A Novel Molecular Method for Simultaneous Identification of Vibrio parahaemolyticus 57 K-Serogroups Using Probe Melting Curve Analysis

Abstract

The serotyping of Vibrio parahaemolyticus, which is crucial to the surveillance and detection of outbreaks of vibriosis infection, has been widely used in many countries. In this study, we developed a molecular assay, named multiplex ligation reaction based on probe melting curve analysis (MLMA), for simultaneous identification of V. parahaemolyticus 57 K-serogroups. Based on the previous genomes of 418 strains including 39 K-serogroups and the 18 K-serogroups sequences from public databases, we obtained 57 K-serogroups specific gene sequences for designing primers and probes. The developed MLMA assay for identifying the V. parahaemolyticus 57 K-serogroups showed high reproducibility, with the intra- and inter-assay standard deviations and coefficients of variation of no more than 1°C and 1%, respectively. The limit of detection for all gene targets ranged from 0.1 to 1.0 ng/µl. We validated the MLMA assay with a double-blind test identifying 595 V. parahaemolyticus isolates using conventional serotyping methods for comparison. The results showed the kappa value between the MLMA assay and the traditional serological method was 0.936 and that there was a 96.97% consistency rate with conventional serotyping methods for all detected isolates. Additionally, five rare K-serogroups were identified using the MLMA assay, as well as 18 strains that could not be identified using the traditional serotyping method. Thus, the MLMA assay provides a rapid, robust, and promising tool for the molecular serotyping of V. parahaemolyticus K-serogroups and has the potential application to the detection of outbreaks and surveillance of V. parahaemolyticus infection.

Universal two‐dimensional labelled probe‐mediated melting curve analysis based on multiplex PCR for rapid typing of Plasmodium in a single closed tube

“…The original sequence of homologous tags was fluorescently labelled for the universal two‐dimensional probe. The function of the probe was the same as that of a fluorescent probe in 2D PCR (Lu et al, 2021 ; Zhan et al, 2020 ). It can bind to homologous cTag, leading to production of different Tm in the MCA process.…”

“…Although some real-time PCR assays have been described (Dos Santos et al, 2020;Rougemont et al, 2004), they cannot distinguish among multiple Plasmodium species in one-pot reactions, mainly due to limited number of fluorescence channels and only can achieve limited detection of one or two Plasmodium species. Plasmodium species detection assays based on isothermal amplification, such as loop-mediated isothermal amplification (LAMP; Hashimoto et al, 2018;Nolasco et al, 2021;Piera et al, 2017;Tambo et al, 2018), recombinase polymerase amplification (RPA; Lai et al, 2018;Lai & Lau, 2020;Lalremruata et al, 2020), showed great convenience for point-ofcare diagnosis, especially for resource-limited settings, but they lack detection throughput and sensitivity in diagnosis, especially for false-positive or false-negative results. The CRISPR-based diagnostic platform (CRISPR-Dx) is a new emerging nucleic acid detection technology used for point-of-care diagnosis, such as Cas12a-mediated Plasmodium detection assay (Lee et al, 2020), showing great sensitivity and specificity.…”