TaqMan Real-Time PCR For Detection Of Neisseria Meningitidis, Haemophilus Influenzae, And Streptococcus Pneumoniae

Bacterial meningitis is a serious infection affecting the central nervous system.It arises rapidly,severely and can lead to high fatality rate without timely treatment.The three major pathogens causing bacterial meningitis are N.meningitidis,H.influenzae and S.pneumoniae.At present,the diagnostic methods that are usually used to diagnose bacterial meningitis are culture and latex agglutination.But extensive using of antibiotics makes culturing very difficult and latex agglutination methods are not enough sensitive and can not detect all N.meningitidis serogroups.So bacterial meningitis has been clinically confirmed without pathogenic results in our country.Traditional PCR method detects specific genes of different pathogens in suspicious specimens and can be used to aid diagnosing of bacterial meningitis.Compared to traditional PCR method,real-time PCR method is more sensitive,specific,and rapid throughput.In recent years,real-time PCR method has been focused on and applied to detect the pathogens causing bacterial meningitis.In this study,real-time PCR method was established for detecting multiple pathogens causing bacterial meningitis.Nine sets of primers and TaqMan probes were designed and synthesized of which three were applied to detect specific genes of N.meningitidis, H.influenzae,and S.pneumoniae species respectively:ctrA(Nm),bexA(Hi),and lytA (Sp);the other six were for detecting N.meningitidis serogroup specific genes including sacB(serogroup A),siaD(serogroup B),siaD(serogroup C),xcbB(serogroup X),synF (serogroup Y) and synG(serogroup W₁₃₅).Reaction concentrations of each set of primers and probe were optimized.Detection results of different isolates showed that the specificities of all nine sets of primers and probes are high among species or serogroups.The sensitivity of each primer set was determined by detecting genome DNAs copies. It is 8 genome copies for ctrA,10 genome copies for bexA,90 genome copies for lytA,8 genome copies for sacB,80 genome copies for siaD(serogroup B),8 genome copies for siaD(serogroup C),8 genome copies for xcbB,80 genome copies for synF and 8 genome copies for synG.Real-time PCR is 10¹ï½ž10² times more sensitive than standard PCR on detecting N.meningitidis species and serogroup A,B,C,Y,W₁₃₅.In this study,the cut-off value of cycle threshold(Ct) were determined.TaqMan real-time PCR and latex agglutination test were simultaneously applied to detect 282 CSF specimens from bacterial meningitis cases.The results were compared with bacterial culture.Forty seven of 282 CSF specimens were positive with at least one method.All culture or latex agglutination positive specimens except one from which N.meningitidis was isolated were positive with real-time PCR.There were four specimens from which two pathogens were simultaneously identified by real-time PCR. H.influenzae was isolated from one of the four specimens and the other three were negative by either culture or latex agglutination.Real-time PCR was also tentatively used to detect blood specimens,but no positive result was obtained.Real-time PCR method established in this study could specifically detect N.meningitidis,H.influenzae,and S.pneumoniae and identify main pathogenic serogroups A,B,C,X,Y and W₁₃₅ of N.meningitidis.With more sensitivity and rapidness,This method can improve the diagnostic rate of suspicious meningitis.