction ◽

Avian Influenza ◽

Real Time ◽

High Throughput ◽

Reverse Transcription ◽

Virus Isolation ◽

Rt Pcr ◽

Chain Reaction ◽

Response Planning ◽

Polymerase Chain

As concerns over the global spread of highly pathogenic avian influenza H5N1 have heightened, more countries are faced with increased surveillance efforts and incident response planning for handling a potential outbreak. The incorporation of molecular techniques in most diagnostic laboratories has enabled fast and efficient testing of many agents of concern, including avian influenza. However, the need for high-throughput testing remains. In this study, the use of a 384–well format for high-throughput real-time reverse transcription polymerase chain reaction (real-time RT-PCR) testing for avian influenza is described. The analytical sensitivity of a real-time RT-PCR assay for avian influenza virus matrix gene with the use of both 96– and 384–well assay formats and serial dilutions of transcribed control RNA were comparable, resulting in similar limits of detection. Of 28 hunter-collected cloacal swabs that were positive by virus isolation, 26 (92.9%) and 27 (96.4%) were positive in the 96– and 384–well assays, respectively; of the 340 hunter-collected swabs that were negative by virus isolation, 45 (13.2%) and 23 (6.8%) were positive in the 96– and 384–well assays, respectively. The data presented herein supports the utility of the 384–well format in the event of an avian influenza outbreak for high-throughput real-time RT-PCR testing.

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Collaborative Trial for Validation of a Real-Time Reverse Transcriptase-Polymerase Chain Reaction Assay for Detection of Central Nervous System Tissues as Bovine Spongiform Encephalopathy RiskMaterial: Part 1

Journal of AOAC International ◽

10.1093/jaoac/89.5.1335 ◽

2006 ◽

Vol 89 (5) ◽

pp. 1335-1340

Author(s):

Amir Abdulmawjood ◽

Holger Schnenbrcher ◽

Michael BÜlte

Keyword(s):

Central Nervous System ◽

Polymerase Chain Reaction ◽

Real Time ◽

Meat Products ◽

Spongiform Encephalopathy ◽

Rt Pcr ◽

Chain Reaction ◽

Pcr Assay ◽

Heat Treated ◽

Polymerase Chain

Abstract A collaborative trial was conducted to evaluate a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection of central nervous system (CNS) tissues in meat products (e.g., sausages). The method is based on the detection of ruminant glial fibrillary acidic protein (GFAP) mRNA by applying real-time RT-PCR. The assay was evaluated through a multicenter trial involving 12 participating laboratories that received coded cDNA obtained from 3 different types of sausages. The participants used 5 different real-time detection systems. The results obtained in this validation revealed that this real-time RT-PCR assay performed well in the different laboratories with a detection limit of at least 0.1% CNS in those test materials that contained strongly heat-treated samples (sausages cooked at 120C) and the medium heat-treated samples (sausages cooked at 80C). The detection limit of liver sausages was determined to be 0.2% of CNS. Neither the samples with no CNS additive nor the bovine DNA and the negative control containing 100% swine brain gave any positive signals. The presented results indicate that the real-time RT-PCR assay was just as reproducible between laboratories, as repeatable within a laboratory, could reliably be used for detection of bovine spongiform encephalopathy risk material in meat and meat products, and signify that it may be used with confidence in any laboratory.

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The utility of real-time polymerase chain reaction in detecting Coccidioides immitis among clinical specimens in the Central California San Joaquin Valley

Medical Mycology ◽

10.1093/mmy/myy111 ◽

2018 ◽

Vol 57 (6) ◽

pp. 688-693 ◽

Cited By ~ 3

Author(s):

Dominic Dizon ◽

Marilyn Mitchell ◽

Bernadette Dizon ◽

Robert Libke ◽

Michael W Peterson

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Pleural Fluid ◽

Clinical Setting ◽

Predictive Value ◽

Coccidioides Immitis ◽

Rt Pcr ◽

Chain Reaction ◽

Acute Pneumonia ◽

Polymerase Chain

AbstractCoccidioidomycosis, the fungal infection caused by dimorphic Coccidioides species, is typically diagnosed by histopathologic identification of spherules, by culture, or by serology. These tests are reliable but time-intensive, delaying diagnosis and treatment. Rapid real-time polymerase chain reaction (RT-PCR) can be performed and was validated to identify Coccidioides immitis using an in-house developed assay for the Becton Dickinson molecular instrument (BD MAXTM). These studies were performed using patient samples that had been shown to be positive on previously set up fungal cultures. To evaluate this new RT-PCR test in the clinical setting, we conducted a retrospective chart review of patients (N = 1160) who underwent Coccidioides PCR (Cocci PCR) on clinical samples between March 1, 2014, and Dec 31, 2016. We abstracted clinical, microbiologic, serologic, radiographic, treatment, and follow-up data. Specimens of cerebrospinal fluid (CSF), bronchioalveolar lavage fluid (BAL), lung tissue biopsy (LTB), sputum, and pleural fluid were evaluated to determine sensitivity and specificity. Of the 113 specimens that tested positive for Cocci PCR, all had clinical disease defined by traditional clinical criteria, yielding 100% specificity. Overall sensitivity was 74% versus 46% for fungal culture and was available in 4 hours rather than 1–2 weeks. Sensitivities varied by source material and clinical setting. CSF had a sensitivity of 59%, BAL for acute pneumonia 91%, sputum for acute pneumonia 94%, pleural fluid 86%, but LTB for lung nodules only 44%. Overall positive predictive value (PPV) was 100%, while negative predictive value (NPV) was 96%, but again this varied by specimen and clinical setting. Our experience with clinical testing of >1160 specimens over 2–3 years shows we can utilize this technology to improve our ability to diagnose disease but that the sensitivity varies by specimen source and clinical setting.

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Improved RNA Extraction from Woody Plants for the Detection of Viral Pathogens by Reverse Transcription-Polymerase Chain Reaction

Plant Disease ◽

10.1094/pdis.1997.81.2.222 ◽

1997 ◽

Vol 81 (2) ◽

pp. 222-226 ◽

Cited By ~ 262

Author(s):

Donald J. MacKenzie ◽

Morven A. McLean ◽

Srima Mukerji ◽

Margaret Green

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Woody Plants ◽

Rna Extraction ◽

Viral Rna ◽

Rt Pcr ◽

Chain Reaction ◽

Apple Stem ◽

Spin Column ◽

Polymerase Chain

An efficient procedure for the extraction of high-quality RNA from woody plants without the use of phenol, organic solvents, or alcohol precipitation is described. The method employs commercially available spin-column matrices and mitigates the inhibitory effects of plant polysaccharides and polyphenolic compounds commonly observed on subsequent polymerase chain reaction amplification when conventional extraction methods are applied to woody plant species. The method described has been successfully used in the development of highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) techniques for the detection of a number of viruses in their woody hosts. The viruses detected included apple stem grooving capillovirus (ASGV), apple stem pitting virus, Prunus necrotic ringspot ilarvirus (PNRSV), grapevine fanleaf and Arabis mosaic nepoviruses, and grapevine leafroll-associated closterovirus type 3. The method described was equally effective for the extraction of viral RNA from either budwood, leaves, or flower blossoms as determined by the equivalent RT-PCR detection of ASGV and PNRSV from these tissues. Detection of viral RNA in samples of total plant RNA prepared using this method was found to be as sensitive as was previously described for the immunocapture RT-PCR technique.

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