Abstract: Salmonella is a kind of food-borne pathogen which belongs to Enterobacteriaceae. However, the content of Salmonella in food is usually low, the routine test is time-consuming, so the detection with high sensitivity, repeatability and accuracy is needed. The nucleic acid reference material of Salmonella was used to verify the real-time fluorescence quantitative PCR (qRT-PCR) method for detecting Salmonella, the applicability, uniformity and stability of nucleic acid reference material of Salmonella were investigated in this study. To optimize the nucleic acid reference materials for verifying qRT-PCR method in detection of Salmonella, various enzyme systems for PCR and protective agents for nucleic acid reference materials were studied. The homogeneity, short-term stability and repeated freeze-thaw stability of nucleic acid reference materials were analyzed based on the linear graph and parameter table. The results showed that qRT-PCR method with relevant nucleic acid reference materials in this study was specific to Salmonella amplification, it was applicable to commonly used PCR instruments and various enzyme systems. The protective agent in reference materials did not inhibit the fluorescence amplification of Salmonella. The quality evaluation of reference material indicated that Salmonella nucleic acid samples were uniform, it remained stable within 6 days in the stability test without dilution. The quantity was stable after repeated freezing and thawing for 10 times. And the stability of diluted Salmonella nucleic acid sample was enhanced after adding protective agent. However, the short-term stability and quantity of diluted Salmonella nucleic acid samples were unstable in repeated freezing and thawing tests. The RT-PCR with reference material has high accuracy and stability in detecting Salmonella. It can be used to verify the detection ability of laboratories in rapidly and accurately detecting microbial nucleic acid with traceability.
王菲,张玲,杨佳怡,等. 基于实时荧光定量PCR检测的沙门氏菌标准物质验证[J]. 食品与发酵工业, 2020, 46(8): 219-225.
WANG Fei,ZHANG Ling,YANG Jiayi,et al. Verification of reference material for detecting Salmonella based on the real-time fluorescence quantitative PCR method[J]. Food and Fermentation Industries, 2020, 46(8): 219-225.
XIONG Dan, SONG Li, PAN Zhiming, et al. Identification and Discrimination of Salmonella enterica serovar gallinarum biovars pullorum and gallinarum based on a one-step multiplex PCR[J]. Frontiers in Microbiology, 2018, 9:1 718.
CARLONJ E,ROTUDOL,BRAND I, et al. Rapid and simultaneous detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes by magnetic capture hybridization and multiplex real-time PCR[J]. Folia Microbiologica, 2018, 63(6):735-742.
[6]
ALFAMAE R G,HESSEL C T,DEOLIVEIRA E S, et al. Assessment of temperature distribution of cold and hot meals in food services and the prediction growth of Salmonella spp. and Listeria monocytogenes[J]. Food Control, 2019,91:501-970.
NADIN-DAVIS S, POPE L, OGUNREMI D, et al. A real-time PCR regimen for testing environmental samples for Salmonella enterica subsp. enterica serovars of concern to the poultry industry, with special focus on Salmonella Enteritidis[J].Canadian Journal of Microbiology, 2019, 65(2): 162-173.
LIU Yuejiao, SINGH P, MUSTAPHA A. Multiplex high resolution melt-curve real-time PCR assay for reliable detection of Salmonella[J].Food Control, 2018, 91(9): 225-230.
LUGERT R, SCHETTLER C, GROSS U. Comparison of different protocols for DNA preparation and PCR for the detection of fungal pathogens in vitro[J]. Mycoses, 2006, 49(4):298-304.
[21]
FEY H, PFISTER H, RÜEGG O. Comparative evaluation of different enzyme-linked immunosorbent assay systems for the detection of staphylococcal enterotoxins A, B, C, and D[J]. Clin Microbiol. 1984, 19(1):34-38.
[22]
VECCIO P D. Effect of osmoregulatory solutes on the thermal stability of calf-thymus DNA[J]. Journal of the Chemical Society, Faraday Transactions, 1996, 92(8):1 361-1 367.
YANG Mengrui, LIU Fang, WANG Min, et al. Development of a whole liquid egg certified reference material for accurate measurement of enrofloxacin residue[J].Food Chemistry, 2019, 309:125 253.
[25]
DJEBBL-SIMMONS D, XU W Q, JANES M, et al. Survival and inactivation of Salmonella enterica serovar Typhimurium on food contact surfaces during log, stationary and long-term stationary phases[J]. Food Microbiology, 2019, 84:103 272.
[26]
HAIM-VILMOVSKY L. High-throughput single-cell real-time quantitativePCR analysis[J]. Methods in Molecular Biology, 2019,1979:177-183.
[27]
NIU D L, ZHAO Y E, GONG X J, et al. Screening of reference genes and quantitative real-time PCR detection and verification in Dermatophagoides farinae under temperature stress[J]. Experimental Parasitology, 2019,206:107 754.
[28]
SAKURIA A,NOMURAN,NANBAR, et al. Rapid typing of influenza viruses using super high-speed quantitative real-time PCR[J]. Journal of Virological Methods, 2011,178(1-2):75-81.
2020, Vol. 46 
