Rapid and sensitive detection of VBNC Escherichia coli O157: H7 in beef by PMAxx and real-time LAMP

Lv, Xinrui; Wang, Li; Zhang, Jingfeng; Zeng, Haiyan; Chen, Xun; Shi, Lei; Cui, Hualing; He, Xiaoxin; Zhao, Linna

doi:10.1016/j.foodcont.2020.107292

Cited by 38 publications

(18 citation statements)

References 29 publications

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“…Although RT-PCR is able to show results via a digital curve, the sensitivity of this method is limited and the procedure is complicated ( Xu et al, 2008a ; Miao et al, 2018 ; Xu et al, 2018 ; Zhao et al, 2018a,b ). RT-LAMP or other isothermal amplification has been used reported to replace RT-PCR to increase the detection limit ( Xu et al, 2008b ; Xu et al, 2009 ; Liu et al, 2018a,b ; Lv et al, 2020 ). However, sophisticated equipment with these technologies has brought certain difficulties to rapid on-site testing ( Xu et al, 2011b ; Xu et al, 2012b ).…”

Section: Discussionmentioning

confidence: 99%

Direct Detection of Viable but Non-culturable (VBNC) Salmonella in Real Food System by a Rapid and Accurate PMA-CPA Technique

Wang

et al. 2021

Front. Microbiol.

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Salmonella enterica is a typical foodborne pathogen with multiple toxic effects, including invasiveness, endotoxins, and enterotoxins. Viable but nonculturable (VBNC) is a type of dormant form preserving the vitality of microorganisms, but it cannot be cultured by traditional laboratory techniques. The aim of this study is to develop a propidium monoazide-crossing priming amplification (PMA-CPA) method that can successfully detect S. enterica rapidly with high sensitivity and can identify VBNC cells in food samples. Five primers (4s, 5a, 2a/1s, 2a, and 3a) were specially designed for recognizing the specific invA gene. The specificity of the CPA assay was tested by 20 different bacterial strains, including 2 standard S. enterica and 18 non-S. enterica bacteria strains covering Gram-negative and Gram-positive isolates. Except for the two standard S. enterica ATCC14028 and ATCC29629, all strains showed negative results. Moreover, PMA-CPA can detect the VBNC cells both in pure culture and three types of food samples with significant color change. In conclusion, the PMA-CPA assay was successfully applied on detecting S. enterica in VBNC state from food samples.

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Section: Discussionmentioning

confidence: 99%

Direct Detection of Viable but Non-culturable (VBNC) Salmonella in Real Food System by a Rapid and Accurate PMA-CPA Technique

Wang

et al. 2021

Front. Microbiol.

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“…LAMP is a novel and efficient isothermal nucleic acid (DNAs and RNAs) amplification method [ [82] , [83] , [84] , [85] ], showing high sensitivity and specificity [ [86] , [87] , [88] ]. LAMP analysis method is often used in clinical detection of coronavirus [ 89 , 90 ].…”

Section: Detection Methodsmentioning

confidence: 99%

Current methods and prospects of coronavirus detection

Deng

Liu

et al. 2021

Talanta

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“…Previous reports have illustrated that genomic DNA can stay in foods or the environment for a long time after bacterial death and can be normally amplified by the proposed real-time recombinase-aided amplification (rRAA; Gao et al, 2021), potentially overestimating the quantity of viable bacteria in samples and even resulting in false-positive signals (Lv et al, 2020). In other studies, some agents such as ethidium bromide monoazide (EMA) and propidium monoazide (PMA) have been applied in reducing interference from dead bacteria (Roumani et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

Real-time recombinase-aided amplification with improved propidium monoazide for the rapid detection of viable Escherichia coli O157:H7 in milk

Zhou²,

Xie

et al. 2022

Journal of Dairy Science

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Escherichia coli O157:H7, the causative agent of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome in humans, generates a effective harm to community health because of its high pathogenicity. A real-time recombinase-aided amplification (rRAA) is an emerging method for nucleic acid detection. However, genomic DNA of bacteria could exist in food and the environment for a long time after death and could be amplified by rRAA assay, resulting in false-positive signal; thus, developing a fast and sensitive method is necessary to detect viable foodborne pathogens in food products. In our research, rRAA assay coupled with an enhanced nucleic acid binding dye named improved propidium monoazide (PMAxx) was established and applied in viable E. coli O157:H7 identification in skim milk. The PMAxx could eliminate interference from dead bacteria by permeating impaired membranes and covalently linking to DNA to prevent DNA amplification. The PMAxx-rRAA assay was performed with high sensitivity and good specificity. The PMAxx-rRAA assay could detect as low as 5.4 × 10 0 cfu/mL of viable E. coli O157:H7 in pure culture, and 7.9 × 10 0 cfu/mL of viable E. coli O157:H7 in skim milk. In addition, the PMAxx-rRAA assay was performed in the presence of a high concentration of dead bacteria or nontarget bacteria in skim milk to verify the capacity to resist interference from dead bacteria and nontarget bacteria. Therefore, the established PMAxx-rRAA assay is a valuable tool for the identification of viable E. coli O157:H7 in complex food matrix.

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Rapid and sensitive detection of VBNC Escherichia coli O157: H7 in beef by PMAxx and real-time LAMP

Cited by 38 publications

References 29 publications

Direct Detection of Viable but Non-culturable (VBNC) Salmonella in Real Food System by a Rapid and Accurate PMA-CPA Technique

Direct Detection of Viable but Non-culturable (VBNC) Salmonella in Real Food System by a Rapid and Accurate PMA-CPA Technique

Current methods and prospects of coronavirus detection

Real-time recombinase-aided amplification with improved propidium monoazide for the rapid detection of viable Escherichia coli O157:H7 in milk

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