Development Of Multiple PCR Assay For Pig-pathogenic Of Streptococci And Their Application In Epidemiological Investigation

Pathogenicity between numerous species and types of Streptococci are strikingly different. Targeting at predominant pig-pathogenic species and types including Streptococcus equi subsp . zooepidemicus and Streptococcus suis type1, 2, 7, 9 (SS1, SS2, SS7, SS9), present study aimed to develop a novel polymerase chain reaction (PCR) method to diagnose Streptococci at genus, species and type levels simultaneously by just a single PCR. And it was applied to carry out epidemiological survey of Streptococcus suis in parts of china by combining with simple PCR, serum agglutination test and MLST typing method. The major results are as follows:(1)Development of multiple PCR assay for pig-pathogenic of Streptococci. Based on genetic sequences in Genebank, 7 pairs of primers respectively targeting at Streptococcus genus-conservative gene (elongation factor EF-TU), SS and Streptococcus equi subsp . zooepidemicus conservative gene (glutamate dehydrogenase, GDH and M-like protein respectively), and type specific genes (capsular polysacchride, CPS, CPS1I, CPS2J, CPS7H, CPS9H respectively for SS1, SS2, SS7, SS9) were designed with expected length of amplified fragments respectively as 197, 943, 1137, 441, 291, 541, 388bp. Multiple PCR was developed based on template mix(SS1, 2, 7, 9 and Streptococcus equi subsp . zooepidemicus) Specificity of the multiple PCR was determined using S. agalactiae and S.Mutans, Staphylococcus aureus, E.coli and water respectively as control. Accuracy was verified by standard strains of SS1, SS2, SS7, SS9 and Streptococcus equi subsp . zooepidemicus strain 555, and by detecting 16 clinical isolates already identified by traditional bacteriological methods and simple PCR. Sensitivity was confirmed by serial dilution of template mix and stability by preparation of template mix each time. The novel multiple PCR assay designed in this study could clearly produce 7 expected bands with correct sizes and sequences at 61℃as annealing temperature. Specificity and accuracy were confirmed by expected bands when detecting standard SS strains and Streptococcus equi subsp . zooepidemicus strain 555, and even further proven by 100% in agreement with bacteriological and PCR results when detecting 16 Streptococcus isolates. Sensitivity reached 0.08ng/μL and stability were verified by same performance in 5 separate tests. The multiple PCR developed in this study, are the only assay up to date which could diagnose at three levels (genus, species, type) for pig-pathogenic Streptococcus by a single PCR with high specificity, accuracy, sensitivity and stability. The striking advantages of this novel PCR are highly efficient, economic and time-saving, suiting for Streptococcus surveillance in pig farm and fast epidemiological survey.(2)The epidemiological survey of Streptococcus suis . 900 samples(tonsil or lymph) were collected from 6 provinces of China in 2010. Streptococcus suis were isolated and purified, followed by identification of suspected species and types by PCR based on gene squences of GDH, CPS1I, CPS2J, CPS7H, CPS9G and serum agglutination test. The results showed: of the 28 strains identified, 3 strains are SS2, 2 SS7, 1 SS9, 2 SS3, 1 SS15, 1 SS19, 1 SS27, 1 SS34. Genetic relationships of isolates were studied by multilocus sequence typing (MLST). Analyzed by MLST, The seven housekeeper genes (aroA, Cpn60, Gki, Dpr, Muts, ThrA, RecA) of some strains, the PCR product negative or not accurate sequencing, so can't determine the corresponding ST type. 13 strains can be identified, the results implied that 3 strains are ST1, 2 ST29, 2 ST168, ST27, ST28, ST45, ST117, ST163, ST174 each one strain. Two strains belonged to new ST types, which was not reported in previous publications. The new ST was submitted to MLST database of Streptococcus suis (http://ssuis.mlst.net).