Optimized protocol for a quantitative SARS-CoV-2 duplex RT-qPCR assay with internal human sample sufficiency control.
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ABSTRACT:
SUBMITTER: Rowan AG
PROVIDER: S-EPMC8108476 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first assays targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level, and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail, and our findings reveal some limitations but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Nevertheless, whilst our data show that some errors can be tolerated, it is always prudent to confirm that the primer and probe sequences complement their intended target, since, when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case, it is unlikely that a mismatch will result in poor specificity or a significant number of false-positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.
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Project description:BackgroundThe spatiotemporal expression patterns of microRNAs (miRNAs) are important to the verification of their predicted function. RT-qPCR is the accepted technique for the quantification of miRNA expression; however, stem-loop RT-PCR and poly(T)-adapter assay, the two most frequently used methods, are not very convenient in practice and have poor specificity, respectively.ResultsWe have developed an optimal approach that integrates these two methods and allows specific and rapid detection of tiny amounts of sample RNA and reduces costs relative to other techniques. miRNAs of the same sample are polyuridylated and reverse transcribed into cDNAs using a universal poly(A)-stem-loop RT primer and then used as templates for SYBR® Green real-time PCR. The technique has a dynamic range of eight orders of magnitude with a sensitivity of up to 0.2 fM miRNA or as little as 10 pg of total RNA. Virtually no cross-reaction is observed among the closely-related miRNA family members and with miRNAs that have only a single nucleotide difference in this highly specific assay. The spatial constraint of the stem-loop structure of the modified RT primer allowed detection of miRNAs directly from cell lysates without laborious total RNA isolation, and the poly(U) tail made it possible to use multiplex RT reactions of mRNA and miRNAs in the same run.ConclusionsThe cost-effective RT-qPCR of miRNAs with poly(A)-stem-loop RT primer is simple to perform and highly specific, which is especially important for samples that are precious and/or difficult to obtain.
| S-EPMC3465266 | biostudies-literature
Project description:With the ongoing COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), there is a need for sensitive, specific, and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR (quantitative reverse transcription PCR), with many commercial kits now available for this purpose. However, these are expensive, and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control (RPP30) and a viral spike-in control (Phocine Herpes Virus 1 [PhHV-1]), which monitor sample quality and nucleic acid extraction efficiency, respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by combined nose and throat swabbing and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false-negative rates. We demonstrate the feasibility of establishing a robust RT-qPCR assay at approximately 10% of the cost of equivalent commercial assays, which could benefit low-resource environments and make high-volume testing affordable.
| S-EPMC7771873 | biostudies-literature
Project description:The current COVID-19 pandemic constitutes a threat to the population worldwide with over 21 million infected people. There is an urgent need for the development of rapid and massive detection tools as well as the identification and isolation of infected individuals. we sought to evaluate different RT-qPCR kits and protocols to evaluate the best approach to be used omitting an RNA extraction step. We have investigated the sensitivity and performance of different commercially available RT-qPCR kits in detecting SARS-CoV-2 using 80 extracted RNA and NSS from COVID-19 diagnosed patients. We evaluated the ability of each kit to detect viral RNA from both kit-extracted or directly from a pre-boiled NSS observing that direct RNA detection is possible when Ct values are lower than 30 with the three kits tested. Since SARS-CoV-2 testing in most locations occurs once COVID-19 symptoms are evident and, therefore, viral loads are expected to be high, our protocol will be useful in supporting SARS-CoV-2 diagnosis, especially in America where COVID-19 cases have exploded in the recent weeks as well as in low- and middle-income countries, which would not have massive access to kit-based diagnosis. The information provided in this work paves the way for the development of more efficient SARS-CoV-2 detection approaches avoiding an RNA extraction step.
| S-EPMC7480275 | biostudies-literature
Project description:The emergence of a novel SARS-CoV-2 B.1.1.7 variant sparked global alarm due to increased transmissibility, mortality, and uncertainty about vaccine efficacy, thus accelerating efforts to detect and track the variant. Current approaches to detect B.1.1.7 include sequencing and RT-qPCR tests containing a target assay that fails or results in reduced sensitivity towards the B.1.1.7 variant. Since many countries lack genomic surveillance programs and failed assays detect unrelated variants containing similar mutations as B.1.1.7, we used allele-specific PCR, and judicious placement of LNA-modified nucleotides to develop an RT-qPCR test that accurately and rapidly differentiates B.1.1.7 from other SARS-CoV-2 variants. We validated the test on 106 clinical samples with lineage status confirmed by sequencing and conducted a country-wide surveillance study of B.1.1.7 prevalence in Slovakia. Our multiplexed RT-qPCR test showed 97% clinical sensitivity and retesting 6,886 SARS-CoV-2 positive samples obtained during three campaigns performed within one month, revealed pervasive spread of B.1.1.7 with an average prevalence of 82%. Labs can easily implement this test to rapidly scale B.1.1.7 surveillance efforts and it is particularly useful in countries with high prevalence of variants possessing only the ΔH69/ΔV70 deletion because current strategies using target failure assays incorrectly identify these as putative B.1.1.7 variants.
| S-EPMC8516907 | biostudies-literature
Project description:Emerging SARS-CoV-2 (SC-2) variants with increased infectivity and vaccine resistance are of major concern. Rapid identification of such variants is important for the public health decision making and to provide valuable data for epidemiological and policy decision making. We developed a multiplex reverse transcriptase quantitative PCR (RT-qPCR) assay that can specifically identify and differentiate between the emerging B.1.1.7 and B.1.351 SC-2 variants. In a single assay, we combined four reactions-one that detects SC-2 RNA independently of the strain, one that detects the D3L mutation, which is specific to variant B.1.1.7, one that detects the 242 to 244 deletion, which is specific to variant B.1.351, and the fourth reaction, which identifies the human RNAseP gene, serving as an endogenous control for RNA extraction integrity. We show that the strain-specific reactions target mutations that are strongly associated with the target variants and not with other major known variants. The assay's specificity was tested against a panel of respiratory pathogens (n = 16), showing high specificity toward SC-2 RNA. The assay's sensitivity was assessed using both in vitro transcribed RNA and clinical samples and was determined to be between 20 and 40 viral RNA copies per reaction. The assay performance was corroborated with Sanger and whole-genome sequencing, showing complete agreement with the sequencing results. The new assay is currently implemented in the routine diagnostic work at the Central Virology Laboratory, and may be used in other laboratories to facilitate the diagnosis of these major worldwide-circulating SC-2 variants. IMPORTANCE This study describes the design and utilization of a multiplex reverse transcriptase quantitative PCR (RT-qPCR) to identify SARS-COV-2 (SC2) RNA in general and, specifically, to detect whether it is of lineage B.1.1.7 or B.1.351. Implementation of this method in diagnostic and research laboratories worldwide may help the efforts to contain the COVID-19 pandemic. The method can be easily scaled up and be used in high-throughput laboratories, as well as small ones. In addition to immediate help in diagnostic efforts, this method may also help in epidemiological studies focused on the spread of emerging SC-2 lineages.
| S-EPMC8510166 | biostudies-literature
Project description:To minimize sample dilution effect on SARS-CoV-2 pool testing, we assessed analytical and diagnostic performance of a new methodology, namely swab pooling. In this method, swabs are pooled at the time of collection, as opposed to pooling of equal volumes from individually collected samples. Paired analysis of pooled and individual samples from 613 patients revealed 94 positive individuals. Having individual testing as reference, no false-positives or false-negatives were observed for swab pooling. In additional 18,922 patients screened with swab pooling (1,344 pools), mean Cq differences between individual and pool samples ranged from 0.1 (Cr.I. -0.98 to 1.17) to 2.09 (Cr.I. 1.24 to 2.94). Overall, 19,535 asymptomatic patients were screened using 4,400 RT-qPCR assays. This corresponds to an increase of 4.4 times in laboratory capacity and a reduction of 77% in required tests. Therefore, swab pooling represents a major alternative for reliable and large-scale screening of SARS-CoV-2 in low prevalence populations.
| S-EPMC7861376 | biostudies-literature
Project description:Testing for the presence of coronavirus is an essential diagnostic tool for monitoring and managing the current COVID-19 pandemic. The only reliable test in current use for testing acute infection targets the genome of SARS-CoV-2, and the most widely used method is quantitative fluorescence-based reverse transcription polymerase chain reaction (RT-qPCR). Despite its ubiquity, there is a significant amount of uncertainty about how this test works, potential throughput and reliability. This has resulted in widespread misrepresentation of the problems faced using this test during the current COVID-19 epidemic. This primer provides simple, straightforward and impartial information about RT-qPCR.
| S-EPMC7215906 | biostudies-literature
Project description:BackgroundMicroscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular methods in detection of malaria. Some of the challenges that continue to limit the widespread utilization of qPCR include lack of assay standardization, assay variability, risk of contamination, and the need for cold-chain. Lyophilization of molecular assays can overcome some of these limitations and potentially enable widespread qPCR utilization.MethodsA recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-Ready™ format (MMSR). MMSR assay contained all the required reagents for qPCR including primers and probes, requiring only the addition of water and sample to perform qPCR. The performance of the MMSR assay was compared to the non-freeze dried, "wet" assay. Stability studies were done by maintaining the MMSR assays at four different ambient temperatures of 4°C, room temperature (RT), 37°C and 42°C over a period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two different concentrations. The CT values and the standard deviations (SD) were used in the analysis of the assay performance.ResultsThe limit of detection for the MMSR assay was 0.244 parasites/?L for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to "wet" assay which was 0.39 and 3.13 parasites/?L for PLU and FAL assay targets, respectively. The MMSR assay performed with high efficiencies similar to those of the "wet" assay and was stable at 37°C for 42 days, with estimated shelf-life of 5 months. When used to analyse field clinical samples, MMSR assay performed with 100% sensitivity and specificity compared to the "wet" assay.ConclusionThe MMSR assay has the same robust performance characteristics as the "wet" assay and is highly stable. Availability of MMSR assay allows flexibility and provides an option in choosing assay for malaria diagnostics depending on the application, needs and budget.
| S-EPMC4026594 | biostudies-literature
Project description:During the COVID-19 pandemic, the reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay has been the primary method of diagnosis of SARS-CoV-2 infection. However, RT-qPCR assay interpretation can be ambiguous with no universal absolute cut-off value to determine sample positivity, which particularly complicates the analysis of samples with high Ct values, or weak positives. Therefore, we sought to analyse factors associated with weak positive SARS-CoV-2 diagnosis. We analysed sample data associated with all positive SARS-CoV-2 RT-qPCR diagnostic tests performed by the Victorian Infectious Diseases Reference Laboratory (VIDRL) in Melbourne, Australia, during the Victorian first wave (22 January 2020-30 May 2020). A subset of samples was screened for the presence of host DNA and RNA using qPCR assays for CCR5 and 18S, respectively. Assays targeting the viral RNA-dependent RNA polymerase (RdRp) had higher Ct values than assays targeting the viral N and E genes. Weak positives were not associated with the age or sex of individuals' samples nor with reduced levels of host DNA and RNA. We observed a relationship between Ct value and time post-SARS-CoV-2 diagnosis. High Ct value or weak positive SARS-CoV-2 was not associated with any particular bias including poor biological sampling.
| S-EPMC9239708 | biostudies-literature