Microsatellite FTA-LAMP And BmSA1 And SRA5 Antigen Diagnostic Techniques

Detection technology is currently a hot spot, key point, and difficult domain in the field of babesiasis control and prevention and scientific research. Pathogenic morphological detection methods of infection with Bahesia are old-fashioned, poor sensitivity, the morphological identification still needs professionals trained and mastered special skills to complete, and easily causes missed and false negative result especially in the low-density parasitemia. In recent years, the most of immune detection techniques for diagnosing babesiosis are in the experimental stage, however, IF AT is currently the only one of the immunodiagnostic teagents commercialized, and difficult to use and spread in the fields, due to need the fluorescence microscopy. In the other hand, molecular detection technology is basically in the experimental stage, more for the research. Immune and molecular diagnostic methods for infection with Bahesia microti are still in the initial stage in our country. And the researchers are still working on diagnostic antigen screening, rapid immune and molecular diagnostic techniques. In this paper, we will develop the early rapid diagnosis method for detecting Babesia microti infection, via diagnostic antigen screening, cloning and expression, and the monoclonal antibody, polycional antibody preparation, detection system optimization, with theories and techniques of modern immunology and molecular biology.Objective:To establish a set of molecular biological technique and immunological methods for early diagnosis of infection with B.microti.Method:1) The gene sequence of the seroreactive antigen 5 (SRA 5) protein obtained via screening the relative proteins of B.microti invading erythrocytes with mass spectrometer was taken by bioinformatics analysis, and cloning and expression of the SRA5 protein were performed. The SRA5 protein purified was used to establish SRA5-based ELISA and colloidal gold test methods for detection of antibodies against B.microti, and to evaluate their diagnostic effect for babesiosis.2) Taking out the cell strains of producing the mAbs of IF 11,2B12 and 6D12 against BmSAI of B. microti from the nitrogen canister, the mAbs of anti-BmSAl were prepared from the ascites of BALB/c mice which were injected with above the cell strains, and the polyclonal antibodies (pAb) of anti-BmSAl were also prepared from the sera of rabbits immunized with purified BmSAI. Via matching and screening of the mAbs and pAb, the MAb-based sandwich-ELISA and colloidal gold immunochrotnatography test strip method were established for detecting circulating antigen of B.microti, and their diagnostic eftects were evaluated.3) According to the published GenBank sequences of B.microti, many pairs of primers were designed targeting the conserved region of Cytochrome B gene of B. microti through the LAMP Real Time Turbidimeter LA-320. A rapid and specific LAMP method for detecting B.microti with optimizing the LAMP primers was established. Meanwhile, the amplified products were colored by Calcein that could be detected with naked eyes, and also detected by agarose gel electrophoresis.Results:1) Establishment of SA5-ELISA method for the detection of 7 days of continuous of mice infected with Babesia microti, found that form 4th that can be detected positive results 70%, and with similar species of Plasmodium, Toxoplasma gondii there is no cross reaction.2) Establishment of SA5-based colloidal gold test strip. For 21 days of continuous infection of mice infected with Babesia microti and it was found that the method can detect the Babesia microti from 4th that can be detected positive results, antibody can be detected at a rate of more than 10%in the early stage of infection, and with similar species of Plasmodium, Toxoplasma gondii there is no cross reaction.30 suspected Babesiasis screened for Changzheng Hospital 5000 sample serum by PCR, whitch diagnosed with colloidal gold test strip method the positive rate was 83,3%.3) The establishment of BmSAl protein with a MAb-based sandwieh-EUSA method and it was able to react with BmSAl and the crude antigen of Babesia microti.the detection limit was O.lmg/uL, However, it is not suitable for the detection of circulating antigens in sera of mice infected with B.microti.4) The establishment of 2B12 monoclonal antibody-based colloidal gold test strip to the test on 21 days of continuous infection of mice infected with B.microti were detected in the peak density of 4-9 days, which the rate of infection is higher than 10%, but before and after that, it could not be detected.5) Establishment of BmSAl-based of colloidal gold test strip. For 21 days of continuous infection of mice infected with Babesia microti and it was found that the method can detect the Babesia microti form 4th that can be detected positive results, and with similar species of Plasmodium, Toxoplasma gondii there is no cross reaction, sensitivity 100%and specificity 100%. 6) FTA-LAMP method for detecting the specific cytochrome B gene of Babesia microti was successfully established, and can detect SBA-ELISA method can detect serum of early infected (7d) mice, which shows no cross reaction with positive serum of Plasmodium falciparum, Plasmodium vivax and Toxoplasma gondii, thus indicating good sensitivity and specialty. ELISA method based on SBAwas established, which could detect the early B.microti infection of mice (at 7d post-infection) with no cross-reaction with the sera of human infected with Plasmodium falciparum, Plasmodium vivwc and Toxoplasma, indicating higher sensitivity and specificity.the mice infected with B. microti, for 21 days of continuous infection of mice infected with B.microti and it was found that the method can detect the B.microti form 1th,The detection limit was 0.687fg/μL higher than PCR, no cross reaction with other parasites.Conclusion:The recombinant proteins of BmSA1 and SRA5 have been successfully prepared with the molecular cloning and expressing techniques, and the mAbs of anti-BmSAl have been also produced. The FTA-LAMP for detecting specific gene of Babesia microti, the sandwish-ELISA and colloidal gold immunochromatography test methods for detecting the specific circulating antibodies and antigens in the blood of the infectors with B. microti have been established, and show more sensitivity, specificity, and early diagnostic valuation for babesiosis, but its diagnostic effects shoμld be further verified in the fields.