AUTHOR=Wang Yi , Wang Yan , Luo Lijuan , Liu Dongxin , Luo Xia , Xu Yanmei , Hu Shoukui , Niu Lina , Xu Jianguo , Ye Changyun 

TITLE=Rapid and Sensitive Detection of Shigella spp. and Salmonella spp. by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique

JOURNAL=Frontiers in Microbiology

VOLUME=6

YEAR=2015

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2015.01400

DOI=10.3389/fmicb.2015.01400

ISSN=1664-302X

ABSTRACT=<p><italic>Shigella</italic> and <italic>Salmonella</italic> are frequently isolated from various food samples and can cause human gastroenteritis. Here, a novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully established and validated for simultaneous detection of <italic>Shigella</italic> strains and <italic>Salmonella</italic> strains in only a single reaction. Two sets of MERT-LAMP primers for 2 kinds of pathogens were designed from <italic>ipaH</italic> gene of <italic>Shigella</italic> spp. and <italic>invA</italic> gene of <italic>Salmonella</italic> spp., respectively. Under the constant condition at 63°C, the positive results were yielded in as short as 12 min with the genomic DNA extracted from the 19 <italic>Shigella</italic> strains and 14 <italic>Salmonella</italic> strains, and the target pathogens present in a sample could be simultaneously identified based on distinct fluorescence curves in real-time format. Accordingly, the multiplex detection assay significantly reduced effort, materials and reagents used, and amplification and differentiation were conducted at the same time, obviating the use of postdetection procedures. The analytical sensitivity of MERT-LAMP was found to be 62.5 and 125 fg DNA/reaction with genomic templates of <italic>Shigella</italic> strains and <italic>Salmonella</italic> strains, which was consist with normal LAMP assay, and at least 10- and 100-fold more sensitive than that of qPCR and conventional PCR approaches. The limit of detection of MERT-LAMP for <italic>Shigella</italic> strains and <italic>Salmonella</italic> strains detection in artificially contaminated milk samples was 5.8 and 6.4 CFU per vessel. In conclusion, the MERT-LAMP methodology described here demonstrated a potential and valuable means for simultaneous screening of <italic>Shigella</italic> and <italic>Salmonella</italic> in a wide variety of samples.</p>