Evaluation of PCR-based diagnostic methods for identifying Babesia in ticks
Objective To evaluate 4 PCR-based diagnostic methods to detect Babesia in ticks from dogs and cattle,respectively.Methods Specimens of ticks were collected from 62 counties of 6 municipals in Guangxi Zhuang Autonomous Region.The 18s rDNA genes of Babesia were specifically amplified by nested PCR.The PCR products of positive samples were purified,sequenced and blasted in GenBank.Duplex PCR and duplex nested PCR,duplex Loop-mediated isothermal amplification (LAMP) were evaluated for detection of Babesia bigemina and B.bovis infections in cattle.Multiplex PCR was evaluated for B.canis canis,B.canis vogeli and B.canis rossi in dogs.The comparison of the diagnostic methods results was taken to evaluate the performance of each method.Results A total of 102 tick samples was collected from cattle.No positive sample was found by nested PCR,duplex PCR and duplex nested PCR,while 5 and 29 positive samples were detected by duplex LAMP of which results determined by turbidity reader and visual color change,respectively.The 5 positive samples by turbidity reader are also positive by visual color change.184 tick samples were collected from dogs.9 B.canis vogeli,positive samples were detected by nested PCR.4 of the 9 samples were B.canis vogeli,positive by multiplex PCR.5 B.canis rossi were determined by multiplex PCR,while sequencing results were totally negative.Conclusion Methods of duplex PCR and duplex nested PCR for bovine babesiosis,showed high specificity,with no false positive claimed.The sensitivity should be further evaluated for the next step due to the lack of positive specimens.Positive rates of 15.76％ and 2.72％ were determined by duplex LAMP with turbidity reader and visual color change,respectively.While 2 of the 29 visual color change were B.microti positive by nested PCR.Despite of easy and quick operation,positive results should be confirmed.And contamination should be prevented efficiently due to the high sensitivity of LAMP.Though the diagnosis of B.canis,B.vogeli,B.rossi infections in dogs could be made at the same time by multiplex PCR,considering the false positive and false negative,it is unreasonable to do so.It is suggested that the detection should be carried individually.
NETL
NSTL
万方数据
维普
