The Early Diagnosis Of HFRS With PCR-ELISA

Objective: To establish a rapid, sensitive and specific method to detect and identify HFRS by a liquid phase hybridizations for PCR-ELISA.Methods:1 The patients sera samples which were diagnosed as HFRS clinically were detected for IgG antibody in sera by indirect immunofluorescence assay(IFA) .The patients sera samples were collected from the local hospitals of HeBei province.2 the position rats lungs by IFA were made virus supernatant after rinising, grinding, filtration .3 The total RNA in Hantan virus was extracted from virus supernatant or HFRS patients sera which had been dectected positive by IFA, primer was designed by refering to HV consensus sequence and the cDNA which depended on total RNA was got through inverse transcribe.4 The samples were amplificated by RT-nested PCR while the upstream primer was labled by biotin;5 The results of amplification were detcted by agarose gel electrophoresis and the product of amplification was solution hybridization with probe labeled digoxin, then the product of hybridization was fixed in the microwell plate which embeded by streptavidin, conjugated with Anti-Digoxigenin-AP and added substrate for the microwell plate coloration, at last the results were identified .Results:1 Establishment experimertal methods1.1 Establishment the best concentration in reaction Refer to the related literature , the best reaction condition were determinded by square matrix.Detertminded the value of OD of different concentration and different material. Ratio between the OD value of virus supernatant and the OD value of blank control ,the best condictions were determined by the maximal of ratio. Finally the best conditions were that concentration of streptavidin was 10 mg/L,concentration of prober was 1.5μmol/L,dilution of Anti-Digoxigenin-AP was 1:2500.1.2 Determination of the time of reactionThe process of the amplifation by PCR-ELISA, the color varied at last by eye ,while the time of incubate increased , the color grow deeper ( from the colorless turned to yellow ) .Prolonging the incubate time , the value of OD increase. The last value in this experiment were that the value of positive were between 0.8～2.0. The blank value were lower than 0.2, and the difference value between the value of the positive and the value of the blank which determined the incubation time .This experiment the incubation time was an hour.1.3Three blank were used control , calculate the average and standard deviation of the value of blank .Cut- off was the average value add tripling standard deviation. In the experiment if the value of OD of samples exceeded than Cut-off , then the sample was determinded positive .If the value of OD of samples lower than cut-off , then the sample was determinded negative. If the value of the sample was nearby the Cut-off, then did three times and the final value wasdeterminded by average of the three OD value.2 Methodology evaluation2.1. The result was ,the amplification products diluted 1000 times the results by microwell plate hybridisation was determinded positive which by agarose gel electrophoresed the motive stripe was turned weaker when the amplification products diluted only10 times , the sensitivity of microwell plate hybridisation was 100 times than that of agarose gel electrophoresed.2.2 The effects of all samples were negative in 20 sera samples of healthy subjects,16 sera samples which were negative by IFA, 2 sera sample of patients with hepatitis had been detected by RT-nested PCR at the same time , the motive strip were not appeared. Then the amplifated products crossed with the probe labling digoxin , the product of hybridization was fixed in the microwell plate which embeded by streptavidin, conjugated with Anti-Digoxigenin-AP and added substrate for the microwell plate coloration, at last the hybridization results were identified .When the OD value lowed the Cut-off value ,the effects were evaluated as negative.The specificity of the method was good when HFRS was detected at earlier period.2.3 The repeativity of microwell plate hybridisation.4 times PCR-ELISA were carried for the same virussupernatant at the same time ,the mean in one group was 1.214, the standard deviation was 0.076, coefficient of variability was 0.063; the same virus supernatant was detected for 6 times in different time such as at the 1st day ,2nd day, 7th day, 1st month, 3rd month and 6th month, the trial mean value in different group was 1.484, the standard deviation was 0.311, coefficient of variability was 0.210. When virus supernatant was used as the sample, the reproducibility was better.3 detection methods and application3.1 89 blood samples which had diagnosed as HFRS inclinical were collected from the different hospitals in 2007 in HeBei, there were 31 positive samlpes by IFA for IgG antibody in sera.3.2 PCR detection of HFRS patients'sera in different course.31seropositive which diagnosed as HFRS clinically were detected and amplification was carried byⅠtype primer andⅡtype primer, then the results were observed by gel electrophoresis agarose. 5 blood sera had apperaed motive strip when usedⅡtype primer, the ratio of positive was 16.13%, while all smples which usedⅠtype primer were not appeared motive strip.3.3 PCR-ELISA detection of HFRS patients'sera in different course.Then the amplifated products crossed with the probe labling digoxin , the product of hybridization was fixed in the microwell plate which embeded by streptavidin, conjugated with Anti-Digoxigenin-AP and added substrate for the microwell plate coloration, at last the hybridization results were identified .The amplification products were detected by ELISA. The OD value of 9 sera which usedⅡtype primer exceeded the Cut-off value, so the 11 sera were evaluated as positive.The ratio of positive was 38.71%.3.4 PCR-ELISA was compared with PCR of HFRS patients'sera in different course.11 sera positive by PCR-ELISA were used byⅡtype primer, in which 5 sera appeared motive stripe by agarose gel electrophoresed that showed two methods were quite consistency.Conclusion:1 This experiment applied successfully to found the method of early dignosed for patients with HFRS by PCR-ELISA through optimization of experimental conditions.2 Sensitivity of detection virus supernatant by PCR-ELISA is 100 times than PCR. The specificity and the repeatability of the method are good.3 The positive ratio of detection patient'sera by PCR-ELISA is higher than PCR especially the early diagnosis HFRS patients.4 PCR-ELISA can used in early diagnosis of HFRS patients,but the methods need further improvement.