A Novel Method for Rapid Screening of Salmonidae Ingredients and Accurate Detection of Atlantic Salmon (Salmo salar) Simultaneously Using Duplex Real-Time PCR Coupled with Melting Curve Analysis.

The substitution of ingredients with Salmonidae, particularly Salmo salar, has led to widespread reports of financial losses and health risks globally, emphasizing the urgent need for the development of a rapid and precise method for species identification. The aim of the present study was to develop a novel method for the rapid screening of Salmonidae ingredients and the accurate detection of S. salar simultaneously using multiplex real-time PCR coupled with melting curve analysis. Specifically, primer sets specific for S. salar and Salmonidae were cross-confirmed. Moreover, the reaction system and conditions of a real-time duplex PCR were optimized, and the proposed methodology was verified, proving that the assay has good specificity and sensitivity. Clear and distinguishable melting peaks, with expected Tm values of around 80 °C (S. salar) and 84 °C (Salmonidae), were observed for twelve products, proving the presence of S. salar. However, four products were not derived from S. salar, but they could have belonged to another species within the Salmonidae family due to the presence of only one specific melting peak at a Tm value of about 84 °C. Therefore, the novel assay in the present study allows for the fast and accurate screening of Salmonidae ingredients and the detection of S. salar simultaneously.

Multigeographic clinical assessment of a molecular diagnostic assay for detection of key codons to predict decreased susceptibility or resistance to cephalosporins in Neisseria gonorrhoeae.

Cephalosporin resistance in Neisseria gonorrhoeae has severely compromised the efficacy of World Health Organization (WHO)-recommended therapies. This study aimed to methodologically evaluate the optimized Six-CodonPlus assay, and additionally conducted a multicenter evaluation to assess its clinical application, especially for predicting antimicrobial resistance (AMR). For methodological evaluation, 397 sequence-known N. gonorrhoeae isolates were evaluated for specificity, 17 nongonococcal isolates were assessed for cross-reactivity, 159 uncultured urogenital swabs and urine samples were evaluated for sensitivity at the clinical level. For multicenter evaluation, 773 isolates with confirmed phenotypic data and 718 clinical urogenital swabs collected from four geographical cities were, respectively, utilized for the evaluation of AMR-prediction strategies and the clinical application of the assay. The assay accurately identified specific single-nucleotide polymorphisms in resistance-associated genes, the detection limits dropped to 10 copies/reaction for individual targets. The specificity reached 100% and no cross-reactivity occurred with double-target confirmation. The assay could be directly applied to clinical samples containing over 20 copies/reaction. Multicenter evaluation formulated two optimal strategies for decreased susceptibility prediction in specific scenarios, and one tactic for prediction of resistance and identification of FC428-like strains. High sensitivity of 86.84% (95% CI, 71.11-95.05) and specificity of 99.59% (95% CI, 98.71-99.89) for resistance prediction were demonstrated for ceftriaxone (CRO). Regarding N. gonorrhoeae identification among multicenter swabs, specificity reached 97.53% (95% CI, 95.49-98.69), and sensitivity reached 93.77% (95% CI, 90.04-96.22). The Six-CodonPlus assay exhibited excellent detection performance and formulated optimal AMR-related prediction strategy with regional adaptability, providing critical information for population screening and clinical treatment.

The pertinence of resistin gene single nucleotide polymorphism G > A and its expression in oral cancer.

BACKGROUND/AIM: Oral cancer (OC) is the leading cause of fatalities in Pakistan among males due to inadequate oral hygiene and chewing habits. However, genetic susceptibility patterns also play a critical role in disease progression. Since the frequency of Resistin (RETN) SNP (Single nucleotide polymorphism) rs3219175 is unknown; there is a requirement for early diagnosis of the OC. Therefore, the current study aims to determine the frequency of targeted SNP and develop a safe, simple, and fast alternative technique for better treatment using a real-time PCR assay with HRM (high-resolution melting curve) analysis. MATERIALS AND METHODS: A case-control study was conducted on 35 Oral squamous cell carcinomas (OSCC) diagnosed patients and 35 healthy individuals. HRM and RT-PCR results were analysed by the bioinformatics analyses. RESULTS: The frequency of RETN SNP rs3219175 genotypes GG and GA in male patients was 16 (46 %) and 5 (14 %) respectively and in females 8 (23 %) and 6 (17 %) respectively. The chi-square test of independence consummated the assessment between males and females in both control and patients. The relation between these variables was significant (p < 0.05). The interaction network of String 8.3 demonstrates strong interactions at a high confidence score, which helps to characterize functional disorders that may be a causative factor for oral pathology. Reactome and KEGG data were acquired to rule out the pathway involvement of the targeted gene. MuPIT software was used to identify the 3D structure or RETN and their expected mutation effect. CONCLUSION: This study provides baseline data regarding the frequency of RETN SNP rs3219175 among the Pakistani population. For further clarification of their stage in cancer emergence and growth, large-scale studies must be conducted. This study might be helpful in the precision medicine approach and provide better therapeutic for OSCC patients.

Rapid identification, pathotyping and quantification of infectious bursal disease virus by high-resolution melting curve quantitative reverse transcription PCR analysis: An innovative technology well-suited for real-time large-scale epidemiological surveillance.

With the virus continuing to evolve, very virulent IBDV (vvIBDV) and novel variant IBDV (nvIBDV) have become the predominant epidemic strains in China, exacerbated by the widespread use of attenuated vaccine strains (attIBDV), making a complex infection situation of IBDV in the field. Therefore, developing a rapid and accurate high-resolution melting curve quantitative reverse transcription PCR (HRM-qRT-PCR) for the identification and pathotyping of IBDV is crucial for clinical monitoring and disease control. Extensive data analysis and genome-screening of the three dominant IBDV pathotypes identified a specific region (nucleotides 2450-2603 in segment A) with distinct GC content as the detection target. Experimental testing of HRM-qRT-PCR revealed distinct melting curves and high sensitivity, with the detection limits of 61.2 copies/µL, 61.1 copies/µL and 67.5 copies/µL for vvIBDV, nvIBDV and attIBDV, respectively. The method exhibited excellent specificity, with no inter-genotypes cross-reactivity among the three pathotypes and no reactivity to other common avian pathogens. Applied to samples with double and triple co-infections of different IBDV pathotypes, the method displayed specific melting peaks corresponding to the viruses present in the samples, with an accuracy rate of 100 %. This method precisely identifies and differentiates all the single or co-infected samples, generating distinct peaks corresponding to the Tm values of each virus pathotype in traditional melting curve plots. Furthermore, the method overcomes the limitations of traditional pathotyping methods, requiring only one reaction to achieve rapid viral pathotyping and facilitating quantitative analysis of viruses within the samples. This study introduces an innovative HRM-qRT-PCR method, offering new technology to rapid and accurate identification, pathotyping and quantification of vvIBDV, nvIBDV, and attIBDV. With strong discriminatory power, user-friendliness and a short processing time, this method is highly attractive for the rapid IBDV pathotyping in real-time large-scale epidemiological surveillance during outbreaks.

Simultaneous Genotyping of Three SNVs, rs5471, rs5472, and rs2000999 Involved in Serum Haptoglobin Levels by Fluorescent Probe-Based Melting Curve Analysis.

Haptoglobin (Hp) is a hemoglobin-binding acute-phase serum protein. Several single nucleotide variations (SNVs) within the Hp gene (HP) or Hp-related protein gene (HPR), such as rs5471 (A > C) and rs5472 (A > G) in HP promoter region and rs2000999 (G > A) in intron 2 of HRP, are suggested to correlate with the serum Hp levels. To determine these three SNVs simultaneously, a genotyping assay based on duplex dual-labeled fluorescent probes was developed. The method was then validated by analyzing genomic DNA from 121 Ghanaian and two Japanese subjects who had been previously genotyped for rs5471, rs5472, and rs2000999. Both rs5471 and rs5472 could be determined as haplotypes with a single FAM-labeled fluorescent probe, and rs2000999 could be genotyped with a HEX-labeled fluorescent probe. The results obtained with the present method were consistent with the previous results except for those of three Ghanaian subjects. All three subjects appear to have multiple HPR copy number variants characteristic of African populations, which may have led to incorrect results during previous genotyping. This method allows us to genotype these three SNVs in a relatively large number of samples, especially in African populations where rs5471 is uniquely distributed.

Highly specific screening of aspirin resistance-related single-nucleotide polymorphisms using ligase chain reaction strategy.

Aspirin (ASP) is currently the drug of choice for antiplatelet therapy. However, approximately 5%-45 % of patients are resistant to ASP and do not achieve the expected result. At present, a few studies have investigated the correlation between ASP resistance (AR) and single-nucleotide polymorphism (SNP). Traditional detection methods are time-consuming and laborious, affecting the accuracy of personalized medicine. This study aimed to establish a new assay to identify four SNPs associated with AR. A large amount of double-stranded DNA was formed after multiple cycles of specific exponential amplification by ligase chain reaction, the specific melting peak of which was visible in the detection curve, with a detection limit of 10-11mol/L. The specificity experiments of different proportions of wild-type and mutant plasmid standards showed that the novel method could detect up to 1 % allele frequency and the specificity was good. Clinical blood samples of 57 patients were tested in this study. The results were consistent with those of sequencing and more accurate and reliable than those of the high-resolution melting method. The technique used in this study was simple, sensitive and specific compared with the traditional method. Statistical analysis revealed that AR was significantly correlated with the rs12041331 site of the PEAR1 gene and the rs1695 site of the GSTP1 gene, providing an important reference value for the study of AR.

Investigation of suspected false positive norovirus results on a syndromic gastrointestinal multiplex molecular panel.

BACKGROUND: Suspected false positive norovirus results occurred after introduction of the BioFire® FilmArray® Gastrointestinal panel (BF-GIP) into 6 laboratory sites, in British Columbia, Canada. OBJECTIVES: The primary objective was to investigate suspected false positive results by performing additional molecular assays and whole genome sequencing (WGS). The second objective was to determine if melting curve review could predict false positive results. STUDY DESIGN: From February 2023 to May 2024, the proportion of potential false positive norovirus results from the BF-GIP was calculated based on confirmatory testing using alternate molecular method. A subset of 65 norovirus BF-GIP positive specimens, including 35 negatives and 30 positives on additional molecular assays, underwent WGS. Melting curves appearances from 150 specimens were reviewed. RESULTS: Overall, 215/784 (27.4 %) BF-GIP norovirus results were suspected to be false positives. When using WGS, 64/65 results were in agreement with confirmatory testing. Notably, 35 specimens negative on confirmatory testing and suspected to be BF-GIP norovirus false positive results were undetectable by WGS. Melting curves did not accurately predict false positives, since 20/72 (27.8 %) had typical appearances upon review. CONCLUSIONS: BF-GIP produces false positive results for norovirus, which cannot be predicted by melting curve review.

Detection Of CCR5 Delta-32 Mutation Using High-Resolution Melting Curve Analysis: Challenges and Facts.

INTRODUCTION: The C-C chemokine receptor type 5 (CCR5) is a major co-receptor for human immunodeficiency virus (HIV). Some individuals carry the CCR5 delta-32 genetic polymorphism. People with homozygous CCR5 delta-32 gene are nearly completely resistant to HIV-1 infection. High-resolution melting curve (HRM) analysis is a post-PCR technique utilized for identifying genetic variations in a quick, affordable, and closed-tube assay. The objective of this study was to develop an HRM assay for easy detection of delta-32 mutations. MATERIALS AND METHODS: DNA was extracted from peripheral blood mononuclear cells. HRM was performed to detect delta-32 mutation. The study investigated the impact of various factors, including annealing temperature, template concentration, touchdown PCR, additives, amplicon size, and program settings, on HRM Tm differentiation. RESULTS: It was expected that there would be a 4°C Tm difference between amplicons with and without delta-32 mutation, but the test showed a difference of only 2.3°C. In attempts to identify heterozygote delta-32 variants, a Tm difference of only 0.4°C could be achieved. Various modifications were applied, such as adjusting the template concentration, using touchdown PCR, and adding DMSO and glycerol. However, none of these changes helped to differentiate the Tm effectively, especially in delta-32 heterozygote samples. CONCLUSION: The HRM test identified four samples with heterozygote mutations in each HIV-infected (8.89%) and control (5.72%) groups. More importantly, this study showed that identifying the delta-32 mutation of the CCR5 gene using HRM assay is not as straightforward as previously suggested in some literature, and it requires special setup conditions.

pH-dependent binding of ATP aptamer to the target and competition strands: Fluorescent melting curve fitting study.

The pH varies in different tissues and organelles and also changes during some diseases. In this regard, the application of molecular switches that use a competition-based aptamer switch design in biological systems requires studying the thermodynamics of such systems at different pH values. In this work, we studied the binding of the classical ATP aptamer to ATP and competition strands under different pH and ionic conditions using fluorescent melting curve analysis. We have developed an original approach to processing source data from a PCR thermal cycler. It is based on constructing a thermodynamic model of the melting profile and the subsequent fit of experimental curves within this model. We have shown that this approach enables us to narrow the temperature region under study to the width of the melting region without a significant loss in the quality of the result. This impressively expands the application area of this approach compared to frequently used techniques that require mandatory measurement of the signal outside the melting region. The results obtained by the method showed that the thermodynamic parameters of the ATP aptamer and its duplexes with competition strands change depending on pH. Therefore, molecular switches that use a competition strand to the ATP aptamer may have a pH-dependent sensitivity that has not been previously considered. This should be taken into account for future rational design of similar systems.

Rapid and specific differentiation of Salmonella enterica serotypes typhi and Paratyphi by multicolor melting curve analysis.

Rapid and accurate identification of Salmonella enterica serotypes Typhi and Paratyphi (A, B and C), the causal agents of enteric fever, is critical for timely treatment, case management and evaluation of health policies in low and middle-income countries where the disease still remains a serious public health problem. The present study describes the development of a multiplex assay (EFMAtyping) for simultaneous identification of pathogens causing typhoid and paratyphoid fever in a single reaction by the MeltArray approach, which could be finished within 2.5 h. Seven specific genes were chosen for differentiation of typhoidal and nontyphoidal Salmonella. All gene targets were able to be detected by the EFMAtyping assay, with expected Tm values and without cross-reactivity to other relevant Salmonella serovars. The limit of detection (LOD) for all gene targets was 50 copies per reaction. The LOD reached 102-103 CFU/ml for each pathogen in simulated clinical samples. The largest standard deviation value for mean Tm was below 0.5 °C. This newly developed EFMAtyping assay was further evaluated by testing 551 clinical Salmonella isolates, corroborated in parallel by the traditional Salmonella identification workflow, and serotype prediction was enabled by whole-genome sequencing. Compared to the traditional method, our results exhibited 100% of specificity and greater than 96% of sensitivity with a kappa correlation ranging from 0.96 to 1.00. Thus, the EFMAtyping assay provides a rapid, high throughput, and promising tool for public health laboratories to monitor typhoid and paratyphoid fever.

Evaluation of the FPMC respiratory panel for detection of respiratory tract pathogens in nasopharyngeal swab and sputum specimens.

OBJECTIVES: The performance of the new Respiratory Pathogen panel (fluorescent probe melting curve, FPMC) for the qualitative detection of 12 organisms (chlamydia pneumoniae, mycoplasma pneumoniae, adenovirus, influenza A virus, influenza B virus, parainfluenza virus, rhinovirus, etc.) was assessed. METHODS: Prospectively collected nasopharyngeal swab (NPS) and sputum specimens (n = 635) were detected by using the FPMC panel, with the Sanger sequencing method as the comparative method. RESULTS: The overall percent concordance between the FPMC analysis method and the Sanger sequencing method was 100% and 99.66% for NPS and sputum specimens, respectively. The FPMC testified an overall positive percent concordance of 100% for both NPS and sputum specimens. The FPMC analysis method also testified an overall negative percent concordance of 100% and 99.38% for NPS and sputum specimens, respectively. CONCLUSIONS: The FPMC analysis method is a stable and accurate assay for rapid, comprehensive detecting for respiratory pathogens.

High-Resolution Melting (HRM) analysis of DNA methylation using semiconductor chip-based digital PCR.

BACKGROUND: Digital PCR (dPCR) technology allows absolute quantification and detection of disease-associated rare variants, and thus the use of dPCR technology has been increasing in clinical research and diagnostics. The high-resolution melting curve analysis (HRM) of qPCR is widely used to distinguish true positives from false positives and detect rare variants. In particular, qPCR-HRM is commonly used for methylation assessment in research and diagnostics due to its simplicity and high reproducibility. Most dPCR instruments have limited fluorescence channels available and separate heating and imaging systems. Therefore, it is difficult to perform HRM analysis using dPCR instruments. OBJECTIVE: A new digital real-time PCR instrument (LOAA) has been recently developed to integrate partitioning, thermocycling, and imaging in a single dPCR instrument. In addition, a new technique to perform HRM analysis is utilized in LOAA. The aim of the present study is to evaluate the efficiency and accuracy of LOAA dPCR on HRM analysis for the detection of methylation. METHODS: In this study, comprehensive comparison with Bio-Rad qRT-PCR and droplet-based dPCR equipment was performed to verify the HRM analysis-based methylation detection efficiency of the LOAA digital PCR equipment. Here, sodium bisulfite modification method was applied to detect methylated DNA sequences by each PCR method. RESULTS: Melting curve analysis detected four different Tm values using LOAA and qPCR, and found that LOAA, unlike qPCR, successfully distinguished between different Tm values when the Tm values were very similar. In addition, melting temperatures increased by each methylation were about 0.5℃ for qPCR and about 0.2 ~ 0.6℃ for LOAA. The melting temperature analyses of methylated and unmethylated DNA samples were conducted using LOAA dPCR with TaqMan probes and EvaGreen, and the result found that Tm values of methylated DNA samples are higher than those of unmethylated DNA samples. CONCLUSION: The present study shows that LOAA dPCR could detect different melting temperatures according to methylation status of target sequences, indicating that LOAA dPCR would be useful for diagnostic applications that require the accurate quantification and assessment of DNA methylation.

Rapid detection of the irinotecan-related UGT1A1 & 5-fluorouracil related DPYD polymorphism by asymmetric polymerase chain reaction melting curve analysis.

BACKGROUND: Determination of DPYD and UGT1A1 polymorphisms prior to 5-fluorouracil and irinotecan therapy is crucial for avoiding severe adverse drug effects. Hence, there is a pressing need for accurate and reliable genotyping methods for the most common DPYD and UGT1A1 polymorphisms. In this study, we introduce a novel polymerase chain reaction (PCR) melting curve analysis method for discriminating DPYD c.1236G > A, c.1679 T > G, c.2846A > T, IVS14 + 1G > A and UGT1A1*1, *28, *6 (G71R) genotypes. METHODS: Following protocol optimization, this technique was employed to genotype 28 patients, recruited between March 2023 and October 2023, at the First Affiliated Hospital of Xiamen University. These patients included 20 with UGT1A1 *1/*1, 8 with UGT1A1 *1/*28, 4 with UGT1A1 *28/*28, 22 with UGT1A1*6 G/G, 6 with UGT1A1*6 G/A, 4 with UGT1A1*6 A/A, 27 with DPYD(c.1236) G/G, 3 with DPYD(c.1236) G/A, 2 with DPYD(c.1236) A/A, 27 with DPYD(c.1679) T/T, 2 with DPYD(c.1679) T/G, 3 with DPYD(c.1679) G/G, 28 with DPYD(c.2846A/T) A/A, 2 with DPYD(c.2846A/T) A/T, 2 with DPYD(c.2846A/T) T/T, 28 with DPYD(c.IVS14 + 1) G/G, 2 with DPYD(c.IVS14 + 1) G/G, and 2 with DPYD(c.IVS14 + 1) G/G, as well as 3 plasmid standards. Method accuracy was assessed by comparing results with those from Sanger sequencing or Multiplex quantitative PCR(qPCR). Intra- and inter-run precision of melting temperatures (Tms) were calculated to evaluate reliability, and sensitivity was assessed through limit of detection examination. RESULTS: The new method accurately identified all genotypes and exhibited higher accuracy than Multiplex qPCR. Intra- and inter-run coefficients of variation for Tms were both ≤1.97 %, with standard deviations ≤0.95 °C. The limit of detection was 0.09 ng/µL of input genomic DNA. CONCLUSION: Our developed PCR melting curve analysis offers accurate, reliable, rapid, simple, and cost-effective detection of DPYD and UGT1A1 polymorphisms. Its application can be easily extended to clinical laboratories equipped with a fluorescent PCR platform.

Screening for thalassemia carriers among the Han population of childbearing age in Southwestern of China.

Purpose: Thalassemia is a severe hereditary blood disorder that poses a significant threat to human health and leads to mortality and disability. It is one of the most prevalent monogenic diseases worldwide. The aim of this study was to analyze the molecular epidemiological data of individuals of childbearing age from the Han ethnic group with thalassemia in Southwest China and to explore the application of next-generation sequencing (NGS) technology in screening thalassemia carriers. Methods: The participants were Han males and females of childbearing age who sought medical advice at the West China Second University Hospital, Sichuan University from June 2022 to June 2023. We detected α- and ß-thalassemia mutations using full-length capture of the thalassemia genes and NGS technology. Results: In a cohort of 1,093 participants, 130 thalassemia carriers were identified, with an overall detection rate of 11.89% (130/1,093). Among these, 0.91% (10/1,093) had mutations that could not be detected using traditional PCR techniques. The proportions of carriers with α-, ß-, and α-complexed ß-thalassemia gene mutations were 7.68% (84/1,093), 3.93% (43/1,093), and 0.27% (3/1,093), respectively. We identified a novel HBA2 c.166del variant that has not been previously reported. Conclusion: Using NGS technology, we found that the mutation-carrying rate of thalassemia genes was 11.89% in the Han population of childbearing age in Southwest China. Compared with the results of traditional PCR techniques, NGS detected an additional 0.91% (10/1,093) rare genetic variants. NGS technology should be utilized as the primary screening method for thalassemia carriers among Han nationality people of childbearing age in Southwest China.

Prediction of tensile modulus based on parameters of crystalline structure in polyethylene terephthalate with cold crystallization ability.

This work aims to adopt a simple modulus prediction method for the crystalline poly(ethylene-terephthalate) (PET), which has strong cold-crystallization ability. Based on a single melting curve generated by calorimetry, crystallinity and average melting temperature can easily be evaluated and consequently, tensile modulus can be predicted. Nonetheless, in the case of polymers with cold crystallization behavior, such as PET, the melting process is affected by cold crystallization, impeding the simple calculation of the aforementioned important parameters. In this paper, the techniques to eradicate cold crystallization during calorimetry are presented. Accordingly, the results of a tensile modulus prediction model are presented and discussed. The crystallization and melting characteristics of PET were measured by differential scanning calorimetry (DSC). The mechanical properties of the specimens were estimated by standardized tensile tests. The specimens, which were used for mechanical tests were fabricated using conventional injection molding. The samples were annealed at different temperatures in order to obtain different crystalline structures. The results clearly indicate that the prediction technique is capable to describe the tensile modulus of PET accurately in the case of very diverse crystalline structures.

Development and validation of multiplex real-time PCR for simultaneous detection of six bacterial pathogens causing lower respiratory tract infections and antimicrobial resistance genes.

BACKGROUND: Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae and Staphylococcus aureus are major bacterial causes of lower respiratory tract infections (LRTIs) globally, leading to substantial morbidity and mortality. The rapid increase of antimicrobial resistance (AMR) in these pathogens poses significant challenges for their effective antibiotic therapy. In low-resourced settings, patients with LRTIs are prescribed antibiotics empirically while awaiting several days for culture results. Rapid pathogen and AMR gene detection could prompt optimal antibiotic use and improve outcomes. METHODS: Here, we developed multiplex quantitative real-time PCR using EvaGreen dye and melting curve analysis to rapidly identify six major pathogens and fourteen AMR genes directly from respiratory samples. The reproducibility, linearity, limit of detection (LOD) of real-time PCR assays for pathogen detection were evaluated using DNA control mixes and spiked tracheal aspirate. The performance of RT-PCR assays was subsequently compared with the gold standard, conventional culture on 50 tracheal aspirate and sputum specimens of ICU patients. RESULTS: The sensitivity of RT-PCR assays was 100% for K. pneumoniae, A. baumannii, P. aeruginosa, E. coli and 63.6% for S. aureus and the specificity ranged from 87.5% to 97.6%. The kappa correlation values of all pathogens between the two methods varied from 0.63 to 0.95. The limit of detection of target bacteria was 1600 CFU/ml. The quantitative results from the PCR assays demonstrated 100% concordance with quantitative culture of tracheal aspirates. Compared to culture, PCR assays exhibited higher sensitivity in detecting mixed infections and S. pneumoniae. There was a high level of concordance between the detection of AMR gene and AMR phenotype in single infections. CONCLUSIONS: Our multiplex quantitative RT-PCR assays are fast and simple, but sensitive and specific in detecting six bacterial pathogens of LRTIs and their antimicrobial resistance genes and should be further evaluated for clinical utility.

Molecular biology of SARS-CoV-2 and techniques of diagnosis and surveillance.

The World Health Organization (WHO) declared coronavirus disease 2019 (COVID-19), a disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a global pandemic in March 2020. Reverse transcription-polymerase chain reaction (RT-PCR) is the reference technique for molecular diagnosis of SARS-CoV-2 infection. The SARS-CoV-2 virus is constantly mutating, and more transmissible variants have emerged, making genomic surveillance a crucial tool for investigating virus transmission dynamics, detecting novel genetic variants, and assessing mutation impact. The S gene, which encodes the spike protein, is frequently mutated, and it plays an important role in transmissibility. Spike protein mutations affect infectivity and vaccine effectiveness. SARS-CoV-2 variants are tracked using whole genome sequencing (WGS) and S-gene analysis. WGS, Sanger sequencing, and many S-gene-targeted RT-PCR methods have been developed. WGS and Sanger sequencing are standard methods for detecting mutations and can be used to identify known and unknown mutations. Melting curve analysis, endpoint genotyping assay, and S-gene target failure are used in the RT-PCR-based method for the rapid detection of specific mutations in SARS-CoV-2 variants. Therefore, these assays are suitable for high-throughput screening. The combinatorial use of RT-PCR-based assays, Sanger sequencing, and WGS enables rapid and accurate tracking of SARS-CoV-2 variants. In this review, we described RT-PCR-based detection and surveillance techniques for SARS-CoV-2.

Existing status and future advancements of adulteration detection techniques in herbal products.

BACKGROUND: Herbal products have been commonly used all over the world for centuries. Its products have gained remarkable acceptance as therapeutic agents for a variety of disorders. However, following recent research disclosing discrepancies between labeling and actual components of herbal products, there is growing concern about the efficacy, quality and safety of the products. The admixture and adulteration of herbal medicinal products pose a risk of serious health compromise and the well-being of the consumers. To prevent adulteration in raw ingredients and final herbal products, it is necessary to use approaches to assess both genomes as well as metabolomics of the products; this offers quality assurance in terms of product identification and purity. The combinations of molecular and analytical methods are inevitable for thorough verification and quality control of herbal medicine. METHODS AND RESULTS: This review discusses the combination of DNA barcoding, DNA metabarcoding, mass spectroscopy as well as HPLC for the authentication of herbal medicine and determination of the level of adulteration. It also discusses the roles of PCR and real-time PCR techniques in validating and ensuring the quality, purity and identity of the herbal products. CONCLUSIONS: In conclusion, each technique has its own pros and cons, but the cumulative of both the chemical and molecular methods is proven to be the best strategy for adulteration detection. Moreover, CRISPR diagnosis tools equipped with multiplexing techniques may be implemented for screening adulteration from herbal drugs, this will play a crucial role in herbal product authentication in the future.

A rapid molecular diagnostic tool to discriminate alleles of avirulence genes and haplotypes of Phytophthora sojae using high-resolution melting analysis.

Effectors encoded by avirulence genes (Avr) interact with the Phytophthora sojae resistance gene (Rps) products to generate incompatible interactions. The virulence profile of P. sojae is rapidly evolving as a result of the large-scale deployment of Rps genes in soybean. For a successful exploitation of Rps genes, it is recommended that soybean growers use cultivars containing the Rps genes corresponding to Avr genes present in P. sojae populations present in their fields. Determination of the virulence profile of P. sojae isolates is critical for the selection of soybean cultivars. High-resolution melting curve (HRM) analysis is a powerful tool, first applied in medicine, for detecting mutations with potential applications in different biological fields. Here, we report the development of an HRM protocol, as an original approach to discriminate effectors, to differentiate P. sojae haplotypes for six Avr genes. An HRM assay was performed on 24 P. sojae isolates with different haplotypes collected from soybean fields across Canada. The results clearly confirmed that the HRM assay discriminated different virulence genotypes. Moreover, the HRM assay was able to differentiate multiple haplotypes representing small allelic variations. HRM-based prediction was validated by phenotyping assays. This HRM assay provides a unique, cost-effective and efficient tool to predict virulence pathotypes associated with six different Avr (1b, 1c, 1d, 1k, 3a and 6) genes from P. sojae, which can be applied in the deployment of appropriate Rps genes in soybean fields.

Highly Sensitive Detection of PIK3CA Mutations by Looping-Out Probes-Based Melting Curve Analysis.

PIK3CA mutations have important therapeutic and prognostic implications in various cancer types. However, highly sensitive detection of PIK3CA hotspot mutations in heterogeneous tumor samples remains a challenge in clinical settings. To establish a rapid PCR assay for highly sensitive detection of multiple PIK3CA hotspot mutations. We described a novel melting curve analysis-based assay using looping-out probes that can enrich target mutations in the background of excess wild-type and concurrently reveal the presence of mutations. The analytical and clinical performance of the assay were evaluated. The developed assay could detect 10 PIK3CA hotspot mutations at a mutant allele fraction of 0.05-0.5% within 2 h in a single step. Analysis of 82 breast cancer tissue samples revealed 43 samples with PIK3CA mutations, 28 of which were confirmed by Sanger sequencing. Further testing of 175 colorectal cancer tissue samples showed that 24 samples contained PIK3CA mutations and 19 samples were confirmed by Sanger sequencing. Droplet digital PCR supported that all mutation-containing samples undetected by sequencing contained mutations with a low allele fraction. The rapidity, ease of use, high sensitivity and accuracy make the new assay a potential screening tool for PIK3CA mutations in clinical laboratories.