Hantaviruses RNA Detection In Sera Of Patients With HFRS By Microplate Hybridization

Hemorrhagic Fever with Renal Syndrome(HFRS) is an acute infectious disease caused by Hantaviruses (HV). It's clinical characteristics include high fever, hypotension, bleeding and renal damage. It is wellknown that HFRS spreads all over the five continents. There is a serious epidemic situation in China and it is a difficult problem to prevent and control the disease. Since an early diagnosis is actually often imposseble, which sometimes will lead to the deterioration of the illness or life-threaten. It is imperative to find a sensitive and reliable experimental method for HV marker detection. At present , thediagnositic method based on anti-HV antibody detection by IFA and Mac-ELISA is prevalent, but serologic result is only an indirect evidence of HV infection and is often false negative in first five days within the disease course. We can detect directly HV RNA from some patients' sera by RT-nested PCR , but in the sera of most earlier sufferers the viruses level is too low to detect and it limits the application of RT-nested PCR. Therefore we attempt to set a new method, microplate hybridization, which is more sensitive than routine RT-PCR. The results were as follows:1. It took only about seventy minutes for RNA extracting. Both RNA extraction and reverse transcription were finished in a single tube, that means less RNA loss and less contamination.2. The method of RT-nested PCR was founded, and the sera samples from 98 HFRS patients (IFA positive) were detected, the positive rates were 71. 8%( ^ 7days) , 62. 8%(8-14days) , 33.3% (15-21days) and 0% (^22 days) respectively and the total positive percentage rate was 59.2%.3. The amplified products were analized by restriction enzyme digestion and 48 positive amplified products from PCR were typed by restriction enzyme digestion. It showsthat 47 were type I and only 1 was type II and it suggests the predominant type of HV in Xi'an was type I , which is similar with the result of epidemiological investigation before.4. The sera samples were detected by double labeled primers and RT-nested PCR, then enzyme-linked immune sorbent assay (ELISA) was conducted. The positive rates were 85. 7%(^7daysK 79.1% (8~14daysK 78.6%(15-21days) and 66.7% ( ^ 22days). The total percentage was 80.6%.5. The HV RNA can not be detected in sera samples from 5 health persons , 2 patients with fever but non-HFRS and 3 patients with hepatitis B by microplate hybridization .6.To confirm the reliability of microplate hybridization the sera samples from 108 HFRS patients (IFA positive)were tested. The positive rates of detection were 87. 2%(^7days) , 83.7% (8-14days) > 73.7%(15-21days) and 57. 1%(^22days) repectively, the total percentagewas 81.5%. All samples that are positive by RT-PCR also are positive by microplate hybridization. It suggests that the microplate hybridization is a more sensitive andspecific method for detecting Hantavirus RNA than RT-PCR.