Preparation of recombinant antigen of O. tsutsugamushi Ptan strain and development of rapid diagnostic reagent for scrub typhus.

Spring scrub typhus has frequently occurred in Pingtan Island, China, since 2000. In this study, we amplified a 1352-bp DNA fragment encoding a truncated 56-kDa outer membrane protein of the Ptan strain, which was isolated from a serum sample of a patient with spring scrub typhus, and cloned it into the pET28a vector for expression. The expression product was a recombinant polypeptide containing a His-tag to facilitate purification on a Ni2+ chromatography column. The recombinant protein was further identified by Western blotting and enzyme-linked immunosorbent assay (ELISA) and appeared to be a good diagnostic antigen candidate. A rapid colloidal gold immunochromatographic assay (CIA) for detecting serum total antibodies, IgG and IgM, which are anti-Orientia tsutsugamushi, was developed, using a mixture of the r56 of the Gilliam and Ptan strains as the diagnostic antigen. CIA performance was tested on a panel of 112 control sera from confirmed cases of scrub typhus. The detection sensitivities of CIA against anti-O. tsutsugamushi total antibodies, IgM, and IgG were 98.2%, 81.2%, and 94.6%, respectively, while that of IFA (using the lysate of the O. tsutsugamushi Gilliam-infected chicken yolk sac as the antigen) against IgG was 85.7%. One hundred five serum samples from healthy individuals and patients with other febrile diseases were tested with CIA as negative controls. Specificities of CIA against anti-O. tsutsugamushi total antibodies, IgM, and IgG were 98.1%, 100%, and 98.9%, respectively, while the specificity of IFA against IgG was 98.9%. These results indicated that CIA was a good assay and could substitute for conventional immunofluorescence assays for diagnosis of scrub typhus.

Design and development of a DNA array for rapid detection and genotyping of seven kinds of pathogenic microbes.

A DNA array for rapid detection and genotyping of the pathogenic microbes of epidemic hemorrhagic fever, tsutsugamushi disease, leptospirosis, malaria, schistosomiasis, cholera, and hemorrhagic colitis was developed. The specific and relatively conserved PCR primers and DNA probes were screened from the characteristic genes of the pathogenic microbes. The PCR or RT-PCR methods were established for amplifying and labeling the DNA fragments of the pathogenic microbes. All the probes with the same Tm value were synthesized chemically and modified with an NH2 at their 5' terminus, they were printed on glass slides for fabrication of a oligonucleotide DNA array. The developed DNA array could be used for detecting and genotyping the pathogenic microbes simultaneously, and they had a high sensitivity and specificity.

[Proliferation and location of Hantaan virus in gamasid mites and chigger mites, a molecular biological study].

OBJECTIVE: To study the proliferation and location of Hantaan virus (HV) in gamasid mites and chigger mites and the significance of gamasid mites and chigger mites as vectors of transmission of hemorrhagic fever with renal syndrome (HFRS). METHODS: Gamasid mites collected from the nests of wild rodents and chigger mites collected from the bodies of wild rodents and thick growth of grass in the field were raised. Two oligonucleotide primers were developed based on the gene fragments of cDNA of HV 76 - 118 strain to be used in RT-PCR. RNA was extracted from the suspension of the gamasid mites from the nests where rodents no HV had been found in whose lungs lived and from the unfed larvae of chigger mites, being formed groups of 5, 10, 30, or 50 individuals. RT-PCR was conducted to detect the HV-RNA in such suspension. The larvae, nymphs, and imagines of both kinds of mites were ground to make suspension at an interval of 20 days. Vero-E(6) cells were inoculated to measure the titer of 50% tissue culture infective dose (TCID(50))/ml of HV. Frozen sections of larvae, nymphs, and imagines of both kinds of mites were made. RT-PCR and in situ hybridization were conducted to detect the distribution of HV-positive particles. Monoclonal antigen technique was used to compare the antigenicity of the HV-RNA from the rodents, mites, and patients from the same epidemic areas. RESULTS: HV-RNA was detected in gamasid mites and chigger mites. Except in the larvae of chigger mites 60 days after collection, titers of HV were detected and increased gradually in mites at different stages of life cycle. HV-RNA positive particles were detected in the epithelial cells of midgut and ovary, with the signal denser and more numerous in nymphs than in larvae. The genotypes of HV from rodents, mites and patients in the same endemic areas were identical: HTN type virus. CONCLUSION: HV can be transmitted transstadially and proliferated in mites, gamasid mites and chigger mites play the role of vectors of transmission for HFRS.