Establishment And Application Of LAMP Detection Of Several Pathogenic Microorganisms In Infant Food

Children nutrition is an important index of nation and social development,and infant food plays a crucial role of nutritional improvement.In recent years,infant foods security accidents occur frequently,of which microbial contamination is the most serious.Pathogenic microorganisms can lead to food-borne disease and zoonosis, causing infant sick even death.The paper selects several pathogenic microorganisms in infant food,and establishes their loop-mediated isothermal amplification methods.Study content is as follows:(1) Establish detection method for Enterobacter sakazakii using loop-mediated isothermal amplification,and apply this method to detect Enterobacter sakazakii in 90 infant food samples.(2) Establish detection method for aflatoxigenic fungi using loop-mediated isothermal amplification,and apply this method to detect aflatoxingenic fungi in 90 infant food samples.(3) Preliminary study on establishment of detection method for Campylobacter Jejuni using loop-mediated isothermal amplification.(4) Preliminary study on establishment of detection method for enterotoxigenic clostridium perfringens using loop-mediated isothermal amplification. (5) Preliminary study on establishment of detection method for Clostridium botulinum type A using loop-mediated isothermal amplification.(6) Preliminary study on establishment of detection method for Candida albicans using loop-mediated isothermal amplification.The paper designs the primers based on 16s rRNA of Enterobacter sakazakii, then screens three sets of primers and optimizes the reaction conditions,finally establishes loop-mediated isothermal amplification methods for Enterobacter sakazakii.The experimental results show that this new method is rapid and simple, and it is easy to observation results.The LAMP method is proved to have a high specificity and sensitivity,it can distinguish Enterobacter sakazakii from other Enterobacteriaceae bacteria,and its detection limit is 0.3pg/tube,which is superior to PCR.Apply this new method to detect Enterobacter sakazakii in 90 infant food samples,the result is consistent with result of traditional method(Vitek-2 Compact), so established loop-mediated isothermal amplification methods for Enterobacter sakazakii is accurate and reliable.The paper designs the primers based on aflR gene of aflatoxigenic fungi,then screens three sets of primers and optimizes the reaction conditions,finally establishes loop-mediated isothermal amplification methods for aflatoxigenic fungi.The experimental results show that this new method is rapid and simple,and it is easy to observation results.The LAMP method is proved to have a high specificity and sensitivity,it can distinguish Aspergillus flavus and Aspergillus parasiticus from other fungi,and its detection limit is 40pg/tube,which lies between PCR and the real-time PCR.Apply this new method to detect aflatoxigenic fungi in 90 infant food samples, the result is consistent with result of traditional method(fungi microculture),so established loop-mediated isothermal amplification methods for aflatoxigenic fungi is accurate and reliable.The paper designs the primers based on hipO gene of Campylobacter Jejuni,then screens two sets of primers and optimizes the reaction conditions,finally establishes loop-mediated isothermal amplification methods for Campylobacter Jejuni.The experimental results show that this new method is rapid and simple,and it is easy to observation results.The LAMP method is proved to have a high specificity and sensitivity,it can distinguish Campylobacter Jejuni from other Campylobacter,and its detection limit is 2.2ng/tube,which lies between PCR and the real-time PCR.The paper designs the primers based on CPE gene of enterotoxigenic clostridium perfringens,then screens two sets of primers and optimizes the reaction conditions, finally establishes loop-mediated isothermal amplification methods for enterotoxigenic clostridium perfringens.The experimental results show that this new method is rapid and simple,and it is easy to observation results.The LAMP method is proved to have a high specificity and sensitivity,it can distinguish enterotoxigenic clostridium perfringens from non- enterotoxigenic clostridium perfringens,and its detection limit is 33.5pg/tube. The paper designs the primers based on botulinum neurotoxin type A gene of Clostridium botulinum type A,then screens two sets of primers and optimizes the reaction conditions,finally establishes loop-mediated isothermal amplification methods for Clostridium botulinum type A.The experimental results show that this new method is rapid and simple,and it is easy to observation results.The LAMP method is proved to have a high specificity and sensitivity,it can distinguish Clostridium botulinum type A from other type Clostridium botulinum,and its detection limit is 11.1pg/tube.The paper designs the primers based on P₄₅₀L₁A₁ gene of Candida albicans,then screens one set of primers and optimizes the reaction conditions,finally establishes loop-mediated isothermal amplification methods for Candida albicans.The experimental results show that this new method is rapid and simple,and it is easy to observation results.The LAMP method is proved to have a high specificity and sensitivity,it can distinguish Candida albicans from other yeast,and its detection limit is 3.8ng/tube,which is equivalent with PCR.The paper establishes loop-mediated isothermal amplification detection methods for several pathogenic microorganisms,these methods are rapid,simple,specific and sensitive,filling a domestic gap in this field and providing a new approach to detect pathogenic microorganisms in infant food.Established loop-mediated isothermal amplification detection methods of Enterobacter sakazakii,aflatoxigenic fungi,Clostridium botulinum type A and Candida albicans have applicated patent,public numbers are CN101319249A, CN101381771A,CN101381774A and CN101381775A.