Simultaneous Detection of Three Genotypes of Gene Methylene Tetrahydrofolate Reductase and Methionine Synthase Reductase Based on Multiplex Asymmetric Real-Time PCR-HRM Biosensing

Yu, Wen; Yao, Juan; Zhang, Zhang

doi:10.1021/acs.analchem.2c02096

Cited by 4 publications

(3 citation statements)

References 26 publications

(34 reference statements)

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“…SNP detection techniques that employ real-time PCR with closed tube systems are currently the most prevalent methods used in clinical practice. Among these techniques, TaqMan probe- 3 and dual-labeled probe-mediated high-resolution melt curve methods 4 are the most commonly utilized. In addition, most commercially available SNP genotyping kits, authorized by China National Medical Products Administration (NMPA), US Food and Drug Administration (FDA), and European Conformity (CE) marking, are based on these techniques.…”

Section: ■ Introductionmentioning

confidence: 99%

RART-LAMP: One-Step Extraction-Free Method for Genotyping within 40 min

et al. 2023

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Loop-mediated isothermal amplification (LAMP) is a commonly used alternative to PCR for point-of-care detection of nucleic acids due to its rapidity, sensitivity, specificity, and simpler instrumentation. While dual-labeled TaqMan probes are widely used in PCR for single-nucleotide polymorphism (SNP) genotyping, real-time LAMP primarily relies on turbidimetry or intercalator fluorescence measurements, which can be non-specific and generate false-positive results. In this study, we propose a closed-tube, dual-labeled RNA-modified probes and RNase H II-assisted real-time LAMP (RART-LAMP) method for SNP genotyping. Our findings indicate that (1) fluorescence signals were predominantly derived from probe hydrolysis rather than hybridization, (2) temperature-controlled hybridization between the probe and template ensured the specificity of SNP analysis, and (3) RNase H II hydrolysis between the target containing SNP sites and probes did not exhibit sequence specificity. Our RART-LAMP approach demonstrated excellent performance in genotyping C677T clinical samples, including gDNA extracted from blood, saliva, and swabs. More importantly, saliva and swab samples could be directly analyzed without any pretreatment, indicating promising prospects for nucleic acid analysis at the point of care in resource-limited settings.

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Section: ■ Introductionmentioning

confidence: 99%

RART-LAMP: One-Step Extraction-Free Method for Genotyping within 40 min

et al. 2023

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“…Multiplex asymmetric PCR based detection has evolved a rapid and convenient tool in wide variety of bioanalyses ( Zhu et al, 2007 ; Yu et al, 2022 ; Kong et al, 2023 ). It uses many pairs of unequal concentrations of the primers for amplification, generating single stranded amplicons.…”

Section: Introductionmentioning

confidence: 99%

“…This facilitates the simultaneous amplification of many targets of interest in one reaction, thus enhancing assay throughput and allowing more efficient use of each sample. Previous study has reported the development of multiplex polymorphism detection in a single tube for screening folate metabolism genes ( Yu et al, 2022 ). Additionally, it has been utilized to identify seven immune-escape RBD mutations of Omicron ( Kong et al, 2023 ), as well as detecting drug resistance genes in Enterobacteriaceae ( Zhu et al, 2007 ).…”

Section: Introductionmentioning

confidence: 99%

A novel assay based on DNA melting temperature for multiplexed identification of SARS-CoV-2 and influenza A/B viruses

Gao,

Fan,

Kong

et al. 2023

Front. Microbiol.

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IntroductionThe severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and influenza viruses can cause respiratory illnesses with similar clinical symptoms, making their differential diagnoses challenging. Additionally, in critically ill SARS-CoV-2–infected patients, co-infections with other respiratory pathogens can lead to severe cytokine storm and serious complications. Therefore, a method for simultaneous detection of SARS-CoV-2 and influenza A and B viruses will be clinically beneficial.MethodsWe designed an assay to detect five gene targets simultaneously via asymmetric PCR-mediated melting curve analysis in a single tube. We used specific probes that hybridize to corresponding single-stranded amplicons at low temperature and dissociate at high temperature, creating different detection peaks representing the targets. The entire reaction was conducted in a closed tube, which minimizes the risk of contamination. The limit of detection, specificity, precision, and accuracy were determined.ResultsThe assay exhibited a limit of detection of <20 copies/μL for SARS-CoV-2 and influenza A and <30 copies/μL for influenza B, with high reliability as demonstrated by a coefficient of variation for melting temperature of <1.16% across three virus concentrations. The performance of our developed assay and the pre-determined assay showed excellent agreement for clinical samples, with kappa coefficients ranging from 0.98 (for influenza A) to 1.00 (for SARS-CoV-2 and influenza B). No false-positive, and no cross-reactivity was observed with six common non-influenza respiratory viruses.ConclusionThe newly developed assay offers a straightforward, cost-effective and nucleic acid contamination-free approach for simultaneous detection of the SARS-CoV-2, influenza A, and influenza B viruses. The method offers high analytical sensitivity, reliability, specificity, and accuracy. Its use will streamline testing for co-infections, increase testing throughput, and improve laboratory efficacy.

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Standardization and application of ARMS TaqMan real‐time PCR for screening of folate metabolism genes in Han Chinese

Deng,

Liu,

et al. 2024

Electrophoresis

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Folate has antioxidant properties, and low concentration in seminal plasma may be associated with increased DNA damage in sperm. Mutations of the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes, including MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131), and MTRR A66G (rs1801394), can lead to decreased activity of the encoded folate metabolic enzymes, thereby affecting male reproduction. The current SNP detection methods commonly used in clinical practice have some shortcomings, such as long time‐consuming, complex detection steps, or high cost. The purpose of this study was to establish a simple, time‐saving, sensitive, accurate, and easy to clinical popularization method for folate metabolism gene detection. We combined ARMS‐PCR with TaqMan fluorescent probe to establish an ARMS TaqMan real‐time PCR detection method. According to the variation of rs1801131, rs1801133, and rs1801394, two specific primers (one wild type and one mutant) were designed. Mismatched nucleotides were introduced at the penultimate or third position to improve the specificity of the primer. Specific TaqMan probe was introduced to detect PCR products to improve the sensitivity of the method. The results showed that the sensitivity of ARMS TaqMan real‐time PCR in SNP genotyping was 1 ng, and the accuracy was 100%. A total of 249 clinical samples were detected by the established method, and the correlation between three SNPs and semen quality was analyzed. We found that individuals carrying the AG + GG genotype of rs1801394 had a lower risk of abnormal semen quality. In conclusion, we developed a highly sensitive, accurate, rapid, and easy to be popularized method for detecting SNPs of rs1801394, rs1801131, and rs1801133. ARMS TaqMan real‐time PCR is a reliable SNP genotyping method in folate metabolism genes.

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Simultaneous Detection of Three Genotypes of Gene Methylene Tetrahydrofolate Reductase and Methionine Synthase Reductase Based on Multiplex Asymmetric Real-Time PCR-HRM Biosensing

Cited by 4 publications

References 26 publications

RART-LAMP: One-Step Extraction-Free Method for Genotyping within 40 min

RART-LAMP: One-Step Extraction-Free Method for Genotyping within 40 min

A novel assay based on DNA melting temperature for multiplexed identification of SARS-CoV-2 and influenza A/B viruses

Standardization and application of ARMS TaqMan real‐time PCR for screening of folate metabolism genes in Han Chinese

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