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