RAPID DIAGNOSIS OF TUBERCULOSIS AND OTHER BACTERIAL INFECTIONS BY MULTIPLEX POLYMERASE CHAIN REACTION

Xiang Zhidan1, Min XQ1, Fang F2, Dong YS2

1Department of pediatrics, Medical College of Three Gorge University,

Yi chang, HuBei, China

2Department of Pediatrics, Tongji Hospital,

Tongji Medical University, Wu Han, HuBei, China

 

Objective: To develop a multiplex polymerase chain reaction (PCR) system to detect fragments of 16S rRNA gene (16S rDNA) of tubercule bacilli and other bacteria simultaneously for diagnosing bacterial infections and differentiating tuberculosis from other bacterial infections.

Methods: We designed two universal primers and a tubercule bacilli species-specific primer based on conserved regions and variable region of bacterial 16S rDNA and developed a multiplex PCR system which could amplify 360-bp fragments of the 16S rDNA for general bacteria, and 360-bp and 210-bp fragments for tubercule bacilli. The lowest detectable level, universality and specificity of this system were determined by detecting 20 species of known-cultured bacteria. 82 different clinical specimens including blood (n=42), cerebrospinal fluid n=22, pleural fluid (n=9), peritoneal fluid (n=9), were tested by this assay, compared with the results of  bacterial culture for assessing the sensitivity and specificity of this system for detecting bacteria and tubercule bacilli respectively.

Results: All 19 species of general bacteria could be multiplied for 360-bp fragments, and only tubercule bacilli were detected out two bands of 360-bp and 210-bp fragments. The lowest detectable level of this PCR system was 10fg of DNA for E.coli, and 100fg DNA for tubercule bacilli. Compared with the results of bacterial culture regarded as "gold standard" of diagnosis, this PCR system showed that sensitivity reached 100% for detecting both general bacteria and tubercule bacilli, and specificity was 76% for examining general bacteria and 94% for determining tubercule bacilli.

Conclusion: This multiplex PCR system had excellent universality and specificity. It could detect as low as 10fg of DNA for E. coli and 100fg of DNA for tubercule bacilli. Compared with bacterial culture, it possessed higher sensitivity and specificity. So, it is a novel, rapid and reliable method for diagnosing bacterial infections and differentiating tuberculosis from other bacterial infections.

 

Key words: 16S rRNA gene Multiplex polymerase chain reaction Bacteria Tubercule bacilli

 

 
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