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Rapid differentiation of influenza A virus subtypes and genetic screening for virus variants by high-resolution melting analysis.

ABSTRACT: We assessed the use of high-resolution melting (HRM) analysis for the rapid identification of influenza A virus subtypes and the detection of newly emerging virus variants. The viral matrix gene was amplified by LightCycler real-time reverse transcription-PCR (RT-PCR) in the presence of the LCGreen I fluorescent dye. Upon optimization of the assay conditions, all the major influenza A virus subtypes, including H1N1, H3N2, H5N1, H7N3, and H9N2, were amplifiable by this method and had a PCR product length of 179 bp. Real-time RT-PCR of in vitro-transcribed H3N2 RNA revealed a standard curve for quantification with a linear range (correlation coefficient = 0.9935) across at least 8 log units of RNA concentrations and a detection limit of 10(3) copies of viral RNA. We performed HRM analysis of the PCR products with the HR-1 instrument and used the melting profiles as molecular fingerprints for virus subtyping. The virus subtypes were identified from the high-resolution derivative plot obtained by heteroduplex formation between the PCR products of the viral isolates tested and those of the reference viral isolates. The melting profiles were consistent with minimal interassay variability. Hence, an HRM database and a working protocol were established for the identification of these five influenza A virus subtypes. When this protocol was used to test 21 clinical influenza A virus isolates, the results were comparable to those obtained by RT-PCR with hemagglutinin-specific primer sets. Sequence variants of the clinical isolates (n = 4) were also revealed by our HRM analytical scheme. This assay requires no multiplexing or hybridization probes and provides a new approach for influenza A virus subtyping and genetic screening of virus variants in a clinical virology laboratory.

SUBMITTER: Lin JH

PROVIDER: S-EPMC2268327 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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Rapid differentiation of influenza A virus subtypes and genetic screening for virus variants by high-resolution melting analysis.

Lin Jih-Hui JH Tseng Ching-Ping CP Chen Yen-Ju YJ Lin Chy-Yung CY Chang Shy-Shin SS Wu Ho-Sheng HS Cheng Ju-Chien JC

Journal of clinical microbiology 20080103 3

We assessed the use of high-resolution melting (HRM) analysis for the rapid identification of influenza A virus subtypes and the detection of newly emerging virus variants. The viral matrix gene was amplified by LightCycler real-time reverse transcription-PCR (RT-PCR) in the presence of the LCGreen I fluorescent dye. Upon optimization of the assay conditions, all the major influenza A virus subtypes, including H1N1, H3N2, H5N1, H7N3, and H9N2, were amplifiable by this method and had a PCR produc ...[more]

PMID: 18174299

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Project description:Current methods for the detection of single nucleotide polymorphisms (SNPs) associated with aberrant drug-metabolizing enzyme function are hindered by long turnaround times and specialized techniques and instrumentation. In this study, we describe the development and validation of a high-resolution melting (HRM) curve assay for the rapid screening of variant genotypes for targeted genetic polymorphisms in the cytochrome P450 enzymes CYP2C9, CYP2C19, and CYP3A5.Sequence-specific primers were custom-designed to flank nine SNPs within the genetic regions of aforementioned drug metabolizing enzymes. PCR amplification was performed followed by amplicon denaturation by precise temperature ramping in order to distinguish genotypes by melting temperature (Tm). A standardized software algorithm was used to assign amplicons as 'reference' or 'variant' as compared to duplicate reference sequence DNA controls for each SNP.Intra-assay (n=5) precision of Tms for all SNPs was ?0.19%, while inter-assay (n=20) precision ranged from 0.04% to 0.21%. When compared to a reference method of Sanger sequencing, the HRM assay produced no false negative results, and overcall frequency ranged from 0% to 26%, depending on the SNP. Furthermore, HRM genotyping displayed accuracy over input DNA concentrations ranging from 10 to 200 ng/?L.The presented assay provides a rapid method for the screening for genetic variants in targeted CYP450 regions with a result of 'reference' or 'variant' available within 2 h from receipt of extracted DNA. The method can serve as a screening approach to rapidly identify individuals with variant sequences who should be further investigated by reflexed confirmatory testing for aberrant cytochrome P450 enzymatic activity. Rapid knowledge of variant status may aid in the avoidance of adverse clinical events by allowing for dosing of normal metabolizer patients immediately while identifying the need to wait for confirmatory testing in those patients who are likely to possess pharmacogenetically-relevant variants.

Project description:The possibility of introducing a reliable assay for a quick identification and differentiation of the main species of Mycobacterium tuberculosis complex (MTBC) supports the improvement of efficient tuberculosis combating strategies worldwide. Commercially available assays are often based on cultured samples; however, due to the long cultivation time of mycobacteria, results are delayed. Developed PCR approaches have been published previously, though, when testing intricate veterinary samples, the complex composition of multiplex qPCRs frequently leads to assay failure. In order to overcome those limits, a paradigm of a three-reaction high-resolution melting (HRM) assay for the simultaneous identification and differentiation of the main members of MTBC was established. The assay is based on single nucleotide polymorphisms within gyrB and gyrA, which have been used as target for the establishment of two highly specific HRM assays (HRM assays 1 and 2) discriminating M. tuberculosis/ Mycobacterium canetti, Mycobacterium bovis/M. bovis BCG, Mycobacterium caprae/rare M. caprae/M. bovis ecotypes, Mycobacterium africanum/Mycobacterium orygis/ Mycobacterium pinnipedii/Clade A1, Mycobacterium microti, and a rare subtype of M. canettii followed by a third HRM assay (HRM assay 3) allowing a further differentiation of M. bovis, M. bovis BCG, and a rare subtype of M. caprae/M. bovis, which is considered to be a novel ecotype. High-resolution melting assay 1 is described in a previously published report. High-resolution melting assay 2 showed 100% correlation of all 39 examined isolates with the results of a commercial identification kit. 96% of the clinical samples tested demonstrated concordant results. High-resolution melting assay 3 showed an accordance of 100% with the results of the commercially available identification kit of all 22 samples analyzed. The proposed strategy of the three-reaction HRM assay can be used for an accurate differentiation of up to seven groups of MTBC and potentially to identify a rare subtype of M. canettii either on isolates or on clinical samples.

Project description:Coronavirus disease 2019, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a considerable threat to global public health. This study developed and evaluated a rapid, low-cost, expandable, and sequencing-free high-resolution melting (HRM) assay for the direct detection of SARS-CoV-2 variants. A panel of 64 common bacterial and viral pathogens that can cause respiratory tract infections was employed to evaluate our method's specificity. Serial dilutions of viral isolates determined the sensitivity of the method. Finally, the assay's clinical performance was assessed using 324 clinical samples with potential SARS-CoV-2 infection. Multiplex HRM analysis accurately identified SARS-CoV-2 (as confirmed with parallel reverse transcription-quantitative PCR [qRT-PCR] tests), differentiating between mutations at each marker site within approximately 2 h. For each target, the limit of detection (LOD) was lower than 10 copies/reaction (the LOD of N, G142D, R158G, Y505H, V213G, G446S, S413R, F486V, and S704L was 7.38, 9.72, 9.96, 9.96, 9.50, 7.80, 9.33, 8.25, and 8.25 copies/reaction, respectively). No cross-reactivity occurred with organisms of the specificity testing panel. In terms of variant detection, our results had a 97.9% (47/48) rate of agreement with standard Sanger sequencing. The multiplex HRM assay therefore offers a rapid and simple procedure for detecting SARS-CoV-2 variants. IMPORTANCE In the face of the current severe situation of increasing SARS-CoV-2 variants, we developed an upgraded multiplex HRM method for the predominant SARS-CoV-2 variants based on our original research. This method not only could identify the variants but also could be utilized in subsequent detection of novel variants since the assay has great performance in terms of flexibility. In summary, the upgraded multiplex HRM assay is a rapid, reliable, and economical detection method, which could better screen prevalent virus strains, monitor the epidemic situation, and help to develop measures for the prevention and control of SARS-CoV-2.

Dataset Information

Rapid differentiation of influenza A virus subtypes and genetic screening for virus variants by high-resolution melting analysis.

Publications

Rapid differentiation of influenza A virus subtypes and genetic screening for virus variants by high-resolution melting analysis.

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