Evaluation of nested polymerase chain reaction in detection of pathogens in cerebrospinal fluid

ObjectiveTo evaluate the feasibility of detecting pathogens in cerebrospinal fluid by nested polymerase chain reaction (PCR), and provide the reference for rapid and accurate diagnosis of diseases in clinical practice.

MethodsA nested-PCR was designed by using the bacterial 16S rRNA gene sequences, including two PCR amplification processes by using two pairs primers. The bacterial DNA, which was extracted from cerebrospinal fluid, was amplified firstly by PCR using first pair primers. And the second PCR amplification was followed using the second pair primers. The DNA template was the first PCR amplification products. The products of the second PCR amplification were sequenced. The pathogen was determined by comparing and analyzing the sequences. The concentration of pure Brucella DNA was determined by DNA spectrophotometry. The sensitivity of nested-PCR was evaluated by using serial dilutions of DNA template. Two-fold serial dilutions were prepared from bacterial suspension.

ResultsThe minimum detection limit of nested-PCR was about one copy number of DNA. The cerebrospinal fluid samples of clinical patients were detected by using nested-PCR. In 40 clinical cerebrospinal fluid samples, about 1 460 bp electrophoresis strips were detected in 37 samples. The sequencing results indicated that 7 cases were Neisseria meningitidis, 1 case was Pseudomonas alcaligenes infections, 22 cases were Pseudomonas poae infections. 2 cases were Stenotrophomonas maltophilia infections. 1 case was Streptococcus pneumoniae infection. 4 cases were unknown bacterial infections, and 3 cases had negative bacterial DNA detection results.

ConclusionThe nested-PCR can be used in rapid detection of bacterial pathogens in cerebrospinal fluid with accurate results.