Research On The Application In Diagnosis Of Fluorescent Quantitation Polymerase Chain Reaction Of Paratyphoid A

Objective:Through observing the positive detect rate of hemoculture, Widal reaction,lipopolysaccha-ride passive hemagglutination assay(LPS-PHA) and fluorescent quantitation polymerase chain reaction(PCR) in those fever patients who have highly suspected infectious with salmonella paratyphi A.We explored the clinical value of early diagnosis of fluorescent quantitation PCR regarding paratyphoid A and evaluated its specificity,sensitivity and repeatability.Methods:(1) We used HiBind memb ground substance which was made up with gel silica materials binds centrifugalization pillar technique to extract the DNA of salmonella paratyphi A, Salmonella paratyphi B,Salmonella paratyphi C,Salmonella typhi,bacterium coli, pseudomonas aeruginosa and Shiga bacillus respectively.Then we added specificity primer and probe of salmonella paratyphi A flagella antigen gene in order to carry out fluorescent quantitation PCR.We carried out sensitive and repetitive reasereh simultaneously in the same condition.(2) The group of 30 fever patients who were highly suspected infectious with paratyphoid A is called paratyphoid A group.The four methods of hemoculture,Widal reaction,LPS-PHA and fluorescent quantitation PCR were used to detect the typhoid A group.Using the method of fluorescent quantitation PCR to detect the control group which was made up of 11 fever patients from other reasons.Results:(1)The result of fluorescent quantitation PCR of salmonella paratyphi A standardization strain was postive.Other six standardization strains were passtive.(2)We carried out fluorescent quantitation PCR with the dilution of DNA of Salmonella paratyphi A standardization strain.The lowest detection line was 1×10~2Copies/ml.DNA of salmonella paratyphi A standardization strain was diluted in three concentrations of 1×10~8,1×10~7,1×10~6Copies/ml in order to carry out fluorescent quantitation PCR.The testing results show:1×10~6Copies/ml 22.22±0.50; and 1×10~7Copies/ml:17.49±0.34;1×10~8Copies/ml:12.69±0.18.(2) We used the method of fluorescent quantitation polymerase chain reaction which detected 30 fever patients who were highly suspected with paratyphoid A,There were 22 positive patients in the paratyphi A group andll patients that had negative results in the control group.The two groups have statistical significance.(P=0.000).The positive rates of four testing methods are27%(8/30),23%(7/30),17%(5/30) and 73 %(22/30) respectively in the paratyphi A group.The sensitivity of fluorescent quantitation PCR was significanly higher than of hemoculture(both P＜0.0083),and no statistical significances were found in the difference of sensitivity among hemoculture,Widal reaction and LPS-PHA.(both P＞0.0083).(3)Four testing methods were used to analize 12 patients in the first week,they were infected with positive rates being 25%(3/12),17%(2/12),17%(2/12)and 67%(8/12)respectively. The four methods have obvious differemces with statistical treatment(P＜0.05).Conclusion:Fluorescent quantitation PCR is a quick,sensitive and specific diagnostic method.It is better than traditional laboratory methods,for example, hemoculture,Widal reaction and LPS-PHA.It supplies an objective proof for early diagnosis of paratyphoid A.