Detection of Brucella abortus by alkB and IS711 based primers
Abstract
BACKGROUND: Brucellosis is a zoonotic disease, which involves both animals and human. Although the conventional methods have been widely used for its laboratory diagnosis, the PCR techniques have proved to be useful due to specificity, sensitivity and the rapidness. Various target sequences of brucella bacterium such as OMP2, 16s RNA and IS711 have been used for the primer designing.. All primer sets have shown different sensitivities and specificities. In present investigation, PCR protocol and primer designated based on IS711 and a fragment of chromosomal DNA all were optimized with standard genome and clinical samples.
METHODS: Numerous tissue samples (liver, kidney, lymph node, and uterus) were prepared and were cultured by the bacteriological standard methods along with the serology positive human samples. PCR protocol was optimized and the primer's sensitivity and the specificity were checked using pure genome of B. abortus. All samples were tested by the standard bacteriological methods. The samples were then subject to PCR amplification and the PCR product was confirmed using the RFLP technique.
RESULTS: The culture results indicated a poor sensitivity as it was previously reported. The PCR product 157 bp was observed on the agarose gel indicating that significant number of clinical samples (human brucellosis cases) were positive by PCR but not by the culture method. Although B. abortus DNA was detected in all the culture positive veterinary specimens, some cross-reactions with close related bacteria were observed that might influence the interpretation of the results.
CONCLUSIONS: The sensitivity of the present PCR protocol was significantly higher when alk B and IS711 based primers were used in compare to each of the alkB and IS711 based primers alone. More research will be needed to improve the specificity and sensitivity of the PCR protocol before recommending for routine laboratory works.
KEY WORDS: Brucella, PCR, RFLP, brucellosis.