Development of a sensitive and specific qPCR assay in conjunction with propidium monoazide for enhanced detection of live Salmonellaspp. in food

BMC Microbiology

Methodology Article

Baoguang Li¹  Jin-Qiang Chen¹ 

[1] Division of Molecular Biology, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 20708, Laurel, MD, USA;

关键词: Salmonella;    qPCR;    Propidium monoazide;    Live cells;    invA;   

DOI  :  10.1186/1471-2180-13-273

 received in 2013-05-24, accepted in 2013-11-23,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundAlthough a variety of methodologies are available for detection of Salmonella, sensitive, specific, and efficient methods are urgently needed for differentiation of live Salmonella cells from dead cells in food and environmental samples. Propidium monoazide (PMA) can preferentially penetrate the compromised membranes of dead cells and inhibit their DNA amplification, however, such inhibition has been reported to be incomplete by some studies. In the present study, we report an efficient qPCR assay targeting a conserved region of the invA gene of Salmonella in conjunction with PMA treatment for detection of DNA from live Salmonella cells in food samples.ResultsWe investigated the relationship between amplicon length and inhibitory effect of PMA treatment to prevent DNA amplification from dead cells while allowing for DNA amplification from live cells, and found that the two factors are well correlated with each other. An amplicon that is 130 bp in length was determined to be optimal for PMA treatment and was selected for further PMA-qPCR assay development. A PMA-qPCR assay was established by utilizing this amplicon and adopting a modified PMA-treatment procedure. The PMA-qPCR assay provided excellent inhibition of DNA amplification from dead cells (a 17-CT-value, or 128,000-fold reduction) while only a slight DNA amplification difference (0.5 CT value) was noted between the PMA-treated and untreated live cells. This assay has been validated through stringent inclusivity and exclusivity studies using a large number of (n = 167) Salmonella, including all strains of SARA and SARB collections, and non-Salmonella strains (n = 36). This PMA-qPCR assay is capable of detecting live Salmonella cells in live/dead cell mixtures, or 30 CFU/g live Salmonella cells from enriched spiked spinach samples as early as 4 h.ConclusionsA 130-bp amplicon in invA gene was demonstrated to be optimal for PMA treatment for selective detection of live Salmonella cells by PCR. This PMA-qPCR assay provides a sensitive, specific, and efficient method for detecting live Salmonella cells in foods and environmental samples and may have an impact on the accurate microbiological monitoring of Salmonella in foods and environment samples.

【 授权许可】

CC BY   
© Li and Chen; licensee BioMed Central Ltd. 2013