To rapidly and accurately detect Mycobacterium avium subsp.paratuberculosis(MAP),this study designed and screened primers and probes using its specific gene F57 as the detection target,established a recombinant enzyme-mediated isothermal amplification(RAA)fluorescence detection method,and applied this method to detect 116 clinical samples from cattle and sheep.The results showed that using the primer and probe combination B12F/B2R(0.4 μmol/L)+Probe B(0.12 μmol/L),MAP could be detected at a constant temperature of 42 ℃ within 20 min;this de-tection method had no cross-reaction with 11 common pathogens such as Escherichia coli,Clos-tridium,and bovine viral diarrhea in sheep and cattle;the lowest detection limit was 1.0×102 cop-ies/μL;the coefficient of variation was 3.77％—5.29％;24 clinical samples were positive,with a co-incidence rate of 88.89％with GBT27637-2011.In summary,this study established a fluorescent RAA detection method for MAP,which is simple,rapid,highly specific,sensitive,reproducible,and has a high coincidence rate with national standards,making it suitable for clinical detection and epi-demiological studies.

To rapidly and accurately detect Mycobacterium avium subsp.paratuberculosis(MAP),this study designed and screened primers and probes using its specific gene F57 as the detection target,established a recombinant enzyme-mediated isothermal amplification(RAA)fluorescence detection method,and applied this method to detect 116 clinical samples from cattle and sheep.The results showed that using the primer and probe combination B12F/B2R(0.4 μmol/L)+Probe B(0.12 μmol/L),MAP could be detected at a constant temperature of 42 ℃ within 20 min;this de-tection method had no cross-reaction with 11 common pathogens such as Escherichia coli,Clos-tridium,and bovine viral diarrhea in sheep and cattle;the lowest detection limit was 1.0×102 cop-ies/μL;the coefficient of variation was 3.77％—5.29％;24 clinical samples were positive,with a co-incidence rate of 88.89％with GBT27637-2011.In summary,this study established a fluorescent RAA detection method for MAP,which is simple,rapid,highly specific,sensitive,reproducible,and has a high coincidence rate with national standards,making it suitable for clinical detection and epi-demiological studies.

Objective To investigate the mutation patterns and phylogenetic relationships of the plo gene in strains from different hosts, and provide feasible methods for characterizing the strain-host association.Methods The plo gene was amplified using PCR from 14 strains isolated from goats, 2 strains from pigs and 2 strains from sheep, and sequenced and spliced.Multiple sequence alignment and phylogenetic analysis were performed on the plo complete gene obtained from 18 strains, as well as the plo gene downloaded from Gen Bank, and their coding products.Results The plo complete gene was obtained from 18 isolates, and the homology was higher than 95%. The plo gene had three types of point mutation. Type Ⅰ was composed of strains from bovines and a horse, with 10 consistent mutation sites; typeⅡwas composed of strains from pigs and a dog, with 22 consistent mutation sites; and type Ⅲ was composed of strains from goats, sheep, a chamois and a forest musk deer, with 32 consistent mutation sites. The mutation sites were distributed dispersedly in most regions of the plo gene. These mutations produced 16 sense mutations, resulting in three corresponding point mutation patterns of PLO protein. Trueperella pyogenes(T.pyogenes) was divided into four branches in the plo complete gene phylogenetic tree established by the neighborjoining method(NJ). The bovine strains and a strain from horse were clustered into GroupⅠ, the strains from pigs and a dog into Group Ⅱ, the strains from goats, sheep, a chamois and a forest musk deer into Group Ⅲ, and the one strain from sika deer into Group Ⅳalone. Different from plo genotyping, the one strain from sika deer was clustered into Group Ⅲ in the PLO phylogenetic tree.Conclusion The plo gene exhibits host-associated mutations. By constructing phylogenetic trees of the plo complete gene and PLO complete sequence, strain-host association can be demonstrated, indicating that the plo gene and PLO sequence are useful molecular markers for characterizing strain-host association.

Objective To analyze the antibody responses of 10 serum reactive antigens(MAP1138c,MAP2121c,MAP0150c,MAP0862,MAP0209c,MAP2120c,MAP0038,MAP3420c,MAP 2154c and MAP2751)of Mycobacterium avium subspecies paratuberculosis(MAP)in naturally infected young sheep and evaluate their diagnostic value.Methods Serum samples and anal swab samples were collected from 6-month-old sheep without obvious PTB symptoms in the flocks with paratuberculosis(PTB)history.The serum samples were tested by PTB antibody detection ELISA kit,and anal swab samples were detected by the fluorescent quantitative PCR based on MAP F57 element.The sheep were grouped according to the test results.PCR was used to detect 10 MAP antigen genes in positive anal swabs.The antigen genes were cloned into pET-28a and induced to be expressed in E.coli BL21(DE3)strain by IPTG.The recombinant antigens were purified by Ni-Sepharose,and then coated on the ELISA plate for testing the collected serum samples to analyze the antibody reaction of the selected antigens in naturally infected sheep.The detection rates of serum antibodies against different antigens were analyzed to evaluate the diagnostic value of the antigens.Results The 72 sheep sampled were divided into three groups:anal swab positive-antibody positive(n = 34),anal swab positive-antibody negative(n = 23),and anal swab negative-antibody negative(n = 15).All 10 antigen genes were detected from positive anal swabs,and sequences of each gene were highly consistent.Through ELISA detection,MAP1138c,MAP2121c,MAP0150c and MAP0862 produced antibody reactions in infe-cted sheep.Antibodies against MAP1138c,MAP2121c,MAP0150c and MAP0862 were detected in 30,34,24 and 31 of the 57infected sheep,respectively.In the anal swab positive-antibody positive group,the detection rate of anti-MAP1138c antibody was the highest(76.47%).In the anal swab positive-antibody negative group,the detection rates of antibodies against MAP2121 and MAP0862(52.17% and 47.83%)were higher than those of the other two proteins.In the detection of 72serum samples,the overlap of ELISA coated with MAP2121c and MAP0862 exceeded 91%.Conclusion MAP1138c,MAP-2121c and MAP0862 may be dominant biomarkers to induce MAP antibody response in naturally infected young sheep.MAP2121c and MAP0862 can make up for the deficiency of sensitivity of commercial ELISA kits in early diagnosis of PTB.

Objective To identify the heparin-binding motif of drug-binding pocket protein 2(Dbpp2)of sacbrood virus(SBV)and determine the anti-SBV infection activity of the heparin-binding motif and its derived peptides.MethodsThe secondary structure prediction and homology modeling of Dbpp2 were carried out using online software to search for potential heparin-binding motifs. The binding ability of SBV,Dbpp2 and their heparin-binding motif mutants to and the ability of heparin-binding motifs to inhibit the binding of Dbpp2 to heparin were tested using heparin agarose beads. The therapeutic effect of heparin-binding motifs and their derived peptides on the larvae of Apis cerana cerana infected with SBV was tested.ResultsSBV and Dbpp2 showed binding with heparin. The flexible ring(KPANRPRR)rich in basic amino acids exposed at the C-end of Dbpp2 was a heparin-binding motif,which inhibited Dbpp2 from binding to heparin. KPANRPRR and its derived peptides KPAARPRR,KPRNRPRR,KPRARPRR,KPRWRPRR and KPRWRPRRW all had the effect of reducing the mortality of larvae caused by SBV infection,among which KPRARPRR showed the best therapeutic effect.Conclusion The research provides a basis for understanding the interaction of SBV-glycosaminoglycan(GAG),and provides a reference for the development of peptides for the treatment of SBV infection based on the interaction.

Objective To analyzed imaging features of testicular and epididymal tumors or inflammatory nodes in children and to improve its diagnostic accuracy.Methods 13 cases underwent ultrasonography,of them,plain CT scan in 10 cases,enhanced CT scan in6 cases.Its signs were retrospectively analyzed and compared with surgical and pathological results.Results On ultrasonography,theinhomogenous and different echo could be viewed in 13 cases,color Doppler flow imaging was abundant in 8 masses,3 cases with littler,empty blood flow in 2 cases and retroperitoneal lymphatic metastasis were viewed in 2 cases.On plain CT scan,masses were mixed density in 4 cases,calcification could be seen within tumor in 3 cases,masses were soft tissue or main soft tissue density in 6 cases.Contrast-enhancedCT scan displayed obviously and inhomogenously in 4 cases,lightly enhancment in 1 case and no enhanced in 1 case.By surgical andpathological confirmed,3 were mature treatomaes,1 was immaturity teratoma,4 were yolk sactumor,3 were inflammatory nodes,1 wasrhabdomyosarcoma and 1 was cystic lymphangioma.Conclusion Each kind of testicular tumors in children has its owns CT and UScharacteristics.In combination of CT and US can carry high diagnostic accuracy.