Establishment And Application Of Multiplex LAMP-LFB Method For Detection Of Clostridium Perfringens β Toxin And ε Toxin

Background:Clostridium perfringens is an important zoonotic pathogen,which is widely distributed in nature.It can be found in soil,sewage,feed,food,feces and human and animal intestines.Under certain conditions,it can cause a variety of serious diseases,such as sudden death of livestock and poultry,necrotizing enteritis,enterotoxemia,human gas gangrene and food poisoning,antibiotic related diarrhea and other diseases.Clostridium perfringens can produce a variety of exotoxins,among which α、β、ε、τ are the main lethal toxins,and α、β、ε、τ toxins are also known as CPA,CPB,ETX and ITX toxins.The latest toxin typing divides the strain into seven toxin types,in which type B produces CPA,CPB and ETX toxins,and type D produces CPA and ETX toxins.CPB and ETX toxins are perforin.CPB acts on nerve cells through gangliosides,so it is also a neurotoxin;After bacterial propagules are produced in the intestine,ETX toxin is absorbed into the blood and acts on brain,lung,kidney and vascular endothelial cells,as well as sheath nerve fibers in the central nervous system and around,resulting in sudden death,which seriously threatens the public health and safety of animals and the development of modern breeding industry.Objective:in order to quickly and sensitively detect CPB toxin and ETX toxin of Clostridium perfringens,a method of double loop mediated isothermal amplification combined with transverse flow biosensor（LAMP-LFB）was established,which can detect CPB toxin and ETX toxin of Clostridium perfringens at the same time,and identify type B and type D of Clostridium perfringens at the same time.Research methods:two sets of primers were designed according to the CPB toxin gene and ETX toxin gene of Clostridium perfringens.The FIP*-CPB and FIP*-ETX primers were labeled with digoxin and fam biotin respectively,and the plasmids containing CPB toxin gene and ETX toxin gene were constructed respectively.The CPB toxin gene and ETX toxin gene were amplified by loop mediated isothermal amplification（LAMP）.The amplification system established in this study includes 2*RMB,Bst DNA polymerase,primer mix,plasmid template and deionized water.The reaction time and temperature are 60min and 63°C respectively.In this study,the lamp amplification products were detected by transverse flow biosensor（LFB）,and the red line indication was positive in the quality control area and the detection area.Results:（1）The detection limits of single LAMP-LFB system for CPB toxin gene and ETX toxin gene were 1.2*10¹⁴ copies/μl and 1.3*10¹⁴ copies/μl,respectively.（2）The detection limits of multiplex LAMP-LFB detection system for CPB toxin gene and ETX toxin gene were1.2*10¹⁴ copies/μL and 1.3*10¹⁶ copies/μl,respectively.（3）The sensitivity of the multiplex LAMP-LFB assay in simulated fecal samples for Clostridium perfringens type B and D is1.4×10²CFU/m L.