查看更多>>摘要：? 2021 The Author(s)Background: Detection of SARS-CoV-2 infections relies on the use of sensitive, accurate and high throughput RT-PCR assays. Objectives: We assessed the analytical performance of the Abbott RealTime SARS-CoV-2 (RT-SARS), Alinity m SARS-CoV-2 (AlinSARS) assays and compared the clinical performance of the RT-SARS, AlinSARS, and Alinity m Resp-4-Plex (Alin4Plex) assays to the Seegene Allplex assay (Allplex) and an inhouse test (Inhouse). Results: We found 100 % positive percent agreement (PPA) and 100 % negative percent agreement (NPA) comparing RT-SARS and Allplex. RT-SARS, AlinSARS and Inhouse showed 100 % NPA and 100 % PPA across all assays, except for the RdRp target of Inhouse (PPA = 84 %). Similarly, Alin4Plex and Allplex showed high agreement with specimens containing either SARS-CoV-2, influenza A, influenza B, or RSV. Detection rates of 100 % for SARS-CoV-2 at 50 copies/mL, high precision, and no cross-reactivity with non-SARS-CoV-2 respiratory pathogens were observed for RT-SARS and AlinSARS. AlinSARS detected SARS-CoV-2 in spiked throat washes and in specimens infected with SARS-CoV-2 Alpha or Beta variants. Conclusions: The newly developed RT-SARS, AlinSARS, and Alin4Plex assays proved to be useful for detecting SARS-CoV-2 RNA in clinical samples.
查看更多>>摘要：? 2021 Elsevier B.V.A novel porcine circovirus 4 has been recently identified in China and Korea. A sensitive and specific diagnostic method is urgently required to detect the virus in field samples. We developed a loop-mediated isothermal amplification (LAMP) the assay for the visual detection of PCV4 and evaluated its sensitivity, specificity, and applicability in clinical samples. This assay's results can be directly visualized by the naked eye using hydroxynaphthol blue after incubation for 40 min at 64 °C. The assay specifically amplified PCV4 DNA and no other viral nucleic acids. The sensitivity of the assay was <50 DNA copies/reaction, which was 10 times more sensitive than conventional polymerase chain reaction (cPCR) and comparable to real-time PCR (qPCR). Clinical evaluation revealed that the PCV4 detection rate in individual pig samples and at the farm level was 39.3 % (57/145) and 45.7 % (32/70), respectively, which were higher than cPCR (46 samples, 24 farms) and qPCR (52 samples, 29 farms) results. Cumulatively, owing to the advantages of high sensitivity and specificity, direct visual monitoring of the results, no possibility for cross-contamination, and being a low-cost equipment, the developed LAMP assay will be a valuable tool for the detection of the novel PCV4 in clinical samples, even in resource-limited laboratories.
查看更多>>摘要：? 2021Tomato chlorosis virus (ToCV), a species of single-stranded RNA virus belonging to the Crinivirus genus, and Tomato yellow leaf curl virus (TYLCV), a species of single-stranded circular DNA virus belonging to the Begomovirus genus, are two major emerging viruses transmitted by whiteflies and are causing huge losses to tomato production worldwide. To facilitate the simultaneous detection of both viruses in co-infected plants for disease control, a duplex reverse-transcription PCR assay was developed. The assay used three primers, a degenerate reverse primer targeting a conserved region of TYLCV and the RNA2 of ToCV, and two virus-specific forward primers targeting the minor coat protein gene of ToCV and the C3 gene of TYLCV, respectively, to amplify a 762-bp and a 338-bp fragment from ToCV and TYLCV, respectively, in a single reaction. The concentration of the primers, annealing temperature and amplification cycles used in the assay were optimized, and the sensitivity of the assay was assessed. Using this assay, 150 tomato leaf samples collected from the field during 2018 were tested. The results showed that both viruses could be detected simultaneously in co-infected field samples. The assay should benefit the rapid detection of these two viruses in tomato crops and would facilitate early warning of infections for the control of the two virus diseases.
查看更多>>摘要：? 2021BK polyomavirus (BKPyV) is a ubiquitous pathogen that typically results in asymptomatic infection. However, in immunocompromised individuals, BKPyV viral shedding in the urine can reach 109 copies per mL. These high viral levels within urine provide ideal samples for next-generation sequencing to accurately determine BKPyV genotype and identify mutations associated with pathogenesis. Sequencing data obtained can be further analyzed to better understand and characterize the genetic diversity present in BKPyV. Here, methods are described for the successful extraction of viral DNA from urine and the subsequent amplification methods to prepare a sample for next-generation sequencing.
查看更多>>摘要：? 2021 Elsevier B.V.Phi 6 (Φ6) bacteriophage is a proposed surrogate to study pathogenic enveloped viruses including SARS-CoV-2—the causative agent of COVID-19—based on structural similarities, BSL-1 status, and ease of use. To determine the role of virus-contaminated hands in disease transmission, an enhanced understanding of buffer and method performance for Φ6 recovery needs to be determined. Four buffer types and three methodologies were investigated for the recovery of Φ6 from human fingerpads over a 30 min duration. Phosphate buffered saline (PBS), PBS + 0.1 % Tween, 0.1 M glycine + 3% beef extract, and viral transport medium were evaluated as buffers for recovery of Φ6 via a dish, modified glove juice, and vigorous swabbing method. Φ6 concentrations on fingerpads were determined at 0-, 5-, 10-, and 30-min post-inoculation. While there were observed differences in virus recovery across buffer and method types depending on the time point, log PFU recovery based on buffer type or methodology was not significantly different at any time point (P > 0.05). The results presented in this study will allow for future work on Φ6 persistence, transfer between hands and surfaces, and efficacy of hand hygiene methods to be performed using a well-characterized and validated recovery method.
查看更多>>摘要：? 2021 Elsevier B.V.Background: Screening of infectious asymptomatic or pre-symptomatic individuals for SARS-CoV-2 is at present a key to controling the COVID-19 pandemic. In order to expand testing capability and limit cost, pool testing of asymtomatic individuals has been proposed, provided assay performance is not significantly affected. Methods: Combined nose and throat (N/T) swabs collected from COVID-19 infected or non-infected individuals were tested using SAMBA II individually and in pools of four (one positive and 3 negative). The evaluation was conducted by the manufacturer and an independent NHS site. Ct cycles of individual positives and pooled positives were determined by qRT-PCR. Results: In 42 pools containing a single positive sample with Ct values ranging between 17 and 36, 41 pools (97.6 %) were found positive by the SARS-CoV-2 SAMBA II test. The false-negative pool by SAMBA was also negative by both reference methods used in this evaluation.The individual positive sample in this pool was positive by SAMBA (Orf only) and by one of the reference methods (S gene only, Ct 35) but negative by the second reference method indicating that the sample itself was very low viral load. All 78 pools containing 4 negative swabs were negative (100 % specificity). Discussion: The preliminary data of the evaluation indicated a high level of performance in both sensitivity and specificity of the SAMBA II assay when used to test pools of 4 patient samples. The implementation of this pooled protocol can increase throughput and reduce cost/test when the prevalence of COVID is low.
查看更多>>摘要：? 2021 Elsevier B.V.A number of RT-qPCR assays for the detection of SARS-CoV-2 have been published and are listed by the WHO as recommended assays. Furthermore, numerous commercial assays with undisclosed primer and probe sequences are on the market. As the SARS-CoV-2 pandemic progresses, the virus accrues mutations, which in some cases – as seen with the B.1.1.7 variant – can outperform and push back other strains of SARS-CoV-2. If mutations occur in primer or probe binding sites, this can impact RT-qPCR results and impede SARS-CoV-2 diagnostics. Here we tested the effect of primer mismatches on RT-qPCR performance in vitro using synthetic mismatch in vitro transcripts. The effects of the mismatches ranged from a shift in ct values from -0.13 to +7.61. Crucially, we found that a mismatch in the forward primer has a more detrimental effect for PCR performance than a mismatch in the reverse primer. Furthermore, we compared the performance of the original Charité RdRP primer set, which has several ambiguities, with a primer version without ambiguities and found that without ambiguities the ct values are ca. 3 ct lower. Finally, we investigated the shift in ct values observed with the Seegene Allplex kit with the B.1.1.7 SARS-CoV-2 variant and found a three-nucleotide mismatch in the forward primer of the N target.
查看更多>>摘要：? 2021 Elsevier B.V.West Nile virus (WNV) and Wesselsbron virus (WSLV) are mosquito-borne viruses belonging to the Flavivirus genus, family Flaviviridae and cause outbreaks in southern Africa after heavy rain. Isothermal assays have been proposed for application in field situations as well as low resource settings and hence we developed a reverse-transcriptase recombinase polymerase amplification (RT-RPA) to detect WNV and WSLV known to occur in South Africa, causing sporadic outbreaks usually associated with good rainfall favouring mosquito breeding. Infectious virus can only be handled within a biosafety level (BSL) 3 facility, hence we opted to validate the assay with transcribed RNA. Specific RT-RPA primers and probes were designed for detection of WNV and WSLV and products detected using a rapid lateral flow device. The assay was performed in 30 min and detected 1.9 × 101 copies of WNV and 3.5 × 10° copies WSLV using noninfectious transcribed RNA controls. In addition, the assay was not inhibited by the presence of mosquito extracts in spiked samples. Mismatches between the WNV and WSLV probes and other flaviviruses will likely prevent cross reactivity. The sensitivity, low RPA incubation temperature and rapid processing time makes assay systems based on RPA technology ideally suited for fieldable diagnostics.
查看更多>>摘要：? 2021 Elsevier B.V.Murine leukemia virus (MLV) and murine stem cell virus (MSCV) and derived retroviral vectors are widely used to study retrovirus biology and as tools for gene delivery. The method described here represents a quantitative real time PCR (qPCR) with hydrolysis probe that can be applied within classical qPCR as well as in digital droplet PCR (ddPCR). The method targets a 60 bp long fragment located within the U5 region of the MLV/MSCV genome sequence. For the here described method a LOD95% of 25 copies per PCR reaction (DNA) and 80 copies per PCR reaction (RNA) was determined, and PCR efficiencies of 92.5 % and 98.5 %, respectively, were observed. This method enables the fast and simple titration of viral genomic RNA present in retroviral vector stocks for accurate and consistent transduction experiments. Furthermore, it enables the detection of proviral and transfer plasmid derived DNA sequences and can be modified to differentiate between retroviral RNA and DNA.
查看更多>>摘要：? 2021 Elsevier B.V.The increasing prevalence of N501Y variants of SARS-CoV-2 has kindled global concern due to their enhanced transmissibility. Genome sequencing is the gold standard method to identify the emerging variants of concern. But it is time-consuming and expensive, limiting the widespread deployment of genome surveillance in some countries. Health authorities surge the development of alternative assay to expand screening capacity with reduced time and cost. In this study, we developed an in-house TaqMan minor groove binder (MGB) probe-based one-step RT-qPCR assay to detect the presence of N501Y mutation in SARS-CoV-2. A total of 168 SARS-CoV-2 positive respiratory specimens were collected to determine diagnostic accuracy of the RT-qPCR assay. As a reference standard, PANGO lineages and the mutation patterns of all samples were characterised by whole-genome sequencing. The analytical sensitivity and the ability of the assay to detect low frequency of N501Y variants were also evaluated. A total of 31 PANGO lineages were identified from 168 SARS-CoV-2 positive cases, in which 34 samples belonged to N501Y variants, including B.1.1.7 (n = 20), B.1.351 (n = 12) and P.3 (n = 2). The N501Y RT-qPCR correctly identified all 34 samples as N501Y-positive and the other 134 samples as wildtype. The limit-of-detection of the assay consistently achieved 1.5 copies/μL on four different qPCR platforms. N501Y mutation was successfully detected at an allele frequency as low as 10 % in a sample with mixed SARS-CoV-2 lineage. The N501Y RT-qPCR is simple and inexpensive (US$1.6 per sample). It enables robust high-throughput screening for surveillance of SARS-CoV-2 variants of concern harbouring N501Y mutation.
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