The Establishment Of Multiplex PCR Method And Detection Kit For Brucella Abortus、Brucella Melitensis、Brucella Suis And Brucella Canis

Brucellosis was a zoonosis caused by Brucella. It was widely distributed aroundthe world. It has been reported more than170countries and regions had brucellosis inthe world. Since2000, the incidence of brucellosis in humans and animals showed anincreasing trend, which brought great harm to human health and animal husbandrydevelopment.Not only domestic animals but also human could infect with B.abortus、B.melitensis、B.suis and B.canis which were the most serious brucella species. Thephenomenon of various infections caused by brucella species was common in China.With the increasing of cattle and sheep farming, brucellosis epidemic in human anddomestic animals was particularly severe. Swine brucellosis epidemic was not asserve as cattle and sheep brucellosis, but there were much reports about peopleinfected with swine brucellosis. With a huge amount of dog breeding, it has beenconfirmed that dog could infect with Brucella. As dogs were closely contact withhuman, canine brucellosis has been posed a potential threat to humans. Currently, theexisting brucellosis etiological diagnosis methods in our country couldn’t effectivelydistinguish B.abortus、B.melitensis、B.suis and B.canis. Therefore, the study of amultiplex PCR method and detection kit which could simultaneous detectionB.abortus、 B.melitensis、 B.suis and B.canis had important implications forepidemiological investigations and prevention of brucellosis.In this study, we firstly analyzed the gene sequence of B.abortus、B.melitensis、B.suis and B.canis. According to a deletion of2653base pairs in the ABC transporterbinding protein (BR0951-BR0955) of B.abortus and B.melitensis, a deletion of351base pairs in wbkF-wbkD area of B.canis, and the differences of IS711in copynumbers and location of B.abortus and B.melitensis, we designed four pairs ofprimers, which were used to distinguish B.abortus、B.melitensis、B.suis and B.canis bythe size of the fragment amplified by PCR. The optimal multiplex PCR reaction wasdetermined by optimizing PCR reaction conditions, such as annealing temperature、extension time、concentration of primers, then we analyzed the specificity, sensitivityand reproducibility of the multiplex PCR. Moreover, we assembled multiplex PCRdetection kit for B.abortus、B.melitensis、B.suis and B.canis by preparing positive quality control、negative quality control、PCR primers、PCR reaction solution andother components, and evaluated the specificity、 sensitivity、 reproducibility andpreservation of the kit. In order to verify the detection efficiency of the kit, we appliedinitially the kit to detect simulated milk samples, enrichment broth of mice’s spleen、aborted fetuses’s spleen of yaks and small tailed han sheep.We designed four pairs of primers which were verified correctly by single PCRreaction detection, and could be used to multiplex PCR reaction. By optimizingconcentration of primers、annealing temperature、annealing time、 extension time andparameter cycle, the optimum multiplex PCR reaction was established, and weanalyzed the specificity，sensitivity and reproducibility of multiplex PCR. The resultsshowed that the strong specificity of the multiplex PCR which had no reaction withYersinia enterocolitica O:9, E. coli O157:H7and other bacteria that had closelyrelationship with brucella; high sensitivity which sensitivity of B.abortus、B.melitensis、 B.suis and B.canis were respectively1.1×102CFU/mL、5.1×102CFU/mL、2.5×102CFU/mL and3.5×102CFU/mL; the reproducibility of themultiplex PCR method was good with consistent results.According to the multiple PCR method, we assembled multiplex PCR detectionkit for B.abortus、B.melitensis、B.suis and B.canis by preparing positive qualitycontrol、negative quality control、PCR primers、PCR reaction solution and othercomponents. The detection kit had a strong specificity which had no reaction withYersinia enterocolitica O:9, E. coli O157:H7and other bacteria that had closelyrelationship with brucella; high sensitivity which reached102CFU/mL above fordetection of B.abortus、B.melitensis、B.suis and B.canis; good repeatability which hadno difference in the same batch and different batches. The preservation period of thekit was eight months when it was stored at-20℃.At last, we used the kit to detect simulated milk samples whose sensitivity ofB.abortus、 B.melitensis、 B.suis reached1.0×103CFU/mL and B.canis reached1.0×104CFU/mL. Afterwards the kit could accurately detect the spleens of mice whichinfected with brucella species. Furthermore, we used the kit and brucella PCR methodwhich was from enery-exit inspection and quarantine industry standards of thePeople’s Republic of China to simultaneously detect the enrichment broth of theaborted fetuses’s spleen of yak and small tailed han sheep, the brucella PCR methodin the industry standard could only identified to the level of Brucella genus, butcouldn’t distinguish Brucella species. The detection kit we assembled could furtherdistinguish brucella species with high accuracy、quick and easy detection and goodvalue.The above results indicated that the multiplex PCR method of simultaneous detection B.abortus、B.melitensis、B.suis and B.canis had strong specificity and highsensitivity. The utility of the detection kit has been further verified by detectingsimulated samples and clinical samples.