[Application of multiplex semi-nested polymerase chain reaction in detection of pathogens in cerebrospinal fluid].
OBJECTIVE
To establish a new method of multiplex semi-nested polymerase chain reaction (PCR) to detect pathogens in cerebrospinal fluid (CSF).


METHODS
According to the analysis of the conservative and variable regions in bacterial 16S rRNA genes, we designed universal primers for all bacteria and specific primers for most gram-positive and gram-negative bacteria. All primers were added into the same reaction systems successively of a two-step PCR assay to amplify the different bacterial DNA in CSF, and the results were compared with common culture method with sensitivity and the specificity both detected at the same time.


RESULTS
Both gram-positive and gram-negative bacteria amplified DNA fragment about 1,032 bp after first-step amplification with universal primers. In the second step, specific fragments of 336 bp and 127 bp were amplified in gram-positive and gram-negative bacteria respectively besides fragments of 1,032 bp; The detection limit for E. coli was 8 cfu/ml. The comparison of 62 CSF samples detected by both multiplex semi-PCR and conventional culture method revealed sensitivity, specificity, positive and negative values of 93.8%, 95.7%, 88.2%, and 97.8% respectively for PCR.


CONCLUSIONS
The result suggested that the multiplex semi-nested PCR we established was sensitive, specific and rapid method for clinical laboratory to detect pathogens in CSF.
Yan
Zhi-yong
ZY
Department of Medical Microbiology, Qingdao University Medical College, Qingdao 266021, China.
Wang
Bin
B
Bi
Chun-xia
CX
D002555:Cerebrospinal Fluid / Q000382:microbiology*; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D004269:DNA, Bacterial / Q000134:cerebrospinal fluid* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000302:isolation & purification*; D006094:Gram-Positive Bacteria / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S