Sensitively and quickly detecting Vibrio vulnificus by real time recombinase polymerase amplification targeted to vvhA gene

Zhu, Peng; Cui, Yan; Pang, Jianhu; Xiong, Zi; Huang, Zuoan; Guo, Shiyu; Zhang, Shun; Cai, Ting

doi:10.1016/j.mcp.2021.101726

Cited by 7 publications

(6 citation statements)

References 32 publications

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“…Considering the sensitivity, the LOD of the PMA-ddPCR in this study was 29.33 CFU/mL, which performed better than those methods in previous studies, such as the LOD of V. vulnificus, was 1 × 10 3 CFU/mL with qPCR (Panicker and Bej, 2005), or 1.2 × 10 2 -1 × 10 3 CFU/mL with recombinase polymerase amplification (RPA) (Yang et al, 2020;Zhu et al, 2021), or 2 × 10 3 CFU/mL with loop-mediated isothermal amplification (LAMP) (Zhang et al, 2018). In accurate detection in artificially spiked plasma samples, the performance of the PMA-ddPCR was also superior to that of PMA-qPCR (Supplementary Figure 2), which means that PMA-ddPCR is more capable in handling clinical samples.…”

Section: Discussionmentioning

confidence: 49%

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An Assay Combining Droplet Digital PCR With Propidium Monoazide Treatment for the Accurate Detection of Live Cells of Vibrio vulnificus in Plasma Samples

Zhang

et al. 2022

Front. Microbiol.

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Vibrio vulnificus (V. vulnificus) is one of the most common pathogenic Vibrio species to humans; therefore, the establishment of timely and credible detection methods has become an urgent requirement for V. vulnificus illness surveillance. In this study, an assay combining droplet digital PCR (ddPCR) with propidium monoazide (PMA) treatment was developed for detecting V. vulnificus. The primers/probes targeting the V. vulnificus hemolysin A (vvhA) gene, amplification procedures, and PMA processing conditions involved in the assay were optimized. Then, we analyzed the specificity, sensitivity, and ability to detect live cell DNA while testing the performance of PMA-ddPCR in clinical samples. The optimal concentrations of primers and probes were 1.0 and 0.3 μM, respectively. The annealing temperature achieving the highest accuracy in ddPCR assay was 60°C. With an initial V. vulnificus cell concentration of 108 CFU/mL (colony-forming units per milliliter), the optimal strategy to distinguish live cells from dead cells was to treat samples with 100 μM PMA for 15 min in the dark and expose them to LED light with an output wavelength of 465 nm for 10 min. The specificity of the PMA-ddPCR assay was tested on 27 strains, including seven V. vulnificus strains and 20 other bacterial strains. Only the seven V. vulnificus strains were observed with positive signals in specificity analysis. Comparative experiments on the detection ability of PMA-ddPCR and PMA-qPCR in pure cultures and plasma samples were performed. The limit of detection (LOD) and the limit of quantitation (LOQ) in pure culture solutions of V. vulnificus were 29.33 and 53.64 CFU/mL in PMA-ddPCR, respectively. For artificially clinical sample tests in PMA-ddPCR, V. vulnificus could be detected at concentrations as low as 65.20 CFU/mL. The sensitivity of the PMA-ddPCR assay was 15- to 40-fold more sensitive than the PMA-qPCR in this study. The PMA-ddPCR assay we developed provides a new insight to accurately detect live cells of V. vulnificus in clinical samples, which is of great significance to enhance public health safety and security capability and improve the emergency response level for V. vulnificus infection.

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Section: Discussionmentioning

confidence: 49%

“…The LOD is the target level that would be detected in 95% of cases (Zhu et al, 2021). In this study, probit regression analysis was used to fit the limit of detection (LOD) of PMA-qPCR and PMA-ddPCR.…”

Section: Discussionmentioning

confidence: 99%

An Assay Combining Droplet Digital PCR With Propidium Monoazide Treatment for the Accurate Detection of Live Cells of Vibrio vulnificus in Plasma Samples

Zhang

et al. 2022

Front. Microbiol.

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“…In our study, the linear calibration curves shown in Figure 3b are indeed not well compared to real-time PCR (Figure 3a), thus the correlation coefficient was only r 2 = 0.8628. However, our previous study (Zhu et al, 2021) gave a hint the secondorder polynomial regressions could be applied to improve this phenomenon. Here, the calibration curve plotted by the second-order polynomial regression showed better than the linear curve (Figure 3b) and the correlation coefficient was raised to 0.9907.…”

Section: Discussionmentioning

confidence: 99%

“…RPA is also recognized as the most potential techniques alternative to PCR in the future. Furthermore, real‐time RPA, due to its characteristics of real‐time monitoring of reaction and avoiding the risk of contamination, has gotten increasing attention and successfully used to detect several Vibiro species (Pang et al, 2019; Zhu et al, 2018; Zhu et al, 2021). In this study, we developed a real‐time RPA method to detect V. mimicus in the plasma samples by targeting the vmh gene.…”

Section: Discussionmentioning

confidence: 99%

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Development and evaluation of real-time recombinase polymerase amplification assay for rapid and sensitive detection of Vibro mimicus in human plasma samples

Zhu

Huang

Xiong

et al. 2022

Journal of Applied Microbiology

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Aim:We aimed at developing a fast and accurate method to detect Vibrio mimicus using real-time recombinase polymerase amplification assay.Methods and Results: Specific primers and probe were designed to target V. mimicus haemolysin (vmh) gene. Target DNA was successfully amplified at 41°C within 20 min. The method exhibited a high level of specificity and the sensitivity was 2.1 × 10 2 copies/25 μl or 8.4 copies/μl, which is in line with real-time polymerase chain reaction (PCR). The calibration curve plotted by the second-order polynomial regression showed better than the linear curve, as the correlation coefficient was raised to 0.9907, which suggested that the second-order polynomial regressions might be considered to apply to the quantification of real-time recombinase polymerase amplification (RPA). The limit of detection (LOD) was predicted to be 77 copies/25 μl or 3 copies/μl by a probit model. The limit of quantification (LOQ) was calculated to be 28 copies /25 μl or 1 copies/μl by a receiver operating characteristic (ROC) curve, which firstly make LOQ could be available to real-time RPA. For the performance of the real-time RPA in plasma samples, the detection sensitivity of real-time RPA was as good as the real-time PCR. For pretreatment of plasma samples, the boiling method was better than using kits, as it further shortened the time of the real-time RPA in detecting V. mimicus. Conclusions:The real-time RPA assay developed in our study shows multiple advantages over currently available DNA diagnostic method, including a quicker timeto-result for a single sample, requiring minimal infrastructure and technical support and being tolerant to inhibitors in plasma samples. Significance and Impact of the Study:The real-time RPA assay developed here is a potentially valuable tool for point-of-care (POC) diagnosis of V. mimicus infection in endemic field, especially in the resources-limited settings, as combined with portable devices. K E Y W O R D Sreal-time recombinase polymerase amplification, the limit of detection (LOD), the limit of quantification (LOQ), Vibro mimicus, vmh gene | 1651 REAL-TIME RPA ASSAY DETECTING VIBRO MIMICUS IN HUMAN PLASMA SAMPLES

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Droplet digital PCR and real-time PCR for the sensitive and specific detection of Vibrio vulnificus based on the novel target genes

Cui,

Zhou,

et al. 2024

Eur Food Res Technol

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Sensitively and quickly detecting Vibrio vulnificus by real time recombinase polymerase amplification targeted to vvhA gene

Cited by 7 publications

References 32 publications

An Assay Combining Droplet Digital PCR With Propidium Monoazide Treatment for the Accurate Detection of Live Cells of Vibrio vulnificus in Plasma Samples

An Assay Combining Droplet Digital PCR With Propidium Monoazide Treatment for the Accurate Detection of Live Cells of Vibrio vulnificus in Plasma Samples

Development and evaluation of real-time recombinase polymerase amplification assay for rapid and sensitive detection of Vibro mimicus in human plasma samples

Droplet digital PCR and real-time PCR for the sensitive and specific detection of Vibrio vulnificus based on the novel target genes

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