关键词: 布鲁菌病；追踪随访；单一PCR实验室方法

Abstract:

Objective
To construct single PCR assay for brucella, and optimize reaction conditions through simulative blood samples and inoculated mice, and provide laboratory evidence for PCR assay application in clinical diagnosis of brucellosis following-up. Methods Primer BP26 on genus level according to BP26 gene and three primers on strain level according to IS711  were designed; simulative samples of serial concentrations were  constructed  and mice were inoculated with M5 or Pps858-MCS2/M5 as experimental group, while PBS as control; brucella DNA was extracted by boiling method. This PCR assay was used to detect bacteria solution with primers BP26 and three strain primers,simulative samples  and  inoculated  mice  were diagnosed with primer BP26 and strain primer Meli. Results  Designed primers on genus and strain level showed good specificity through brucella standard strains and vaccine strains;  DNA was extracted from simulative samples and blood samples of the inoculated mice by the boiling method and  specific bands were obtained; the PCR positive numbers of the inoculated mice increaseed early and desceased later, and increasing sample quantities  increased the  positive rate; the PCR positive rate of the boiling method was  higher than chemical method at the same time point, and the PCR positive rate was  more sensitive than traditional culture method. Conclusion The boiling method is convenient to extract DNA from blood. The PCR assay demonstrates higher sensitivity than traditional culture method; this PCR assay is very specific and available to be used for following-up of brucellosis patients.

Key words: Brucellosis；follow-up, single PCR laboratory method