Commercial RT-PCR Kits Research Articles

Comparison of seven commercial RT-PCR kits with the NIV kit for the diagnosis of Covid-19

Objectives Coronavirus disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spread across the globe in an unprecedented manner and was declared a pandemic on March 11, 2020 by the World Health Organization (WHO). This study was carried out with the aim to compare the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and agreement of the eight different RT-PCR kits for the diagnosis of COVID-19. Material and Methods This observational cross-sectional study was carried out in the Department of Microbiology of a tertiary care hospital in Central Delhi from July to October 2021. A total of 45 nasopharyngeal and/or oropharyngeal swabs in Viral Transport Medium (VTM) from suspected COVID-19 patients were received in the laboratory for RT-PCR. These samples were tested by eight different Indian Council of Medical Research (ICMR)-approved RT-PCR kits with different gene targets. The comparison was made with the National Institute of Virology (NIV), the Pune COVID-19 RT-PCR kit. Statistical analysis: sensitivity, specificity, PPV, and NPV were calculated for each kit and compared using the McNemar test. Agreement of different kits was evaluated using Kappa analysis. Results The results of the 45 samples of suspected COVID-19 cases were recorded as per the cycle threshold (Ct) provided in the kit insert. Of these, 15 samples detected both E and RdRp genes and 30 were negative for both the genes of SARS CoV-2 by NIV, the Pune COVID-19 RT-PCR kit. All kits showed 100% sensitivity and had 100% NPV when compared with the NIV kit. However, specificity, PPV, and agreement were variable as compared to the NIV kit. Conclusion The reporting should be carried out as per the manufacturer’s instructions. However, positive results with Ct values ≥ 36 showed variable results with different RT PCR kits and hence should be interpreted with caution.

Spike S2 Subunit: Possible Target for Detecting Novel SARS-CoV-2 Variants with Multiple Mutations.

Novel SARS-CoV-2 variants have multiple mutations that may impact molecular diagnostics. The markedly conserved S2 subunit may be utilized to detect new variants. A comparison of 694 specimens (2019-2022) in Thailand using a commercial RT-PCR kit and the kit in combination with S2 primers and a probe was performed. Delayed amplification in ORF1ab was detected in one BA.4 omicron, whereas no amplification problem was encountered in the S2 target. There were no statistically significant differences in mean Ct value between the target genes (E, N, ORF1ab, and S2) and no significant differences in mean Ct value between the reagents. Furthermore, 230,821 nucleotide sequences submitted by 20 representative counties in each region (Jan-Oct 2022) have been checked for mutations in S2 primers and probe using PrimerChecker; there is a very low chance of encountering performance problems. The S2 primers and probe are still bound to the top five currently circulating variants in all countries and Thailand without mismatch recognition (Jun-Nov 2023). This study shows the possible benefits of detecting S2 in combination with simultaneously detecting three genes in a kit without affecting the Ct value of each target. The S2 subunit may be a promising target for the detection of SARS-CoV-2 variants with multiple mutations.

Establishment and Application of a Triplex Real-Time RT-PCR Assay for Differentiation of PEDV, PoRV, and PDCoV.

Porcine viral diarrhea is very common in clinical practice and has caused huge losses to the pig industry. Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are important pathogens of porcine viral diarrhea. Co-infection situations among these three viruses in clinics are common, which increases the difficulty of differential diagnosis. Currently, polymerase chain reaction (PCR) is commonly used to detect pathogens. TaqMan real-time PCR is more sensitive than conventional PCR and has better specificity and accuracy. In this study, a triplex real-time RT-PCR assay based on TaqMan probes was developed for differential detection of PEDV, PoRV, and PDCoV. The triplex real-time RT-PCR assay developed in this study could not detect unrelated pathogens and showed satisfactory specificity, sensitivity, repeatability, and reproducibility with a limit of detection (LOD) of 6.0 × 101 copies/μL. Sixteen clinical samples were used to compare the results of the commercial RT-PCR kit and the triplex RT-PCR for PEDV, PoRV, and PDCoV detection, and the results were completely consistent. A total of 112 piglet diarrhea samples collected from Jiangsu province were next used to study the local prevalence of PEDV, PoRV, and PDCoV. The positive rates of PEDV, PoRV, and PDCoV detected by the triplex real-time RT-PCR were 51.79% (58/112), 59.82% (67/112), and 2.68% (3/112), respectively. The co-infections of PEDV and PoRV were frequent (26/112, 23.21%), followed by the co-infections of PDCoV and PoRV (2/112, 1.79%). This study established a useful tool for simultaneous differentiation of PEDV, PoRV, and PDCoV in practice and provided valuable information on the prevalence of these diarrhea viral pathogens in Jiangsu province.

Evaluation of RT-PCR assays for detection of SARS-CoV-2 variants of concern

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic has been considered with great importance on correct screening procedure. The detection efficiency of recent variants of concern were observed by comparing 5 commercial RT-PCR kits and a SYBR-green method developed and validated in our laboratory. The RNA was extracted from nasopharyngeal samples from suspected COVID-19 patients and RT-PCR assay was performed according to the instruction of the respective manufacturers. The specificity and sensitivity of Maccura kit was 81.8% and 82.5%, A*Star kit was 100% and 75.4%, Da An Gene kit was 100% and 68.4%, Sansure kit was 54.5% and 91.2% and TaqPath kit was 100% and 70.2% respectively. Our in house SYBR-Green method showed a consistent detection result with 90.9% specificity and 91.2% sensitivity. We also found that detection kits targeting more genes showed better accuracy which facilitates less false positive results (< 20%). Our study found a significant difference (p < 0.005) in Ct value reported for common target genes shared by the RT-PCR kits in relation with different variants of COVID-19 infection. Recent variants of concerns contain more than 30 mutations in the spike proteins including 2 deletion and a unique insertion mutation by which makes detection of these variants difficult and these facilitates the variants to escape from being detected.

A report on preparation, expansion and future outlook of COVID-19 testing in Gambia.

BackgroundThe outbreak of COVID-19 disease and rapid spread of the virus outside China led to its declaration as a Public Health Emergency of International Concern (PHEIC) in January 2020. Key elements of the early intervention strategy focused on laboratory diagnosis and screening at points of entry and imposition of restrictions in crossborder activities.ObjectiveWe report the role the Medical Research Council Unit, The Gambia (MRCG) played in the early implementation of molecular testing for COVID-19 in The Gambia as part of the national outbreak response.MethodsLaboratory staff members, with experience in molecular biology assays, were identified and trained on COVID-19 testing at the Africa CDC training workshop in Dakar, Senegal. Thereafter risks assessments, drafting of standard operating procedures (SOPs) and inhouse training enabled commencement of testing using commercial RTPCR kits. Subsequently, testing was expanded to the National Public Health Laboratroy and also implemented across field sites for rapid response across the country.ResultsCapacity for COVID-19 testing at MRCG was developed and can process aproximately 350 tests per day, which can be further scaled up as the demand for testing increases.ConclusionThe long presence of the Unit in The Gambia and strong collaborative relationship with the National Health Ministry, allowed for a synergistc approach in mounting an effective response that contributed in delaying the establishment of community transmission in the country.

Comparison between five PCR techniques for the diagnosis of SARS-CoV-2.

IntroducciónDesde que aparecieron los primeros casos de SARS-CoV-2 son numerosas las técnicas que se han desarrollado para el diagnóstico o seguimiento de la infección, tanto técnicas directas como serológicas. La elección de una buena herramienta diagnóstica es fundamental para el control epidemiológico. El objetivo ha sido comparar cinco técnicas comercializadas de RT-PCR a tiempo real, en sensibilidad, especificidad y concordancia para la detección del SARS-CoV-2.Material y métodosSe compararon cinco kits comerciales de RT-PCR para la detección del SARS-CoV-2. Se tomaron ocho muestras positivas conocidas que se sometieron a siete diluciones o concentraciones diferentes y otras 135 muestras negativas para determinar valores de sensibilidad, especificidad y concordancia.ResultadosLa sensibilidad, especificidad, valor predictivo positivo (VPP) y valor predictivo negativo (VPN) para las técnicas de Palex, Roche y GeneXpert respecto a Seegene fueron idénticas, correspondientes a 98,21%, 100%, 100% y 99,26% respectivamente. Para Becton Dickinson la sensibilidad fue del 89,28%, la especificidad del 100%, el VPP del 100% y el VPN del 95,74%. La concordancia mediante el índice Kappa para Palex, Roche y GeneXpert fue del 0,9892, mientras que la concordancia para Becton Dickinson fue con un índice Kappa de 0,9215.ConclusiónTodos los kits de RT-PCR comerciales presentaron elevadas sensibilidades y especificidades, así como VPP, VPN y concordancia.

RPA-Cas12aDS: A visual and fast molecular diagnostics platform based on RPA-CRISPR-Cas12a method for infectious bursal disease virus detection

Infectious bursal disease (IBD), a major disease of birds, is caused by infectious bursal disease virus (IBDV). The disease can lead to immunosuppression, resulting in huge economic losses in the poultry industry. A specific, rapid, and simple detection method is important for the early diagnosis and prevention and control of IBDV. In this study, we established a naked-eye visual IBDV detection method, named “RPA-Cas12aDS”, by combining recombinase polymerase amplification (RPA) with CRISPR-Cas12a-based nucleic acid detection. The detection process can be accomplished in 50 min, and uncapping contamination can be avoided. The detection results can be observed under blue or UV light. We used the RPA-Cas12aDS method to detect IBDV in bursa of Fabricius tissue samples of chickens, and the results were consistent with those obtained using commercial RT-PCR kits. This method presents great potential for visual, rapid, and point-of-care molecular diagnostics of IBDV in poultry.

Rational Programming of Cas12a for Early-Stage Detection of COVID-19 by Lateral Flow Assay and Portable Real-Time Fluorescence Readout Facilities.

Coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has led to a global pandemic with a high spread rate and pathogenicity. Thus, with limited testing solutions, it is imperative to develop early-stage diagnostics for rapid and accurate detection of SARS-CoV-2 to contain the rapid transmission of the ongoing COVID-19 pandemic. In this regard, there remains little knowledge about the integration of the CRISPR collateral cleavage mechanism in the lateral flow assay and fluorophotometer. In the current study, we demonstrate a CRISPR/Cas12a-based collateral cleavage method for COVID-19 diagnosis using the Cas12a/crRNA complex for target recognition, reverse transcription loop-mediated isothermal amplification (RT-LAMP) for sensitivity enhancement, and a novel DNA capture probe-based lateral flow strip (LFS) or real-time fluorescence detector as the parallel system readout facility, termed CRICOLAP. Our novel approach uses a customized reporter that hybridizes an optimized complementary capture probe fixed at the test line for naked-eye result readout. The CRICOLAP system achieved ultra-sensitivity of 1 copy/µL in ~32 min by portable real-time fluorescence detection and ~60 min by LFS. Furthermore, CRICOLAP validation using 60 clinical nasopharyngeal samples previously verified with a commercial RT-PCR kit showed 97.5% and 100% sensitivity for S and N genes, respectively, and 100% specificity for both genes of SARS-CoV-2. CRICOLAP advances the CRISPR/Cas12a collateral cleavage result readout in the lateral flow assay and fluorophotometer, and it can be an alternative method for the decentralized field-deployable diagnosis of COVID-19 in remote and limited-resource locations.

Evaluation of commercial RT-PCR kits for pooled clinical specimens in COVID-19 patients.

Evaluation of commercial RT-PCR kits for pooled clinical specimens in COVID-19 patients.

SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass Surveillance

Objectives: Limitations of widespread current COVID-19 diagnostic testing exist in both the pre-analytical and analytical stages. To alleviate these limitations, we developed a universal saliva processing protocol (SalivaSTAT) that would enable an extraction-free RT-PCR test using commercially available RT-PCR kits. Methods: We optimized saliva collection devices, heat-shock treatment, and homogenization. Saliva samples (879) previously tested using the FDA-EUA method were reevaluated with the optimized SalivaSTAT protocol using two widely available commercial RT-PCR kits. A five-sample pooling strategy was evaluated as per FDA guidelines. Results: Saliva collection (done without any media) showed performance comparable to that of the FDA-EUA method. The SalivaSTAT protocol was optimized by incubating saliva samples at 95 °C for 30-min and homogenization, followed by RT-PCR assay. The clinical sample evaluation of 630 saliva samples using the SalivaSTAT protocol with PerkinElmer (600-samples) and CDC (30-samples) RT-PCR assay achieved positive (PPA) and negative percent agreements (NPAs) of 95.0% and 100%, respectively. The LoD was established as ~60–180 copies/mL by absolute quantification. Furthermore, a five-sample-pooling evaluation using 250 saliva samples achieved a PPA and NPA of 92% and 100%, respectively. Conclusion: We have optimized an extraction-free RT-PCR assay for saliva samples that demonstrates comparable performance to FDA-EUA assay (Extraction and RT-PCR).

Comparison of an in house and a commercial real-time polymerase chain reaction targeting Toxoplasma gondii RE gene using various samples collected from patients in Turkey

BackgroundToxoplasma gondii is an opportunistic protozoan parasite that can infect all warm-blooded animals including humans and cause serious clinical manifestations. Toxoplasmosis can be diagnosed using histological, serological, and molecular methods. In this study, we aimed to detect T. gondii RE gene in various human samples by in house and commercial real time polymerase chain reactions.MethodsA total of 38 suspected cases of toxoplasmosis [peripheral blood (n:12), amnion fluid (n:11), tissue (n:9), cerebrospinal fluid (n:5), and intraocular fluid (n:1)] were included to the study. An in house and a commercial RT-PCR were applied to investigate the T. gondii RE gene in these samples.ResultsThe compatibility rate of the two tests was 94.7% (37/38). When the commercial RT-PCR kit was taken as reference, the sensitivity and specificity of in house RT-PCR test was 87.5 and 100%. When the in house RT-PCR test was taken as reference, the commercial RT-PCR kit has 100% sensitivity and 96.8% specificity. Incompatibility was detected in only in a buffy coat sample with high protein content.ConclusionsBoth the commercial and in house RT-PCR tests can be used to investigate T. gondii RE gene in various clinical specimens with their high sensitivity and specificity. In house RT-PCR assay can be favorable due to cost savings compared to using the commercial test.

Experience on the Application of a Commercial RT-PCR Kit on the Diagnosis of 28 Chromosomal Translocations Associated with Acute Leukemia in Adults

BACKGROUND:The cytogenetic aspects of acute leukemia have been studied for over 30 years. Genomic sequencing has permitted the identification of recurrent mutations that have been associated with the heterogeneous behavior of acute leukemia.Currently, the genomic profile of patients with acute leukemia is important regarding risk classification and therapeutic approach. At this moment there are scarce studies of the genomic profile in adult patients with acute leukemia in Mexico.OBJECTIVES:To describe genomic alterations found in adult patients with acute leukemia in a cancer reference hospital in Mexico.Evaluate the efficacy of a commercial multiple PCR and nested qualitative PCR kit (HemaVision DNA Technology A/S, HV01-28N), for the detection of 28 translocations on acute leukemia.To assess by a quantitative method with RT-PCR the detection of the most frequently reported mutations in acute myeloid leukemia(38%) [t(15;17) PML-RARa, inv(16) CBF-MYH11, t(8;21) RUNX1-RUNX1T1 and acute lymphoid leukemia (32%) [t(9;22) BCR/ABL, t(4;11) MLL-AFF1, t(1;19) TCF3-PBX1].Compare the results between the commercial kit, RT-PCR and conventional cytogenetic studies.METHODS:Patients with recent diagnosis of acute leukemia (myeloid and lymphoid) according to World Health Administration (WHO) 2008 criteria were included between the period of 03/25/2013 and 12/15/13.All patients were given informed consent form and approval of the local Ethics CommitteeWe performed bone marrow smear and bone marrow biopsy of all patients. 5 mL of bone marrow were collected on heparin for cytogenetic study including karyotipe and FISH analysis. 5 mL of bone marrow were collected for molecular biology studies including PCR-kit and genome sequencing technique.5mL of peripheral blood with heparin were obtained for karyotype in case of not finding metaphases for the analysis on bone marrow specimen.RESULTS:24 patients were enrolled on the study. The mean age was 42 years (17-69). 8/24 were male (33.3%) and 16/24 were female (66.6%). Acute myeloid leukemia 11/24 (45.8%), acute lymphoblastic leukemia 8/24 (33.3%), acute promyeolocytic leukemia 3/24 (12.5%), acute biphenotypic leukemia 1/24 (4.2%) and one case of blastic phase of chronic myeloid leukemia (4.2%).The commercial PCR platform detected only two of the 28 translocations in 4/24 patients (16.6%). 2/24 (8.3%) BCR-ABL and 2/24 (8.3%) PML-RARa.In contrast to RT-PCR that detected 7/24 patients (29%). 4/24 (16%) BCR-ABL, 2/24 (8.3%) PML-RARa and 1/24 (4.1%) CBFB-MYH11.FISH analysis detected the 4/24 (16%) translocations of the commercial kit. Karyotype detected structural chromosomal anomalies in 9/24 patients (37.5%). After the analysis of the 24 samples by quantitative RT-PCR for the six most frequent rearrangements, we also identified 2 additional cases of BCR-ABL.CONCLUSIONS:The commercial qualitative PCR kit was inferior on detecting genomic alterations (16.6%) than quantitative PCR (29%) or conventional cytogenetic study (37.5%).The RT-PCR quantitative method with specific probe for the most frequent genomic alterations in acute leukemia in addition to cytogenetic analysis seems to be a better cost-effective strategy than the commercial multiple PCR platforms. DisclosuresNo relevant conflicts of interest to declare.

An Endogenous Murine Leukemia Viral Genome Contaminant in a Commercial RT-PCR Kit is Amplified Using Standard Primers for XMRV

During pilot studies to investigate the presence of viral RNA of xenotropic murine leukemia virus (MLV)-related virus (XMRV) infection in sera from chronic fatigue syndrome (CFS) patients in Japan, a positive band was frequently detected at the expected product size in negative control samples when detecting a partial gag region of XMRV using a one-step RT-PCR kit. We suspected that the kit itself might have been contaminated with small traces of endogenous MLV genome or XMRV and attempted to evaluate the quality of the kit in two independent laboratories. We purchased four one-step RT-PCR kits from Invitrogen, TaKaRa, Promega and QIAGEN in Japan. To amplify the partial gag gene of XMRV or other MLV-related viruses, primer sets (419F and 1154R, and GAG-I-F and GAG-I-R) which have been widely used in XMRV studies were employed. The nucleotide sequences of the amplicons were determined and compared with deposited sequences of a polytropic endogenous MLV (PmERV), XMRV and endogenous MLV-related viruses derived from CFS patients. We found that the enzyme mixtures of the one-step RT-PCR kit from Invitrogen were contaminated with RNA derived from PmERV. The nucleotide sequence of a partial gag region of the contaminant amplified by RT-PCR was nearly identical (99.4% identity) to a PmERV on chromosome 7 and highly similar (96.9 to 97.6%) to recently identified MLV-like viruses derived from CFS patients. We also determined the nucleotide sequence of a partial env region of the contaminant and found that it was almost identical (99.6%) to the PmERV. In the investigation of XMRV infection in patients of CFS and prostate cancer, researchers should prudently evaluate the test kits for the presence of endogenous MLV as well as XMRV genomes prior to PCR and RT-PCR tests.