Fast PCR Assay Research Articles

Evaluation of Rapid Multiplex Reverse Transcription-Quantitative Polymerase Chain Reaction Assays for SARS-CoV-2 Detection in Individual and Pooled Samples.

Although coronavirus disease 2019 (COVID-19) is no longer a Public Health Emergency of International Concern (PHEIC), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has had a vast impact to date. Hence, continuous management is required, given the uncertainty caused by the potential evolution of SARS-CoV-2. Reverse transcription-quantitative PCR (RT-qPCR) diagnosis has been fundamental in overcoming this issue. In this study, the performances of two rapid RT-qPCR assays (Real-Q Direct SARS-CoV-2 Detection Kit and Allplex™ SARS-CoV-2 fast PCR Assay) with short PCR times were comparatively evaluated using a STANDARD M nCoV Real-Time Detection Kit (STANDARD M, conventional RT-qPCR assay). All kits showed a limit of detection values (102-103 copies/reaction). The evaluation showed that the two rapid assay tests had ≥97.89% sensitivity and ≥99.51% specificity (κ = 0.98) for individual samples and ≥97.32% sensitivity and ≥97.67% specificity for pooled samples compared to STANDARD M. These results indicate that the two rapid RT-qPCR kits, which showed significant time reduction in performance, are as effective as a conventional RT-qPCR assay. They are likely to increase not only the number of tests that can be performed but also the efficiency of sustainable management of COVID-19 in the long term.

Diagnostic performances of four commercially available assays for the identification of SARS-CoV-2, influenza type A/B virus and RSV

We evaluated the diagnostic performance of 4 commercially NAAT for detecting SARS-CoV-2 RNA, Influenza type A/B virus and RSV. Included tests were the Allplex™ SARS-CoV-2 fast PCR Assay (RNA extraction-free), Allplex™ RV Master Assay, Allplex™ SARS-CoV-2 fast MDx Assay (LAMP) and Aptima™ SARS-CoV-2/Flu Assay (RT-TMA). The assays’ performance characteristics were determined using nasopharyngeal swabs from 270 patients with suspected SARS-CoV-2 infection. A total of 215 SARS-CoV-2 positive, 55 negative nasopharyngeal swabs and 19 bacteria strains were included. The sensitivities and specificities for detecting SARS-CoV-2, Influenza type A virus and RSV ranged between 81.8% and 100% with extremely good agreements (κ ≥ 86.8 %). The Aptima™ SARS-CoV-2/Flu Assay introduced a new result parameter, that is, TTime. Here, we showed that TTime may be used as a surrogate for Ct-value. We concluded that all assays assessed in this study can be used for routine detection of SARS-CoV-2, Influenza type A virus and RSV.

Rapid and highly specific screening for NPM1 mutations in acute myeloid leukemia

NPM1 mutations, the most frequent molecular alterations in acute myeloid leukemia (AML), have become important for risk stratification and treatment decisions for patients with normal karyotype AML. Rapid screening for NPM1 mutations should be available shortly after diagnosis. Several methods for detecting NPM1 mutations have been described, most of which are technically challenging and require additional laboratory equipment. We developed and validated an assay that allows specific, rapid, and simple screening for NPM1 mutations. FAST PCR spanning exons 8 to 12 of the NPM1 gene was performed on 284 diagnostic AML samples. PCR products were visualized on a 2% agarose E-gel and verified by direct sequencing. The FAST PCR screening method showed a specificity and sensitivity of 100%, i.e., all mutated cases were detected, and none of negative cases carried mutations. The limit of detection was at 5-10% of mutant alleles. We conclude that the FAST PCR assay is a highly specific, rapid (less than 2h), and sensitive screening method for the detection of NPM1 mutations. Moreover, this method is inexpensive and can easily be integrated in the routine molecular diagnostic work-up of established risk factors in AML using standard laboratory equipment.

Molecular characterization of a new waxy allele with partial expression in spelt wheat

Starch composition which is dependent on the waxy protein, the enzyme responsible for amylose synthesis in the grain, is an important aspect of the wheat quality. In this report, we describe the characterization of a novel Wx-A1 allele (Wx-A1g formerly known as -Wx-A1a) in Spanish spelt wheat lines which is responsible for a remarkable decline in the concentration of Wx-A1 protein found in the endosperm. Comparison of the DNA sequences in the Wx-A1a and Wx-A1g alleles showed the presence of a 160-bp insertion within the fourth intron in the latter. This insertion had some characteristics of a transposable-like element. RT-PCR analysis showed the presence of normal and aberrant mRNA transcripts in the Wx-A1g lines, indicating that the aberrant transcripts are un-spliced and contained the longer fourth intron. This may be related to the low level of Wx-A1 protein in these lines. In addition, a simple and fast PCR assay was designed for differentiating among different Wx-A1 alleles (a, b, f and g). The mutation described here is not related to either of the Wx-A1 mutations identified previously in common and durum wheats and could help to extend the range of amylose content of wheats.