| Clostridium perfringens(C.perfringens)is a Gram-positive pathogen,which exists in nature and the gastrointestinal tract of human and animals.It can cause human food poisoning,gas gangrene,gastrointestinal diseases,liver and kidney injury of human,also can cause gangrenous dermatitis of animals,necrotizing enteritis(NE)of cattle,sheep,pigs and birds,as well as enterotoxemia of cattle and sheep.Further induced serious economic losses to the global animal husbandry in recent decades.Type F strain can cause human food poisoning and many non-foodborne human gastrointestinal(GI)diseases.Type F strain with Clostridium perfringens enterotoxin(CPE)gene located on chromosome causes human food poisoning,while type F strain with cpe located on plasmid are related to non-foodborne human GI diseases.Therefore,it is of great significance to determine the location of cpe on chromosome or plasmid for the diagnosis and control of diseases.When cpe is located on chromosome,the downstream of cpe carries IS1470 sequence;when cpe is located on plasmid,the downstream of cpe carries IS1470-like or IS1151 sequence.The cpe of some cpe~+isolates can be located by the detection of IS1470,IS1470-like,IS1151 and cpe sequence.Superoxide dismutase(SOD)gene is conservative and pathogenic,it is far away from cpe and will not be affected by cpe movement.The location of cpe gene of isolates can be determined by PCR amplification of sod and sod inner primer pair(sod FPF)and further analyzed by single-locus sequence typing(SLST).To establish a sextuple PCR typing detection method and locate cpe position for C.perfringens isolates in this study,results were obtained as follow:1.Development of the sextuple PCR typing detection method of C.perfringensThe primers of six toxin genes were synthesized,and the PCR template were used the genomic DNA of type A～G strain,respectively,following the annealing temperature and the primer concentration of six toxin genes were optimized.The results showed that the optimal annealing temperature was 57.3℃,and the optimal concentrations of cpa,cpb,etx,ia,cpe and net B primers were 0.8μmol/L,1.2μmol/L,1.8μmol/L,1.8μmol/L,1.0μmol/L and 1.2μmol/L,respectively.The specificity test showed that the target bands could be amplified by type A～G strain,while the detection results of Clostridium putrificum,Bacucilius cereus,Staphylococcus aureus,Escherichia coli and Klebsiella pneumoniae were negative.The thresholds of detection of the sextuple PCR assay were 445 pg/μL,41.6 pg/μL,380 pg/μL and 3.75 ng/μL of genomic DNA,as well as 4.9×10~5CFU/m L,5.8×10~4CFU/m L,6.5×10~5CFU/m L and 2.4×10~5CFU/m L of bacterial culture of type B,E,F and G strain,respectively.The stability test showed good repeatability.The toxin genotypes were consistent with the known types of C.perfringens isolates by using the sextuple PCR.A sextuple PCR typing method for C.perfringens was successfully established.2.Localization of cpe of C.perfringens isolatesThe double PCR method for IS1470,IS1470-like,IS1151 and cpe sequence was used to detect whether the downstream cpe of the isolates carried IS1470 with amplification length of 1 300 bp,IS1470-like with amplification length of 1 600 bp or IS1151 sequence with amplification length of 800 bp.The results showed that only 600 bp bands were amplified from 29 cpe~+C.perfringens isolates,the above three sequences were not detected,and the position of cpe could not be located.sod was detected by conventional PCR.The results showed that all C.perfringens isolates amplified 347 bp bands.SLST analysis results showed that the isolates were divided15 clades,of which 2 clades were contained 6 and 7 isolates,respectively.It speculated that cpe of 27 isolates may be located on the chromosome,except cpe of A190926-2 on the plasmid and cpe of A190925 couldn’t be inferred.sod was detected by sod FPF.The results showed that all C.perfringens isolates amplified 350 bp bands.SLST analysis showed that the isolates were divided 4 clades,including 13,8,5 and 3 isolates,respectively.The isolates carrying cpe on chromosome and the strains carrying cpe on plasmid were located in different cluster groups,27 isolates were close to the reference strains carrying cpe on chromosome.A190926-2 and A190925 were located in the same clades,were close to the reference strains that carried cpe on chromosome,and far away from the four reference strains that carried cpe on plasmid.Overall,the cpe of 29 isolates were located on the chromosome by sod FPF detection and SLST comprehensive analysis.To sum up,a sextuple PCR typing method for C.perfringens is successfully established and cpe position of C.perfringens isolates is located in this study.It provides materials and scientific basis for the epidemiological investigation,prevention and control of diseases caused by C.perfringens,diagnosis and control of food poisoning and non-foodborne diseases caused by type F strain as well as the study of biological characteristics of cpe~+strains. |
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