Establishment Of Multiplex PCR Method For Simultaneous Detection Of Five Kinds Of Nontuberculous Mycobacteriums Of The Hand

Objective: To establish simultaneous multiplex PCR method to detect the five common types of nontuberculous mycobacterium(NTM)that cause infection of hand(mycobacterium avium,mycobacterium marinum,mycobacterium fortuitum,mycobacterium kansasii and mycobacterium ulcerans),and provide the experimental foundation and theoretical evidence for diagnosis and treatment of NTM hand infection in the early stages.Methods:Based on the previous research results,firstly,we searched for the unique gene sequence of the five common types of NTM hand infection(mycobacterium avium,mycobacterium marinum,mycobacterium fortuitum,mycobacterium kansasii and mycobacterium ulcerans)from Gene Bank database and literatures.Then,we designed primers with MFEprimer-5.0 and Olige6.0 softwares,which were then evaluated by DNAMAN.Secondly,these standard bacteria were sequenced after PCR amplification with the specific primer to validation standard bacteria is in line with the requirements of this institute to use.Thirdly,we selected out the specific primers which had no or few complementary segments for multiple PCR construction.Through using the optimization of annealing temperature of the multiple PCR to evaluate its specificity and sensitivity.Lastly,the multiple PCR was used to detect and identify 12 specimens of NTM infection in hand.Results: 1.Successfully designed for the above five kinds of mycobacteria five specific primers;2.Single PCR detection of this institute standards accord with the requirement of standard bacteria;3.Established a simultaneous multi-PCR method to detect the five common types of NTM hand infection(mycobacterium avium,mycobacterium marinum,mycobacterium fortuitum,mycobacterium kansasii and mycobacterium ulcerans);4.The optimal annealing temperature for this multi-PCR is57.5℃,with high specificity and sensitivity;5.This multi-PCR examined 12 clinical specimens,in which 10 specimens were amplified positive bands,the detection rate was 83%;6.The time will be reduced to about four hours by using the five PCR to detection NTM.Conclusions:This study established a multiplex PCR method to detect the five common types of NTM hand infection,(mycobacterium avium,mycobacterium marinum,mycobacterium fortuitum,mycobacterium kansasii and mycobacterium ulcerans);It can early and accurately detect hand NTM infection,and identify the level of subspecies,which can provide certain test basis for best clinical treatment.