Study On Fast Detection Technology Of QPCR And LAMP In Paocai Production

Paocai originated in China and has become the business card of fermented vegetables in China after thousands of years of accumulation.The fermentation of Paocai mainly includes traditional fermentation and starter culture fermentation.It is a fermentation process in which fresh vegetables are affected by various microorganisms in brine.However,due to the long production cycle of Paocai and lots of bacteria attached to the raw materials,the growth of pathogenic bacteria such as Escherichia coli will occur during fermentation,causing problems such as food contamination and excessive nitrite,and excessive lactic acid bacteria and yeast may also cause the spoilage of Paocai.Therefore,it is necessary to establish a fast detection method for various microorganisms in Paocai.In this paper,Paocai was used as the research object,and the microbial community dynamics of fermentation process of Chinese Paocai was analyzed by PMA-qPCR.The mechanism of nitrite change during fermentation was studied,and the LAMP detection method of Escherichia coli and yeast in Paocai was established.The research contents and conclusions are as follows:(1)Dynamic analysis of main microorganisms during starter culture fermentation of Paocai by PMA-qPCR:The concentration of total LAB,L.plantarum NCU116,yeast and E.coli increased rapidly during the early stage of fermentation,the concentration of LAB and L.plantarum NCU116 maintained around 10~9 CFU/mL,and the yeast slowly proliferated to 10~6 CFU/mL.E.coli was rapidly extinct.Traditional plate count assay was time-consuming and cannot quantify the target strains NCU116.PMA-qPCR assay can quickly quantify the target strains,and showed no significant difference(P>0.05)from the results of traditional methods.The PMA-qPCR assay provides a rapid and accurate way to quantitatively detect the dynamic changes of the microbial community during the fermentation of Paocai.(2)Changes of nitrite and other related indicators during Paocai fermentation:During traditional fermentation and starter culture fermentation,the pH value of system decreased continuously,during starter culture fermentation,pH value of Paocai declined faster and reached the end point of fermentation earlier;the content of lactic acid bacteria in Paocai increased steadily to 10~8CFU/mL,and the number of E.coli increased first,then decreased gradually to extinction.;The nitrite concen-tration climbed to the“Nitrite Peak”in the early stage of fermentation,and then rapidly decreased to a safe level,which the peak value of starter culture fermented Paocai was much lower than that of traditional fermented Paocai,and its nitrite concentration was reduced to 2.5 mg/kg earlier than that of traditional fermented Paocai;the transcription level of NAR and NiR genes continued to decrease during1~7 days of fermentation,and the transcription level of NAR gene in traditional fermented Paocai was higher than that in starter culture fermented Paocai.Meanwhile,lactic acid bacteria could effectively inhibit the transcription of NAR gene in E.coli,thereby reducing the production of nitrite;the transcription level of NiR gene in starter culture fermented Paocai is higher than that in traditional fermented Paocai,Low pH environment could inhibit transcription of NiR gene,the enzymatic degradation of LAB in starter culture fermentation was higher than that in traditional fermentation.(3)Establishment of LAMP fast detection method:Optimized the detection system,designed the primer group for LAMP detection of E.coli and yeast,which can specifically identify and detect E.coli and yeast.In this study,the minimum detection limit of E.coli DNA in the sample is 100 fg/?L,the sensitivity is 100 times higher than that of PCR technology,and the detection limit of yeast DNA in the sample is 100 fg/?L,the sensitivity is 10 times higher than that of PCR technology;Through the LAMP and routine PCR detection of 0~7 d starter culture fermented and traditional fermented Paocai samples,it was confirmed that E.coli died earlier during intensive fermentation of Paocai.However,due to the low detection sensitivity of conventional PCR,it is impossible to detect some microorganisms in the sample,which may lead to missed detection.In contrast,the LAMP fast detection method is more reliable;LAMP detection results can be visually observed whether there is white suspension or precipitation,or by dropping SYBR Green I to see whether there is yellow-green fluorescence,it can be used for fast detection at the sampling site or for accurate analysis by electrophoresis or quantitative PCR.(4)Establishment of Multiplex LAMP fast detection method:Optimized the Multiplex LAMP fast detection system to reduce the original primer concentration by half,while the other substances have the same concentration;the minimum detection limit of this method is 100 fg/?L,which is same as the single LAMP method.The detection result of double LAMP can be analyzed by melting curve method.If the Tm value of the amplified product is about 92.7°C,it means that E.coli is present in the sample;if the Tm value of the amplified product is about 93.2°C,it means that Yeast is present in the sample;if the Tm value of the amplified product is between 92.7 and93.2°C,it means that E.coli and yeast are both present in the sample;if no amplification reaction occurs(no melting temperature peak),it means that there is no E.coli and yeast in the sample.