Dynamic Changes Of Pathogens In The Bacteria Type Of Douchi

In recent years,bacteria-type Douchi that have exceeded the standard for the consumption of pathogens have triggered many poisoning incidents at home and abroad.It seriously affects public health.Safety problems in Douchi have increasingly become a focus of social concern.At present,the main detection method is the traditional microbiological identification method and PCR,the shortcoming of the former is low sensitivity and specificity,long detection cycle,easy to miss detection,and even false detection events caused by contamination of the detection process,the disadvantage of latter is prone to false positives.They have no way of actually achieving quantitative detection.At present,the detection of the pathogen of Douchi is mainly concentrated on the product.In order to understand the source of the pathogen in Douchi and its reproduction during the fermentation process,using TaqMan qPCR and ddPCR detect the bacteria type of Douchi in the fermentation period of the factory.Designed specific primers and probes for ureR of Proteus mirabilis,NRPS of Bacillus cereus,ompA of Enterobacter sakakii,hlyA of Listeria monocytogenes,amplified objective gene fragment by conventional PCR.The recombinant plasmid was prepared by pGEM-T Easy Vector for single/double qPCR detection.The results of single qPCR specificity test were non-target strain amplification.The sensitivity of ureR,NRPS,ompA and hlyA positive recombinant plasmids was1.65 copies/?L,4.5 copies/?L,2.2 copies/?L,and 8.6 copies/?L respectively by the single qPCR.The sensitivity of ompA positive recombinant plasmids was 2.2×10copies/?L by the double qPCR.The other sensitivity test results were the same as those of the single qPCR.The singlet qPCR standard curves were y_PM=-3.204x+50.797,amplification efficiency is 105.2%,R²=0.998,y_BC=-3.159x+45.038,amplification efficiency is 107.3%,R²=0.999,y_ES=-3.287x+49.074,amplification efficiency was 101.5%,R²=0.999,y_LM=-3.188x+44.725,amplification efficiency was105.9%,R²=1.000.The double qPCR standard curves were y_PM=-3.2x+39.278,amplification efficiency was 105.4%,R²=0.981,y_BC=-3.212x+39.559,amplification efficiency was 104.8%,R²=0.995,y_ES=-3.425x+38.51,amplification efficiency was95.9%,R²=0.987,y_LM=-3.357x+43.335,amplification efficiency was 98.5%,R²=0.996.The results showed that the specificity and sensitivity of qPCR were higher,and the calibration curve had a good linear relationship,which could be used to detect the dynamic changes of Douchi.Double qPCR and double qPCR were used to detect different fermentation time of the same batch,the results of double qPCR were that BC and PM existed during the whole fermentation period,the maximum values of BC being 18 h,which is 1.79×10³ copies/?L,and the maximum of PM being 66 h,which is 8.07×10² copies/?L.ES only appears in 6 h of the fermentation period,which is 3.8×10² copies/?L,LM was not detected during the whole fermented fermentation cycle in Douchi.The results of double qPCR were that BC,PM and ES existed during the whole fermentation period,the maximum values of BC being 18 h,which is 1.83×10³copies/?L,the maximum value of PM being 66 h,which is 7.50×10² copies/?L,and the maximum value of ES being 6 h,which is 3.80×10¹ copies/?L,LM is present in the post-fermentation,with a maximum value of 1 d,which is 3.0 copies/?L.The detection rate of double qPCR is 6.3%,the detection is more accurate and the sensitivity is higher.The analysis of the dynamic changes of pathogenic bacteria showed that BC and PM breed significantly in pre-fermented,and no obvious law.It is presumably that the difference in fermentation environment,and antagonism of the main fermentation Bacillus subtilis.The post-fermentation proliferation stopped,and the quantity gradually decreased.It is presumed that the inhibition of Bacillus subtilis metabolites,the addition of seasonings after the fermentation,the oxygen content,the fermentation temperature,and other conditions,resulting in its normal growth and reproduction,and dying over time.ES is extremely low in the whole fermentation period and does not proliferate,showing a steady decline after a gradual decline,and is presumed to be an inactivated bacterial cell carried by the raw material of soybean before fermentation.LM appeared for the first time in post-fermentation,and was extremely low in number and did not proliferate.It was presumed to be inactivated bacteria carried by fermented ginger,chilli,and prickly ash added in post-fermentation.In this study,double ddPCR and double qPCR assays were used to obtain the dynamic changes of the four pathogenic bacteria during the fermentation process of Douchi.It has practical significance for the prevention and control of the bacterial pathogen contamination,the selection of microbial detection methods,and the safe production of Douchi.