Establishment and application of a qPCR method for differential detection of Brucella S2 vaccine strain.

Abstract

Background: Brucellosis is one of the most serious zoonotic bacterial diseases in the world. The disease has caused serious harm to people and livestock, hindered the healthy development of the breeding industry, and led to serious economic losses.At present, the prevention and control of this kind of disease is still based on vaccine immunization. However, after the widely used vaccine is inoculated to livestock, there is no widely used differential diagnosis method to distinguish vaccine immune antibodies from natural infection antibodies. Quarantine and purification work is difficult to carry out. In addition, there are few studies using real-time PCR(qPCR) methods in the differential diagnosis of natural virulent strains and vaccine strains of brucellosis.The purpose of this study is to establish a rapid, sensitive and accurate differential diagnosis method for Brucella S2 vaccine strain, and to solve the problem of lack of identification of Brucella S2 vaccine strain and natural virulent strain in clinical detection.It avoids the killing of some livestock due to the positive antibody of the Brucella S2 vaccine strain, and can also identify sick animals from immune herds, reducing the economic losses of farms, and providing certain technical support for the quarantine and purification of epidemic diseases.

Results: In this study, combined with TaqMan probe-based qPCR technology, specific primers and probes were designed according to the specific deletion genes of the Brucella S2 vaccine strain,which could be used as marker genes.The qPCR and duplex qPCR detection methods of Brucella were successfully established.The method has good specificity, sensitivity and repeatability, the lowest limit of detection can reach 1 × 10¹ copies/μL, the sensitivity is about 100 times higher than that of conventional PCR, and there is no cross-reaction with Escherichia coli,Salmonella,streptococcus and other common strains.The coefficient of variation between groups was less than 0.6%, and the coefficient of variation within groups was less than 0.55%.Subsequently, this method was used to monitor the antibody levels in goat inoculated with different doses of Brucella S2 vaccine strain, and the method could also detect the corresponding nucleic acid signals in goat milk samples, and the clinical samples were detected. In summary, this method has good specificity, sensitivity and repeatability, and can be used for the differential diagnosis of clinical brucellosis.

Conclusions: This study successfully established a duplex qPCR detection method for the differential diagnosis of the Brucella S2 vaccine strain. From the establishment of the method to the clinical application of the method, it shows that the method can be used for the differential diagnosis of clinical brucellosis.