Study On Real-time Fluorescence Quantitative SRCA Technology Detection Of Shigella In Food

Shigella is a kind of intestinal infection pathogenic bacteria,and it is also the main pathogen causing bacterial food poisoning in developing countries.In recent years,food poisoning incidents caused by Shigella bacteria have occurred frequently,which not only endangers human health,but also has a serious impact on public health safety.At present,microbial detection methods still focus on separation culture and biochemical identification in conventional methods.This detection method is laborious and time-consuming,with low sensitivity,and cannot meet the needs of rapid detection.Consequently,it is very imperative to develop a rapid,sensitive,simpleness and efficient method for Shigella detection.In this research,a real-time fluorescence quantification saltatory rolling circle amplification(qSRCA)technology was established to quantitatively detect Shigella in food.This method designed specific primers according to the ipaH gene as the target gene of Shigella,and the reaction conditions and reaction system of qSRCA were optimized in view of orthogonal experiments.The whole qSRCA process was monitored in real time by real-time fluorescent curve,the result was determined according to initial amplification number.In this study,the qSRCA reaction conditions and reaction system were optimized.The suitable reaction temperature was 64?,the suitable system was obtained:0.75?M of forward and reverse primers,0.50 mM of dNTPs,2.00 mM of Mg2+,2.50 ?L of 10 ×Thermopol Reaction Buffer,0.48U/?L Bst DNA polymerase(large fragement),1.00 ?L of DNA template,1.00 ?L of EvaGreen(20 ×),sterile deionized water to make up the system to 20.00 ?L.Recombination plasmid was constructed in view of the above-mentioned suitable reaction system and reaction conditions,the qSRCA reaction was carried out with the recombinant plasmid as a template,and the amplified products were sequenced and analyzed,which fully confirmed the accuracy of the amplification results.The qSRCA standard curve was established,its expression was y=-1.9489 × x+26.7642,and the correlation coefficient R2 value was 0.9972,indicating that the Ct value of this method has a good linear correlation with the logarithm of the recombinant plasmid copy number.In this study,50 strains were selected to verify the specificity of the qSRCA method,including 16 strains of Shigella and 34 strains of non-Shigella.The results showed that:16 strains of Shigella showed positive results and 34 strains of non-Shigella showed negative results.Hence,qSRCA method has good specificity and accuracy.The recombinant plasmid was diluted by 10-fold gradient,and the qSRCA reaction was performed on each gradient dilution solution to determine the sensitivity of the method and compare it with the qPCR method.The result showed that the sensitivity of qSRCA was 8.87 × 101 copies/?L,and the sensitivity of qPCR method was 8.87 × 102 copies/?L.The genomic DNA from artificially contaminated milk samples was extracted and 10-fold diluted to determine the limit of quantification of the qSRCA method and compared with the qPCR method.The limit of quantification of the qSRCA method for artificially contaminated milk samples was 6.7 × 100 CFU/mL(4.268 × 102 copies/?L),and the limit of quantitation of the qPCR method was 6.7 × 101 CFU/mL(4.975 × 103 copies/?L).In summary,the sensitivity of qSRCA was 10 times higher than that of qPCR,and the limit of quantification of qSRCA was 10 times lower than that of qPCR.In this work,60 food samples were detected for Shigella using the qSRCA method and the GB 4789.5-2012 method.2 positive results were found by the qSRCA method and 1 positive result was found by the GB method.The positivity detection rate of the qSRCA method was 3.33%and that of the GB method was 1.67%.The sensitivity of the qSRCA method was 100%,the specificity was 98.31%,and the compliance rate was 98.33%.It showed that the qSRCA method is suitable for the quantitative detection of Shigella in actual food samples.In summary,this study established a qSRCA method for quantitative detection of Shigella in food for the first time.This method can complete the quantitative detection of Shigella under isothermal conditions,which is easy to operate,has the characteristics of high efficiency,short time,specificity and high sensitivity,and can achieve rapidly detection.