scholarly journals Assessment of 12 Quantitative RT-PCR Commercial Kits for the Detection of SARS-CoV-2

2020 ◽  
Author(s):  
Asmaa M. Altamimi ◽  
Dalia A. Obeid ◽  
Taghreed A. Alaifan ◽  
Moroje T. Taha ◽  
Marwa A. Alhothali ◽  
...  
Keyword(s):  
Clinical Samples ◽  
Rt Pcr ◽  
Gene Target ◽  
Global Response ◽  
Routine Diagnosis ◽  
Purification System ◽  
Clinical Detection ◽  
Public Emergency ◽  
Commercial Kits ◽  
Good Plan

Abstract BackgroundThe emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) in the final months of 2019 had the health officials declare a public emergency raising a global response. In response to the burden of the current pandemic, strict measurements were globally implemented to stop further transmission of the virus. These Measurements rely on an accurate diagnosis of individuals infected with the virus by using real-time reverse transcriptase PCR (RT-PCR). The goal of our study is to relate the fundamental clinical and analytical performance of chosen kits of RT-PCR from distinct manufacturers. Methods A total of 94 nasopharyngeal and oropharyngeal clinical samples were selected randomly, these samples were previously confirmed as 64 positives, and 30 negatives for SARS-COV-2. Generally, 400 µl of each respiratory specimen was subjected to extraction using ExiPrep 96 Viral RNA Kit with the ExiPrep 96 Lite Automated NA Purification System. All kits master mix preparation, cycling protocol, and results interpretation were carried out according to the manufactures’ instructions of use and recommendations. Results In our study, we were able to evaluate the performance of 12 commercial kits in detecting SARS-COV-2 using 5 different targets. The performance of the kits was comparable except for LYRA kit as it was less sensitive (F=67, P <0.001). Particularly, the performance of RealStar, BGI, DiaPlexQ, Genesig, LightMix, TaqPath, and RADI kits were the most sensitive. Moreover, the performance of these commercial kits by gene target showed no significant change in Ct values which indicates that kits disparities are mainly linked to the choice of the gene target (F=0.49, P=0.73).ConclusionWe believe that most of the commercially available RT-PCR kits included in this study can be used for routine diagnosis of SARS-COV-2 patients. Moreover, we recommend that regardless of the laboratory choice of diagnostic commercial kit for the clinical detection of COVID-19 patients the need a for good plan for validation and collaboration with exterior laboratories is essential in order to monitor the virus changes overtime, procedures, technicians, and the different kits performances.

scholarly journals Comparative analysis of the diagnostic performance of five commercial COVID-19 qRT PCR kits used in India

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
J. Singh ◽  
A. K. Yadav ◽  
A. Pakhare ◽  
P. Kulkarni ◽  
L. Lokhande ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Diagnostic Performance ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Diagnostic Process ◽  
Control Group ◽  
Rt Pcr ◽  
Short Period ◽  
Commercial Kits

AbstractTo meet the unprecedented requirement of diagnostic testing for SARS-CoV-2, a large number of diagnostic kits were authorized by concerned authorities for diagnostic use within a short period of time during the initial phases of the ongoing pandemic. We undertook this study to evaluate the inter-test agreement and other key operational features of 5 such commercial kits that have been extensively used in India for routine diagnostic testing for COVID-19. The five commercial kits were evaluated, using a panel of positive and negative respiratory samples, considering the kit provided by National Institute of Virology, Indian Council of Medical Research (2019-nCoV Kit) as the reference. The positive panel comprised of individuals who fulfilled the 3 criteria of being clinically symptomatic, having history of contact with diagnosed cases and testing positive in the reference kit. The negative panel included both healthy and disease controls, the latter being drawn from individuals diagnosed with other respiratory viral infections. The same protocol of sample collection, same RNA extraction kit and same RT-PCR instrument were used for all the kits. Clinical samples were collected from a panel of 92 cases and 60 control patients, who fulfilled our inclusion criteria. The control group included equal number of healthy individuals and patients infected with other respiratory viruses (n = 30, in each group). We observed varying sensitivity and specificity among the evaluated kits, with LabGun COVID-19 RT-PCR kit showing the highest sensitivity and specificity (94% and 100% respectively), followed by TaqPath COVID-19 Combo and Allplex 2019-nCoV assays. The extent of inter-test agreement was not associated with viral loads of the samples. Poor correlation was observed between Ct values of the same genes amplified using different kits. Our findings reveal the presence of wide heterogeneity and sub-optimal inter-test agreement in the diagnostic performance of the evaluated kits and hint at the need of adopting stringent standards for fulfilling the quality assurance requirements of the COVID-19 diagnostic process.

scholarly journals DO INDONESIAN GOVERNMENT DEPLOY RELIABLE AMMUNITION FOR COVID-19 MASS TEST? A COMPARISON OF REAL-TIME PCR KITS

2021 ◽  
pp. 75-77
Author(s):  
NELLY MARISSA ◽  
SALMIATY ◽  
SARI HANUM ◽  
EVAN FEBRIANSYAH ◽  
NUR RAMADHAN ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Great Ability ◽  
Commercial Kits ◽  
Mass Test ◽  
Amplification Procedure

Objective: This study aimed to compare the level of reliability of three commercial real-time PCR kits in determining clinical samples. Methods: A total of 40 swabs samples which were previously tested positive, were re-test using the BioCov-19 RT-PCR kit, Sansure coronavirus disease 19 (COVID-19) nucleic acid diagnostic kit, and Kogen PowerCheck with Thermocycler (Roche). The amplification procedure is carried out based on the manual for each kit. Results: Sansure COVID-19 nucleic acid diagnostic was able to detect 40 samples with positive severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) results detected in both genes, while the PowerChek™ 2019-nCoV real-time PCR kit able to detect 35 samples showed that SARS-COV-II was detected in both genes, and the BioCoV-19 RT-PCR Kit brand kit able to detect 34 samples showed positive SARS-COV-2 results in both genes. Conclusion: The three commercial kits show great ability detection, so that they can be used to detect the presence of SARS-COV-II in clinical samples, and also in mass screening.

scholarly journals Lack of evidence of porcine reproductive and respiratory syndrome virus (PRRSV) infection in domestic swine in Brazil

2004 ◽  
Vol 34 (2) ◽  
pp. 449-455 ◽  
Author(s):  
Janice Reis Ciacci-Zanella ◽  
Cristiano Trombetta ◽  
Ildara Vargas ◽  
Denise Euclydes Mariano da Costa
Keyword(s):  
Genetic Material ◽  
Elisa Test ◽  
Respiratory Syndrome Virus ◽  
Rt Pcr ◽  
Serum Samples ◽  
Viral Isolation ◽  
Culture Cells ◽  
Commercial Kits ◽  
Swine Herds ◽  
Domestic Swine

This report describes the first prevalence of antibodies and experimental inoculation of suspected samples of porcine reproductive and respiratory syndrome virus (PRRSV) from ELISA positive pigs from swine herds in Brazil. Based on the hypothesis that this agent is present in swine herds worldwide, the objective of this work was to establish a diagnostic methodology and to investigate the occurrence of PRRSV in Brazilian swine herds. Fifty-four swine herds, the total number which imported genetic material (live pigs or swine semen) from countries where PRRS was endemic from 1990 to December 2000, from eight Brazilian States all included in this study. The sampling used was such as to detect a prevalence of infection of 5%, with a confidence level of 95%. A total of 3785 serum samples were tested for PRRSV antibodies by ELISA. Following the ELISA test, which was performed with two different commercial kits, all serum positive pigs were retested, examined and additional materials were collected. Viral isolation in permissive tissue culture cells and swine bioassays were performed. Additionally, reverse transcriptase polymerase chain reaction (RT-PCR) and nested RT-PCR were also performed. We could not demonstrate the presence of PRRSV or RNA of PRRSV by viral isolation or RT-PCR (or nested RT-PCR), respectively in all of the analyzed samples. Furthermore, the pigs inoculated with PRRSV suspicion samples did not seroconvert nor produce characteristic PRRS lesions in the swine bioassay. Thus, our results indicate no evidence of PRRSV in the samples analyzed from swine herds in this study.

scholarly journals Artifacts associated with mithramycin fluorescence in the clinical detection and quantitation of aneuploidy by flow cytometry.

1982 ◽  
Vol 30 (4) ◽  
pp. 317-322 ◽  
Author(s):  
R E Cunningham ◽  
K S Skramstad ◽  
A E Newburger ◽  
S E Shackney
Keyword(s):  
Flow Cytometry ◽  
Room Temperature ◽  
Human Melanoma ◽  
Time Dependent ◽  
Clinical Samples ◽  
Fixation Time ◽  
C Cells ◽  
Temperature Dependent ◽  
Clinical Detection

Ethanol-fixed cells stored at 4 degrees C exhibit fixation time-dependent hyperchromatism in comparison with freshly fixed cells when stained with mithramycin and examined by flow cytometry. This hyperchromatism has been found to be temperature-dependent, developing fully within 72 hr at room temperature, and within 2 hr at 37 degrees C. Cells from normal donors that are stained with mithramycin exhibit spurious aneuploid peaks. These spurious aneuploid peaks can be eliminated by incubating ethanol-fixed cells at 37 degrees C for 2 hr prior to staining; true aneuploidy is not affected by this procedure. In rare instances, cytoplasmic fluorescence can be observed in mithramycin-stained cells. In addition, unexplained hypochromatism and hyperchromatism can be observed in some clinical samples, particularly in human melanoma. The effects of these unexplained staining artifacts can be minimized or eliminated by adopting strict criteria for the clinical detection of aneuploidy by flow cytometry.

scholarly journals Applications of Flow Cytometry to Clinical Microbiology

2000 ◽  
Vol 13 (2) ◽  
pp. 167-195 ◽  
Author(s):  
Alberto Álvarez-Barrientos ◽  
Javier Arroyo ◽  
Rafael Cantón ◽  
César Nombela ◽  
Miguel Sánchez-Pérez
Keyword(s):  
Flow Cytometry ◽  
Clinical Microbiology ◽  
Ex Vivo ◽  
Analytical Techniques ◽  
Clinical Samples ◽  
Clinical Microbiology Laboratory ◽  
Commercial Kits ◽  
Antimicrobial Treatments ◽  
Near Future

SUMMARY Classical microbiology techniques are relatively slow in comparison to other analytical techniques, in many cases due to the need to culture the microorganisms. Furthermore, classical approaches are difficult with unculturable microorganisms. More recently, the emergence of molecular biology techniques, particularly those on antibodies and nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. Flow cytometry (FCM) allows single- or multiple-microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometric parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Furthermore, this technique allows the monitoring of in vitro antimicrobial activity and of antimicrobial treatments ex vivo. The most outstanding contribution of FCM is the possibility of detecting the presence of heterogeneous populations with different responses to antimicrobial treatments. Despite these advantages, the application of FCM in clinical microbiology is not yet widespread, probably due to the lack of access to flow cytometers or the lack of knowledge about the potential of this technique. One of the goals of this review is to attempt to mitigate this latter circumstance. We are convinced that in the near future, the availability of commercial kits should increase the use of this technique in the clinical microbiology laboratory.

scholarly journals Quantitative measurement of infectious virus in SARS-CoV-2 Alpha, Delta and Epsilon variants reveals higher infectivity (viral titer:RNA ratio) in clinical samples containing the Delta and Epsilon variants.

2021 ◽  
Author(s):  
Hannah W Despres ◽  
Margaret G Mills ◽  
David J Shirley ◽  
Madaline M Schmidt ◽  
Meei-Li Huang ◽  
...  
Keyword(s):  
Quantitative Measurement ◽  
Infectious Virus ◽  
Viral Rna ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Genome Copy ◽  
Live Virus ◽  
High Degree ◽  
The Relationship ◽  
Rna Levels

ABSTRACT Background Novel SARS-CoV-2 Variants of Concern (VoC) pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between viral RNA and infectious virus for individual variants is unknown. Methods We measured infectious viral titer (using a micro-focus forming assay) as well as total and subgenomic viral RNA levels (using RT-PCR) in a set of 165 clinical samples containing SARS-CoV-2 Alpha, Delta and Epsilon variants that were processed within two days of collection from the patient. Results We observed a high degree of variation in the relationship between viral titers and RNA levels. Despite the variability we observed for individual samples the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (6 and 4 times as much, p=0.0002 and 0.009 respectively) or subgenomic E RNA (11 and 7 times as much, p<0.0001 and 0.006 respectively). Conclusion In addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may also be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity of the Delta variant may further explain increased spread and suggests a need for increased measures to prevent viral transmission.

scholarly journals Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252687
Author(s):  
Sukalyani Banik ◽  
Kaheerman Saibire ◽  
Shraddha Suryavanshi ◽  
Glenn Johns ◽  
Soumitesh Chakravorty ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Viral Rna ◽  
Clinical Samples ◽  
Sample Collection ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Guanidinium Thiocyanate ◽  
Upper Respiratory ◽  
Polymerase Chain ◽  
The Stability

Background Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. Methods We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. Results SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. Conclusion eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

scholarly journals Improved Genotyping Vaccine and Wild-Type Poliovirus Strains by Restriction Fragment Length Polymorphism Analysis: Clinical Diagnostic Implications

2000 ◽  
Vol 38 (12) ◽  
pp. 4337-4342 ◽  
Author(s):  
Amalia Georgopoulou ◽  
Panayotis Markoulatos ◽  
Niki Spyrou ◽  
Nicholas C. Vamvakopoulos
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Clinical Samples ◽  
Polymorphism Analysis ◽  
Rt Pcr ◽  
Restriction Fragment Length

The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5′ noncoding region of polioviruses was selected for RT-PCR amplification by the UC53-UG52 primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (HaeIII, DdeI, NcoI, andAvaI). To compensate for potential genetic drift within the recognition sites of HaeIII, DdeI, orNcoI in atypical clinical samples, the RFLP patterns generated with HpaII and StyI as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success.

scholarly journals High speed large scale automated isolation of SARS-CoV-2 from clinical samples using miniaturized co-culture coupled with high content screening

2020 ◽  
Author(s):  
Rania Francis ◽  
Marion Le Bideau ◽  
Priscilla Jardot ◽  
Clio Grimaldier ◽  
Didier Raoult ◽  
...  
Keyword(s):  
High Speed ◽  
Large Scale ◽  
Vaccine Development ◽  
Work Load ◽  
Drug Susceptibility Testing ◽  
High Content Screening ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Positive Elements

AbstractSARS-CoV-2, a novel coronavirus infecting humans, is responsible for the current COVID-19 global pandemic. If several strains could be isolated worldwide, especially for in-vitro drug susceptibility testing and vaccine development, few laboratories routinely isolate SARS-CoV-2. This is due to the fact that the current co-culture strategy is highly time consuming and requires working in a biosafety level 3 laboratory. In this work, we present a new strategy based on high content screening automated microscopy (HCS) allowing large scale isolation of SARS-CoV-2 from clinical samples in 1 week. A randomized panel of 104 samples, including 72 tested positive by RT-PCR and 32 tested negative, were processed with our HCS procedure and were compared to the classical isolation procedure. Isolation rate was 43 % with both strategies on RT-PCR positive samples, and was correlated with the initial RNA viral load in the samples, where we obtained a positivity threshold of 27 Ct. Co-culture delays were shorter with HCS strategy, where 80 % of the positive samples were recovered by the third day of co-culture, as compared to only 25 % with the classic strategy. Moreover, only the HCS strategy allowed us to recover all the positive elements after 1 week of co-culture. This system allows rapid and automated screening of clinical samples with minimal operator work load, thus reducing the risks of contamination.

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

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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