Comparative Analysis Of Four Detection Methods For Trichinella Spiralis In Mice

Trichinosis is a very serious food-borne zoonotic parasitic disease,which not only causes huge economic losses to animal husbandry production,but also poses a great threat to human health.It is listed as a compulsory inspection disease for slaughter animals in the world.At present,the diagnosis of trichinosis in China is mainly through the inspection of slaughter animals in the downstream of livestock and poultry production,but the inspection methods are still relatively backward,such as microscopic examination and sampling digestion.Due to the low rate of intensive breeding,high rate of infected animals and large slaughter volume in China,the pressure of inspection and the rate of missed detection are high.Because the characteristics of different development stages in the life history of Trichinella spiralis,this study focused on the early rapid diagnosis of trichinosis in vivo,which was suitable for the breeding site,and provided theoretical basis and basic data for the early rapid diagnosis of trichinosis in vivo.In this study,comparison analysis of COX1 and NAD5 genes of T.spiralis mitochondrial DNA from 9 geographical strains in China,showed that T.spiralis were isolated from Harbin,Shandong,Xi’an,Yunnan,Tianjin,Henan and Tibet belonged to T1.Harbin geographical strain of Trichinella spiralis nativa belongs to T.nativa（T2）.Norwegian geographic strain of Trichinella raccoon belongs to T.pseudospiralis（T4）.The results showed that these two genes were stable and conserved,and could be used effectively in the molecular taxonomy and identification of T.spiralis.In this study,a real-time fluorescence quantitative PCR（qPCR）method was established for the detection of T.spiralis.Nine geographical strains of T.spiralis,Ascaris suum,Taenia solium and Fasciola hepatica DNA were tested for specificity.The results showed that all geographical strains of T.spiralis had amplification curves,while A.suum,T.solium and F.hepatica DNA and control group had no fluorescence signals,indicating that the specificity of this method was very strong,and it could be used to detect Trichinella T1,T2 and T4 in the genus Trichinella.The amplified large subunit ribosomal RNA（Ls-r RNA）gene was ligated and transformed by the vector,and then the positive sample was sent for sequencing.A standard curve was established by positive plasmid.The amplification equation was Y=（31.90-x）/3.830,and the correlation coefficient was R²=0.998,which showed a good linear relationship.After the extraction of 25 T.spiralis genomic DNA,the DNA was diluted in multiples of 10 and named Ln（n=1～5）.Then the sensitivity of the method was determined.The results showed that 10^-4T.spiralis were detected,indicating high sensitivity.When the same sample was tested for 10 times,the coefficient of variation was CV%=1.28%,showing no significant difference.L1～L5 samples were stored at-20℃for 60 days and tested again.There was no significant difference in comparison hypothesis test（P>0.05）.The results of intra-group and inter-group repeatability experiments indicated that the method had good repeatability.In this study,recombinant T.spiralis ornithine decarboxylase（Ts ODC）protein was used as the coating antigen to establish an indirect enzyme-linked immunosorbent assay（ELISA）method to detect antibodies of T.spiralis.The conditions such of the quantity of antigen coating,the dilution of serum,the condition of antigen coating,the selection and time of blocking solution,the dilution of enzyme-labeled antibody,incubation time and TMB action time were optimized in this study.The results showed that the best antigen package was 100 ng,the best serum dilution degrees was 1:640,the best antigen package was the condition for 4℃overnight,the best sealing fluid was 5%skim milk,the optimal closed time was 37℃closed 120 min,enzyme mark of resistance to the optimal dilution degrees was 1:8000,optimal incubation time was 37℃closed 60min,the reaction time of TMB was 15 min.The cut-off value of indirect ELISA method was 0.814.The results of repeatability experiment showed that the CV%of intra-and inter-batch repeatability were less than 6%,indicating a good repeatability.The positive serum of T.spiralis was 1:1280,the P/N value was>2.1.There was no cross reaction in the positive sera of A.suis,C.sinensis and S.sinensis,which indicated that the method had good specificity.Forty-five mice were infected with 10 muscle larvae orally.Small intestine,diaphragm,serum and feces samples were collected at different time points.Then qPCR,indirect ELISA,tablet compression and artificial gastric juice digestion were used to detect different samples.The results of qPCR showed that the nucleic acid of T.spiralis could be detected in the mouse small intestine at the earliest 1 d after infection,and the detection rate increased in a wavy manner with the extension of time.Nucleic acid could be detected in the serum in 3 d after infection,and the detection rate fluctuated with the increase of time.T.spiralis nucleic acid was detected for the first time in the diaphragm in 4 d after infection.The nucleic acid of T.spiralis was detected in feces at 3 h after infection,and the detection rate reached the highest at 21 d after infection.The indirect ELISA method showed that Trichinella antibody could be detected in mouse serum at 14 d after infection,and then the detection rate increased with time.The antibody to T.spiralis was detected in the small intestine grinding fluid and diaphragm abrasive fluid of mice at the earliest28 d after infection,and then the detection rate showed a wavy increase with time.T.spiralis was detected at 21 d after infection by tablet compression method.T.spiralis was detected by artificial gastric juice digestion at 28 d after infection.By comparing the above four diagnostic method results,according to the adult,newborn larvae and muscle larvae of T.spiralis different development periods,it can be detected in small intestine,feces,blood,skeletal muscle.The characteristics of qPCR method on the basis of T.spiralis life comprehensive collecting the above parts for testing could make an early and rapid diagnosis of trichinosis in vivo.As mentioned above,molecular classification of 9 geographical strains of T.spiralis by mitochondrial COX1 and NAD5 genes,optimization of qPCR detection method,establishment of indirect ELISA method for recombinant protein of Ts ODC and comparison of four detection methods were compared in this study.It can be confirmed that the qPCR method has the characteristics of different development stages for trichinosis,and the all-around detection of each sampling site can be used as a technique for the early rapid diagnosis of trichinosis in vivo,which not only provides a theoretical basis for the early rapid diagnosis of trichinosis in vivo,but also has practical application value.