| Objective:Establishing two high efficient nucleic acid detection methods for detecting Babesia spp.:real-time fluorescent PCR and droplet digital PCR.Then the two methods were evaluated and applied to detecting the blood samples.The blood samples of voluntary blood donors in Mudanjiang City were detected by indirect immunofluorescence(IFA),real-time fluorescence PCR(Q-PCR)and digital PCR(ddPCR)respectively and the results were used evaluating the risk of blood transfusion safety in Mudanjiang city.Methods:Method establishment:IFA testing method in our laboratory was established with the help of American Red Cross(ARC)Holland Laboratory.Downloading the 18S rRNA conservative gene sequence of Babesia spp.from GenBank and the primers were designed according the common part of the sequence.Then the positive plasmid were synthesized and verified in proper order.And the repeatability and sensitivity were evaluated after optimizating the experimental condition.Blood samples pretreatment:The whole blood samples of 1971 voluntary blood donors were centrifuged and separated into two parts with red blood cells and plasma.Then mixed the red blood cells with 10 samples/pool and kept all of them in-20 ? for reserve.Screening:IFA test:in this part,1000 of 1971 blood donors' plasma samples were detected.Q-PCR test:all 1971 samples of red blood cell were detected by mixed sample and the positive one were spliting detection;the samples of IFA positive blood donors were tested by single sample.ddPCR test:1000 samples of 1971 red blood cells were detected by mixed sample and the positive one were spliting detection;the samples of IFA positive blood donors were tested by single sample.Results:The methods of Q-PCR and ddPCR have been successfully established.Both methods have good reproducibility,but the sensitivity of ddPCR is about 10 times higher than that of Q-PCR.A total of 13 positive samples were detected by IFA in 1,000 plasma samples,of which 8(0.8%)were 1:64 titer positive and 5(0.5%)cases were 1:128 titer positive.The statistical analysis of demographic information of positive samples showed that there was no statistical difference in ethnic,sex,age and other most groups(P<0.05).The results of Q-PCR testing were all negative.A total of 2 positive samples were detected by ddPCR in 1,000 samples of red blood cells.And the statistical analysis of demographic information wasn't made for the few positive samples.Besides,2 cases out of 13 samples with positive IFA were positive for ddPCR nucleic acid,both 2 cases were positive for antibody titer of 1:128.Conclusion:The two methods for the detection of Babesia spp.established in this study could be directly used relavant reaserch,such as blood donor screening,epidemiological studies,clinical diagnosis,monitoring and so on.The B.microti serological infection was found in Mudanjiang City for the first time;the Babesia spp.serological positive rate of voluntary blood in Mudanjiang City was 1.3%and the nucleic acid positive rate was 0.2%.The results showed that Babesia spp.did pose a threat to the blood safety in the region,suggesting that local blood donors should be consulted and screened.IFA is an effective detection method which has high specificity and sensitivity.However,the development of ELISA detection kit is also a significant and valuable research direction.The area which is similar with Mudanjiang City in Babesia spp.epidemiology,like Yunnan province,Zhejiang province and Xinyang City,it is suggested that a survey of Babesia spp.in blood donors should be carried out in tick epidemic season to assess its risk to local blood transfusion safety. |
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