| Mycoplasma ovipneumoniae(Mo) is one of pathogen causing chronic non-progressive and interstitial pneumonia in sheep and goats. Until now it is prevalent worldwide and causes huge economic loss. However, a highly heterogeneity of genome sequence for Mo, it is difficult to development a effective molecule method for detection. Although PCR and real-time PCR for detection had been reported, it is still need to development a more sensitivity molecule method for this pathogen.Elongation factor Tu(EF-Tu) is a protein encoded by tuf which catalyzes this elongation process of polyppeptide chain during the synthesis of protein. It is widespread in fungi, epiphyte and archaebacteria. It was identified a molecule target which was used in a vary of phylogeny and molecular tapping. However, until now little is known about genetic structure and genetic diversity of tuf in Mo. This study aimed at analyzing its molecular characterizations and designing primers to development a more sensitive real-time PCR for detection. It had been used in molecular epidemiology of Mo at last. Analysis molecular characterization of tuf for MoBased on the tuf sequence of Mo in GenBank, a pair of primers was designed to amplify CDS of Mo. 12 strains of Mo was used in this study, the results of sequencing showed that the CDS of tuf were 1209 bp in size in the all strains, encoding 402 amino acids.The nucleotide identity shared 93%~100% and the amino acid identity 93%~100%. Their G+C content was 39.45%~40.12 %. Analysis the sequence of CDS of Mo revealed that there were conserved areas in 3’ and 5’ ends. i.e 138~309bp and 1047~1196bp are highly conserved but variable in other Mycoplasma species. However a large variation in the region 21~960bp(mutation rate was 8.93%)and in the region 961 ~ 1189bp( mutation rate was 26.9%).There were 117 single nucleotide polymorphisms(SNPs), consisting of 41 synonymous polymorphisms and 76 non-synonymous polymorphisms leading to 53 amino acid changes, mainly in 325~354 region near the carboxyl region. There were two TGA codons in two isolates, while only one in all the other isolates tested in this study. TGA was stop codon in procaryote while was codon translated into tryptophan in Mycoplasma.Phylogenetic analysis based on tuf revealed that Mo had the closest evolutionary relationship with Mycoplasma hyopneumoniae which consistent with whole genome, but based on 16 S rRNA was not correspond with based on whole genome. In concluded that tuf of Mo was a better target than 16 S rRNA gene for phylogenetic analysis. 12 strains of Mo in this paper were divided in two clusters(clusterⅠ, clusterⅡ). Cluster Ⅰ include Y98 and clusterⅡ include 11 isolates. ClusterⅡ divided in two sub-clusters. Interestingly, all 3 isolates from Z area were clustered together into a Cluster and all 4 isolates from J area were clustered together into a cluster. Five isolates from L area fall into the two sub-clusters. This projected that there were tendency of regional distribution. It implicated the potential molecular tapping in Mo.. 2 Development of real-time PCR target to tuf for detecting MoPrevious investigations have indicated that nucleotide and protein sequences of Mo exhibit high heterogeneity. However the G+C content of mycoplasma genome is generally low and there is highly heterogeneity among the different strains, which reduce the molecular methods for detection. It is difficult to development a more sensitivity molecule method for this pathogen. Until now there are PCR molecular methods based on 16 S r RNA and Hsp70 gene which sensitivity is low and lead to false-negative results easily. However the more sensitivity detecting method were still needed. tuf CDS of Mo during 138~309bp and 1047~1196bp region was conserved but different from others mycoplasma species in previous studies. It might be ideal target for molecular detection. The G+C content of tuf CDS was 39.54%~40.12% and 11% more than its whole genome. Relatively high G+C content is more conducive to design PCR primers. So primers was designed by Primer 5.0 and screened to a pair of the best primers to amplify fragments of 150 bp fragment in tuf CDS(1047~1196bp). In order to obtain the optimal reaction conditions and the best reaction system, The primer concentration, template concentration, annealing temperature and cycle number were optimized. The results showed that, the real-time PCR present only one peak on melting curve with melting temperature of 81.5℃±0.3 ℃. Non-specific amplification and primer dimmers were not observed.The amplification efficiency was 94.1% with a linear range from 5×109 to 5×104 copies and a correlation coefficient of 0.996. The standard equation was y=-3.473x+43.411. The lowest detection limit was 5 copies the same as 169 CCU/ml.This study established the real-time PCR assay successfully.33 strains of clinically positive Mo were all identified, but the unrelated pathogens, such as mycoplasmas infection and the common respiratory bacterial of sheep were all negative. As it as a new method which was specific, sensitive. 50 of sheep nasal swabs and 134 sheep lung tissue were used to compared the real-time PCR and the real-time PCR target to P113 gene for detection rates. It showed that the detection rate of lung tissue was the same but the nasal swabs were two more than it. However They were determined by sequenced. The detection rate of nasal swabs and lung tissue was higher than 12%~42%,9%~42% respectively. It can not only qualitatively but also quantitatively for Mo which provides a new method for Mo epidemiology. 3 Application of the real-time PCR for detecting Mo 3.1 Comparing the detecting rate of different lesions in lung tissue by Mo artificial infectionSix of the 3-month-old healthy goats were inoculated by in tracheal injection method of 5 × 109 CCU Y98 strains. After 7d, Sheep all appeared typical clinical symptoms like cough,tears,nose running. After sick sheep were killed at 30 d and it showed that the typical lesion like fibrinous pneumoniae and yellow clear liquid in chest. The different lesions of lung tissues were sampled. The lung tissues were detected by the real-time PCR.The results showed that the detecting rate is the highest in the health-junction sample(6/6),the next is the health(4/6),the complete lesion is least or undetectable(1/6). It provides that the health-junction site is the best place for detecting. 3.2 Epidemiological investigation of Mo in Sichuan、Xinjiang、Qinhai provinceThere were many scholars have been used the methods of indirect hem-agglutination for seroepidemiology but little about the pathogen epidemiological data. The trial of 439 samples of lung tissue were collected form Sichuan(n=126),Xinjiang(n=163),Qinhai(n=113) province in 10 th 2013~10th 2014. The results showed that the positive rate of pathogen was 56% 、 63% 、 75% respectively.The results showed that these areas were infected Mo seriously. |
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