A Novel Sero-Specific-Gene Dependent Multiplex PCR Enhances the Discrimination of Major Listeria monocytogenes Serovars.

IF 2.5 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Jing Wang, Jingliang Qin, Bin Hu, Zixu Zhang, Boyang Cao, Xi Guo
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Abstract

Listeria monocytogenes is a foodborne bacterial pathogen distributed worldwide. Serotyping is extensively applied in the classification of L. monocytogenes and is crucial in the early stage of epidemiological tracing. Among the 13 serovars, 1/2a, 1/2b, 1/2c, and 4b are the ones most frequently isolated. Numerous PCR-based methods have been presented, however, their target genes, lmo0737, ORF2110 and ORF2819, are prone to horizontal transfer or loss in certain strains, thus leading to incorrect serovar designation. Herein, we selected novel sero-specific genes and developed an improved multiplex PCR assay. The specificity of our assay was confirmed by the use of target and nontarget Listeria reference strains, as well as by the use of isolates yielding incorrect profiles in previous studies. Sensitivity tests indicated that a minimum of 5 ng of genomic DNA or approximately 3 × 106 CFU of pure culture could be detected. Many collected isolates and genomes of global isolates were used to evaluate the specificity and reproducibility of our assay. The agreement between our assay and the agglutination test was 95%, and the one between our assay and the Doumith scheme was 97%. However, our assay overcomes the drawbacks of currently used PCR-based approaches by exhibiting 100% accuracy for certain strains and clones, for instance, ST782 within the hypervirulent CC2 and ST218 that were incorrectly assigned by the Doumith scheme. In conclusion, the developed assay herein will be a powerful tool and an alternative for the classification of L. monocytogenes strains in foodborne outbreak investigations and surveillance programs.

单核细胞增生李斯特菌是一种食源性细菌病原体,分布于世界各地。血清分型被广泛应用于单增李斯特菌的分类,在流行病学追踪的早期阶段至关重要。在 13 个血清型中,1/2a、1/2b、1/2c 和 4b 是最常分离到的血清型。然而,这些方法的目标基因 lmo0737、ORF2110 和 ORF2819 容易发生水平转移或在某些菌株中丢失,从而导致血清型的错误判断。在此,我们选择了新的血清特异性基因,并开发了一种改进的多重 PCR 检测方法。通过使用目标和非目标李斯特菌参考菌株,以及使用以往研究中产生错误图谱的分离物,证实了我们的检测方法具有特异性。灵敏度测试表明,至少可以检测到 5 纳克的基因组 DNA 或大约 3 × 106 CFU 的纯培养物。许多收集到的分离物和全球分离物的基因组被用来评估我们检测方法的特异性和可重复性。我们的检测方法与凝集试验的一致性为 95%,与 Doumith 方案的一致性为 97%。不过,我们的检测方法克服了目前使用的基于 PCR 方法的缺点,对某些菌株和克隆(例如杜密斯方案中被错误归类的高侵染性 CC2 中的 ST782 和 ST218)的准确性达到了 100%。总之,在食源性疾病爆发调查和监控项目中,本文所开发的检测方法将成为单核细胞增生症菌株分类的有力工具和替代方法。
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来源期刊
Journal of microbiology and biotechnology
Journal of microbiology and biotechnology BIOTECHNOLOGY & APPLIED MICROBIOLOGY-MICROBIOLOGY
CiteScore
5.50
自引率
3.60%
发文量
151
审稿时长
2 months
期刊介绍: The Journal of Microbiology and Biotechnology (JMB) is a monthly international journal devoted to the advancement and dissemination of scientific knowledge pertaining to microbiology, biotechnology, and related academic disciplines. It covers various scientific and technological aspects of Molecular and Cellular Microbiology, Environmental Microbiology and Biotechnology, Food Biotechnology, and Biotechnology and Bioengineering (subcategories are listed below). Launched in March 1991, the JMB is published by the Korean Society for Microbiology and Biotechnology (KMB) and distributed worldwide.
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