Establishment of a triplex TaqMan quantitative real-time PCR assay for simultaneous detection of Cymbidium mosaic virus, Odontoglossum ringspot virus and Cymbidium ringspot virus.

Orchids are significant ornamental plants whose viral infection results in substantial economic damage. Cymbidium mosaic virus (CymMV), Odontoglossum ringspot virus (ORSV), and Cymbidium ringspot virus (CymRSV) represent three important and prevalent orchid viruses. The detection system proposed in this study uses a triplex TaqMan quantitative real-time PCR assay to identify CymMV, ORSV, and CymRSV in a simultaneous manner. We designed specific primers and probes for CymMV, ORSV, and CymRSV, with amplified sequences of 156 bp, 148 bp, and 145 bp, respectively. The minimum detection limit of the triplex qRT-PCR assay for CymMV and CymRSV was 1 copy/assay, and the minimum detection limit was 10 copies/assay for ORSV. The minimum stable detection limits for CymMV, ORSV, and CymRSV were 10, 102, and 102 copies/assay, respectively. Therefore, this system exhibited higher sensitivity (approximately 10 to 104-fold) than RT-PCR. The intra-and interassay CVs of Cq values are less than 0.55 and 0.95%, respectively, indicating that the triplex assay is highly reliable and accurate. In addition, 66 samples from five different orchid genera were analyzed using the established assay and gene chip. The detection results demonstrated that the triplex probe qRT-PCR demonstrated higher sensitivity than the gene chip, indicating that the triplex real-time PCR assay could be used for the detection of field samples. Our findings suggest that the triplex real-time RT-PCR detection system represents a rapid, simple, and accurate tool for detecting CymMV, ORSV, and CymRSV on orchids.

Dumbbell-type triplex molecular switch-based logic molecular assays of SARS-CoV-2.

Accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of great importance to control the COVID-19 pandemic. The gold standard assays for COVID-19 diagnostics are mainly based on separately detecting open reading frame 1ab (ORF1ab) and nucleoprotein (N) genes by RT-PCR. However, the current approaches often obtain false positive-misdiagnose caused by cross-contamination or undesired amplification. To address this issue, herein, we proposed a dumbbell-type triplex molecular switch (DTMS)-based, logic-gated strategy for high-fidelity SARS-CoV-2 RNA detection. The DTMS consists of a triple-helical stem region and two-loop regions for recognizing the ORF1ab and N genes of SARS-CoV-2. Only when the ORF1ab and N gene are concurrent, DTMS experiences a structural rearrangement, thus, bringing the two pyrenes into spacer proximity and leading to a new signal readout. This strategy allows detecting SARS-CoV-2 RNA with a detection limit of 1.3 nM, independent of nucleic acid amplification, holding great potential as an indicator probe for screening of COVID-19 and other population-wide epidemics.

Telomere G-triplex lights up Thioflavin T for RNA detection: new wine in an old bottle.

Few reports are found working on the features and functions of the human telomere G-triplex (ht-G3) though the telomere G-quadruplex has been intensely studied and widely implemented to develop various biosensors. We herein report that ht-G3 lights up Thioflavin T (ThT) and establish a sensitive biosensing platform for RNA detection by introducing a target recycling strategy. An optimal condition was selected out for ht-G3 to promote ThT to generate a strong fluorescence. Accordingly, an ht-G3-based molecular beacon was successfully designed against the corresponding RNA sequence of the SARS-CoV-2 N-gene. The sensitivity for the non-amplified RNA target achieves 0.01 nM, improved 100 times over the conventional ThT-based method. We believe this ht-G3/ThT-based label-free strategy could be widely applied for RNA detection.

Clinical Evaluation of BD Veritor SARS-CoV-2 and Flu A+B Assay for Point-Of-Care System.

Differential diagnosis of COVID-19 and/or influenza (flu) at point of care is critical for efficient patient management and treatment of both these diseases. The study presented here characterizes the BD Veritor System for Rapid Detection of SARS-CoV-2 and Flu A+B ("Veritor SARS-CoV-2/Flu") triplex assay. The performance for SARS-CoV-2 detection was determined using 298 specimens from patients reporting COVID-19 symptoms within 7 days from symptom onset (DSO) in comparison with the Lyra SARS-CoV-2 RT-PCR (reverse transcriptase PCR) assay ("Lyra SARS-CoV-2") as the reference. The performance for flu A and flu B detection was determined using 75 influenza-positive and 40 influenza-negative retrospective specimens in comparison with the previously FDA-cleared BD Veritor System for Rapid Detection of Flu A+B assay ("Veritor Flu") as the reference. The Veritor SARS-CoV-2/Flu assay met the FDA EUA acceptance criteria (86.7%; 95% confidence interval [95% CI]: 75.8 to 93.1) for SARS-CoV-2 testing compared to Lyra SARS-CoV-2. The Veritor SARS-CoV-2/Flu assay also demonstrated 100% agreement with the Veritor Flu for Flu A+B assay. For flu A detection, the lower bound of the 95% CI was 91.2%; for flu B detection, the lower bound was 90.0%. The dual detection capability of Veritor SARS-CoV-2/Flu for the etiologic agents causing COVID-19 and flu will allow efficient differentiation between the two illnesses, inform disease management, and facilitate optimal treatment. IMPORTANCE COVID-19 and flu are two respiratory illnesses which share similar clinical symptoms. The BD Veritor SARS-CoV-2/Flu assay has high sensitivity and specificity for detecting the SARS-CoV-2 and influenza A/B, the two etiologic agents causing COVID-19 and flu, respectively. This dual detection capability is critical when overlap occurs between the COVID-19 pandemic and the flu season. This triplex assay will allow efficient differentiation between the two respiratory illnesses and support a point-of-care physician diagnosis to facilitate the proper treatment and disease management for patients exhibiting overlapping symptoms.

Evaluation of two RT-PCR screening assays for identifying SARS-CoV-2 variants.

BACKGROUND: The recent emergence of new SARS CoV-2 variants (variants of concern, VOC) that spread rapidly and may lead to immune escape has emphasized the urgent need to monitor and control their spread. METHODS: We analyzed 2018 SARS-CoV-2 positive specimens collected between February 9 and March 22, 2021 using the Thermofisher® TaqPath™ COVID-19 CE-IVD RT-PCR kit (TaqPath) and the ID solutions® ID™ SARS-CoV-2/UK/SA Variant Triplex RT-PCR (ID triplex) assay to screen for VOCs. RESULTS: The ID triplex assay identified 62.8% of them as VOCs: 61.8% B.1.1.7 and 0.9% B.1.351/P.1. The agreement between the ID triplex results for B.1.1.7 and the TaqPath S gene target failure (SGTF)/ S gene target late detection (SGTL) profile for this variant agreed very well (k = 0.86). A low virus load was the main cause of discrepancies. Sequencing discordant results with both assays indicated that the TaqPath assay detected the B.1.1.7 lineage slightly better. Both assays suggested that the virus loads of B.1.1.7 variants were significantly higher than those of non-B.1.1.7 strains. Only 10/20 B1.351/P.1 strains detected with the ID triplex assay were confirmed by sequencing. CONCLUSIONS: We conclude that the SGTF/SGTL profiles identified using the TaqPath assay and ID triplex results are suitable for detecting the B.1.1.7 lineage. The ID triplex assay, which rapidly determines all three current VOCs simultaneously, could be a valuable tool for limiting virus spread by supporting contact-tracing and isolation.

G-triplex: A new type of CRISPR-Cas12a reporter enabling highly sensitive nucleic acid detection.

CRISPR-Cas12a (Cpf1) trans-cleaves ssDNA and this feature has been widely harnessed for nucleic acid detection. Herein, we introduce a new type of Cas12a reporter, G-triplex (G3), and a highly sensitive biosensor termed G-CRISPR. We proved that Cas12a trans-cleaves G3 structures in about 10 min and G3 can serve as an excellent reporter based on the cleavage-induced high-order structure disruption. G3 reporter improves the analytical sensitivity up to 20 folds, enabling the detection of unamplified and amplified DNA as low as 50 pmol and 0.1 amol (one copy/reaction), respectively. G-CRISPR has been utilized for the analysis of 27 PCR-amplified patient samples with HPV infection risk based on both fluorescence and lateral flow assays, resulting in 100% concordance between the two. In comparison with the clinical results, it achieved overall specificity and sensitivity of 100% and 94.7%, respectively. These results suggest that G-CRISPR can serve as a rapid, sensitive, and reliable biosensor, and could further expand the CRISPR toolbox in biomedical diagnostics.

Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR.

BACKGROUND: The novel respiratory virus SARS-CoV-2, responsible for over 380,000 COVID-19 related deaths, has caused significant strain on healthcare infrastructure and clinical laboratories globally. The pandemic's initial challenges include broad diagnostic testing, consistent reagent supply lines, and access to laboratory instruments and equipment. In early 2020, primer/probe sets distributed by the CDC utilized the same fluorophore for molecular detection - requiring multiple assays to be run in parallel - consuming valuable and limited resources. METHODS: Nasopharyngeal swabs submitted to UW Virology for SARS-CoV-2 clinical testing were extracted, amplified by our laboratory developed test (LDT) - a CDC-based quantitative reverse transcriptase PCR reaction - and analyzed for agreement between the multiplexed assay. Laboratory- confirmed respiratory infection samples were included to evaluate assay cross-reaction specificity. RESULTS: Triplexing correctly identified SARS-CoV-2 in 98.4% of confirmed positive or inconclusive patient samples by single-plex LDT (n = 183/186). All 170 SARS-CoV-2 negative samples tested by single-plex LDT were negative by triplexing. Other laboratory-confirmed respiratory infections did not amplify for SARS-CoV-2 in the triplex reaction. CONCLUSIONS: Multiplexing two virus-specific gene targets and an extraction control was found to be comparable to running parallel assays independently, while significantly improving assay throughput.