| The Listeria genus is a Gram positive, non-spore-forming, motile, rod shaped, faculta-tive-anaerobic bacteria that was found in a variety of foods and environments. The genus Lis-teria is comprised of 15 species, six species (Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Listeria welshimeri, Listeria seeligeri and Listeria grayi) are the most commonly iso-lated member form the cases of listeriosis. food stuffs and environments, and the other nine species (Listeria rocourtiae, Listeria weihenslephanensis. Listeria fleischmannii, Listeria mar-thii, Listeria floridensis. Listeria aquatica. Listeria cornellensis, Listeria riparia and Listeria grandensis) are described as rare Listeria isolates.The conventional detection methods for Listeria species is obstained by initial enrichment, subsequent colony formation on selective agar medium and biochemical tests. The major drawback of routine laboratory tests is time-consuming, labor-intensive and higher cost, and the biochemical tests are not always reliable. To overcome the disadvantages of the conventional tests, a novel multiplex PCR assay, which rapidly and preciously differentiates six common Listeria species in only one multiplex PCR step with easily interprotable results, were devel-oped. This multiplex PCR method was successfully evaluated with 511 strains of Listeria spe-cies and 15 strains of common bacteria. The assay has the advantage of extensive applications in laboratory diagnosis and epidemiological surveillance of Listeria species.Serotype 4b strains of L. monocytogenes are overreprosented in sporadic and outbreak lis- teriosis cases that are separated into four subgroups (EC?. EC?, EC?, non-EC). A multiplex PCR assay was developed to rapidly detect four 4b subgroups. The PCR test was successfully evaluated with 16 serotype 4b isolates, with 11 isolates belonging to ECI. one belonging to EC?, two belonging to ECIII and two belonging to non-EC. The novel one-step molecular method will be valuable and reliable for rapid discrimination of the distinct subgroups strains of L. monocytogenes serotype 4b from food samples, processing environments and clinical sam-ples, which would facilitate the source tracing of the outbreak of L. monocytogenes in future.L. monocytogenes, on the basis of various phenotypic and genotypic methods, can be di-vided into four phylogenetic lineages. Lineages ? and ? are responsible for most listeriosis cas-es, while lineages ? (including sublineages ?A and ?C) and ? strains are rarely associated with human morbidity but providing significant clues for Listeria evolution. We developed a multiplex PCR for rapid and simultaneous detection of four lineages and two sublineages in only one multiplex PCR step with easily interpretable results. This multiplex PCR strategy was successfully evaluated with 392 Listeria strains, and the classification of lineages and subline-ages was consistent with others phenotypic and genotypic methods. This established multiplex PCR assay provides rapid and reliable results and will be useful for the detection of L. mono-cytogenes lineages and sublineages in contaminated food products and clinical samples.In this study, a loop-mediated isothermal amplification (LAMP) assay for rapid and sensi-tive detection of the L. ivanovii strains had been developed and evaluated in this study. Oligo-nucleotide primers specific for L. ivanovii species were designed corresponding to smcl gene sequences. The primers set comprise six primers targeting eight regions on species-specific gene smcl. The LAMP assay could be completed within 1h at 64? in a water bath. Amplifica-tion products were directly observed by the Loopamp Fluorescent Detection Reagent (FD) or detected by agarose gel electrophoresis. Moreover, the LAMP reactions were also detected by real-time measurement of turbidity. The exclusivity of 77 non-L. ivanovii strains and the inclu-sivity of 17 L. ivanovii strains were both 100% in the assay. Sensitivity of the LAMP assay was 250 fg DNA and 16 CFU per reaction for detection of L. ivanovii in pure cultures and simulated human stool. The LAMP assay was 10 and 100-fold higher sensitive than that of quantitative PCR (qPCR) and the conventional PCR assays, respectively. When applied in human stool samples spiked with low level (8 CFU/0.5 g) of L. ivanovii strains, the established LAMP assay achieved positive detection after 6 hours enrichment. In conclusion, the new LAMP assay in this study can be used as a valuable, rapid and sensitive detection tool for the detection of L. ivanovii in field, medical and veterinary laboratories.We devised a novel model of loop-mediated isothermal amplification (LAMP), termed multiple inner primers-LAMP (MI-LAMP), which offered an isothermal, rapid, highly specific and sensitive assay for detection of L. monocytogenes. A set of 10 specific MI-LAMP primers, which recognized 14 different regions of target gene, was designed to target a sequence in the hlyA gene which is specific to L. monocytogenes. The MI-LAMP assay efficiently amplified the target element within 45 min at 64 ? and was evaluated for sensitivity and specificity. Thirty four non-L. monocygenes strains and thirty nine L. monocytogenes strains were employed for the method verification, and the analytical specificity was 100%. In a pure culture of L. mono-cytogenes. the limit of detection (LoD) of MI-LAMP assay was 62.5 fg genomic DNA per tube and 2.4 CFU per reaction. Moreover, the analytical sensitivity of the novel MI-LAMP approach was as few as 24 CFU per reaction in artificially contaminated milk samples. The amplified products were analyzed by real-time monitoring of changes in turbidity, and visualized by add-ing Loop Fluorescent Detection Reagent (FD), or as a ladder-like banding pattern on gel elec-trophoresis. A total of 48 pork samples were investigated for L. monocytogenes by the novel MI-LAMP method, and the diagnostic accuracy was shown to be 100% when compared to the culture-biotechnical method. In conclusion, the MI-LAMP methodology was demonstrated to be a reliable, sensitive and specific tool for rapid detection of L. monocytogenes strains.The loop-mediated isothermal amplification (LAMP) is restricted to detect a single target, limiting the utility of this method. To achieve multiplex LAMP-based detection, we developed a novel approach we called the MERT-LAMP (Multiple endonuclease restriction real time-Loop-mediated isothermal amplification) assay. In this system, the LAMP forward or back interior primers (FIP or BIP) contain 5'end short sequences (Es) that are recognized by re-striction endonuclease and the new FIP or BIP primers were named EFIP or EBIP. The EFIP or EBIP was modified at the 5'end with a fluorophore and in the middle with a dark quencher. The endonuclease digests the newly synthesized double-stranded terminal sequences (Es and its complementary sequence), and this releases the quenching resulting in a gain of signal. The as-say permitted real-time detection of a single or multiple target sequences in a single tube, and the positive results can be obtained in as short as 12 minutes. The novel methodology was highly efficient and specific, detecting down to 250 fg DNA per reaction of Listeria DNA tested, and successfully evaluated by raw meat samples. The MERT-LAMP technology, which is an extension of LAMP to accommodate robust, target-specific, and multiplex detection, provides a molecular diagnostic tool with less detection time, high sensitivity and specificity compared to LAMP and qPCR.This study reports the development of single cross priming amplification (S-CPA) and double CPA (D-CPA) assays targeting species-specific gene lmo0733 for identifying L. mono-cytogenes strains. The CPA assays were performed at a constant temperature 64? using seven specific primers and evaluated for specificity and sensitivity. The color change of positive amplification was directly observed by Loopamp Fluorescent Detection Reagent (FD) and the DNA products were visualized as a ladder-like banding pattern on 2.5% gel electrophoresis. Moreover, the positive reactions were also detected by real-time measurement of turbidity.50 L. monocytogenes and 46 non-L.monocytogenes strains were used for the method verification and the specificity was 100%. The limit of detection (LoD) of the S-CPA and D-CPA assays was 2.5 pg DNA per reaction and 10-fold more sensitive than that of PCR. A total of 60 pork samples were tested for L. monocytogenes using the S-CPA assay developed in the study, and the accu-racy of the S-CPA and the culture-biotechnical method was 100% identical. The results sug-gested that the S-CPA assay was a rapid, sensitive and valuable tool for detection of L. mono-cytogenes in food products.In conclusion, three multiplex PCR methodologies were successfully developed and eval-uated for differention of 6 Listeria species.4 Listeria monocytogenes serotype 4b subgroups,4 phylogenetic lineages and 2 sublineages. which were valuable for the screening test to identifiy the Listeria strains, lineages, sublineages or subgroups of Listeria monocytognes strains, espe-cially for the surveillance of the Listeria strains related food safety and foodborne disease cases. Moreover, the novel isothermal amplification approaches. including LAMP. MI-LAMP, MERT-LAMP and CPA strategies, were devised and subsequently applied to detect Listeria strains, which provided the advantages on rapidity, sentitivity. specificity, time consumption, and easiness in operation. Thus, those technologies can be used as potential screening tools for Listeria in basic, medical and field laboratories. |
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