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

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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Real-time reverse transcription-polymerase chain reaction for detection of SYT-SSX translocation in synovial sarcoma

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.9553 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 9553-9553

Author(s):

J. Liu ◽

K. Qu ◽

C. Chai ◽

H. Li ◽

A. Sferruzza ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Synovial Sarcoma ◽

Internal Control ◽

Reverse Transcription ◽

Gel Electrophoresis ◽

Rt Pcr ◽

Chain Reaction ◽

Pcr Assay ◽

Polymerase Chain

9553 Background: Synovial sarcoma is the most common non-rhabdomyosarcomatous soft tissue sarcoma in children and adolescents. A specific translocation, t(X;18), induces fusion of the SYT gene on chromosome 18 to an SSX gene on chromosome X. The resulting fusion gene consists of at least 2 subtypes with different breakpoints: SYT-SSX1(X;18)(p11.23;q11.2) and SYT-SSX2 (X;18)(p11.21;q11.2). Because t(X;18) transcripts occur in >90% of synovial sarcoma subtypes, this marker may be useful for diagnosis. We evaluated the accuracy of a multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for detection of the primary SYT-SSX fusion transcript types in formalin-fixed, paraffin-embedded (FFPE) tissues and frozen tissues. Methods: 17 tumors (7 synovial sarcomas, 4 Ewing’s sarcomas, 5 rhabdomyosarcomas, 1 small round blue-cell tumor), 4 normal tissues, and 4 control samples were tested for SYT-SSX translocations using real-time RT-PCR. Results were compared to those obtained with gel electrophoresis detection of amplified transcripts; discrepant results were confirmed by sequencing. Results: Concordance between real time RT-PCR and gel electrophoresis was 100% (25/25) for internal control genes and SYT-SSX1, and 92% (23/25) for SYT-SSX2. Of the 2 samples with discordant SYT-SSX2 results, 1 was positive by real-time RT-PCR but not gel electrophoresis and 1 was positive by electrophoresis but not real-time RT-PCR; in both cases, DNA sequencing confirmed the real-time RT-PCR results. The minimum percentage of tumor to normal cells required for detection of SYT-SSX fusion transcripts by real-time RT-PCR was 6.25%. Conclusions: This real-time RT-PCR assay appears to provide greater accuracy than gel electrophoresis for identification of SYT-SSX translocation and fusion types. No significant financial relationships to disclose.

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Comparison of SARS-CoV-2 viral load in saliva samples in symptomatic and asymptomatic cases

10.1101/2021.02.12.21251229 ◽

2021 ◽

Author(s):

Anuja Bhatta ◽

Rebecca Henkhaus ◽

Heather L. Fehling

Keyword(s):

Polymerase Chain Reaction ◽

Viral Load ◽

Reverse Transcription ◽

Specific Gene ◽

Rt Pcr ◽

Chain Reaction ◽

Gene Target ◽

Viral Loads ◽

Ct Value ◽

Polymerase Chain

AbstractSARS-CoV-2 infections can be symptomatic as well as asymptomatic. In this study, we analyzed 460,814 saliva samples collected from July 2020 to January 2021 for a SARS-CoV-2-specific gene target using the FDA EUA test, CRL Rapid Response™, based on reverse transcription polymerase chain reaction (RT-PCR). We measured SARS-CoV-2 viral loads using cycle threshold (Ct) values. A total of 17,813 samples tested positive for COVID-19 using self-collected saliva samples. The Ct values ranged from 11 to 40, 91.3% distributed between 22 to 38 Ct. We then compared Ct values for symptomatic and asymptomatic cases for all positive saliva samples. A total of 8,706 cases were symptomatic with an average Ct value of 29.24, and 9,107 cases were asymptomatic with an average Ct value of 30.99. Hence, SARS-CoV-2 viral loads (Ct) in saliva samples for both symptomatic and asymptomatic cases are similar.

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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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Detection of specific mRNAs in single nephron segments by use of the polymerase chain reaction

AJP Renal Physiology ◽

10.1152/ajprenal.1990.258.5.f1470 ◽

1990 ◽

Vol 258 (5) ◽

pp. F1470-F1474 ◽

Cited By ~ 4

Author(s):

T. Moriyama ◽

H. R. Murphy ◽

B. M. Martin ◽

A. Garcia-Perez

Keyword(s):

Polymerase Chain Reaction ◽

Aldose Reductase ◽

Collecting Duct ◽

Specific Gene ◽

Rt Pcr ◽

Chain Reaction ◽

Nephron Segments ◽

Single Nephron ◽

Specific Mrnas ◽

Polymerase Chain

We have developed a procedure to detect specific mRNAs in single renal nephron segments. This approach combines microdissection, reverse transcription (RT) of the target mRNA, and amplification of the resulting cDNA using the polymerase chain reaction (PCR). After microdissection, the sample is placed in a tube where it is permeabilized and where all reactions are performed directly without the need for isolation of the RNA. Our model target was the mRNA for aldose reductase. This enzyme catalyzes the conversion of glucose to sorbitol. Its expression is modulated by changes in extracellular osmolality in the renal medulla. RT-PCR of inner medullary collecting duct (1 mm) and glomeruli (6-10) yielded a product of the predicted length (670 base pairs) defined by the PCR primers. Its identity was confirmed by a specific oligonucleotide probe that differed from the primers. RT-PCR of proximal tubules (1 mm) resulted in no aldose reductase-specific amplification product. RT-PCR is generally applicable for measuring specific gene expression in single nephron segments or small numbers of cultured cells. Utility, limitations, and refinements of this approach are discussed.

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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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