A RAPID AND SENSITIVE PCR STRATEGY EMPLOYED FOR AMPLIFICATION AND SEQUENCING OF PORA FROM A SINGLE COLONY-FORMING UNIT OF NEISSERIA-MENINGITIDIS

The predicted amino acid sequence was determined for the class-1 outer membrane protein, PorA, from a B:15:P1.7,3 strain of Neisseria ia men ingitidis that is currently causing an epidemic of meningitis in North ern Chile. The P1.7,3 PorA showed a unique sequence in the exposed loo p 4 of the putative porin structure that is different from all the rep orted PorA sequences. Based on the nucleotide (nt) sequence of the P1. 7,3 porA, we designed two sets of PCR (polymerase chain reaction) prim ers that specifically amplified porA from any N. meningitidis strain, and a third set of primers that amplified por A only from the P1.7,3 s train. Using these primers, we developed a sensitive double hot-start nested PCR (HNPCR) strategy that could amplify porA and generate nt se quence from as low as a single colony-forming unit. This strategy cons isted of three phases of PCR. The first two phases were designed to ge nerate amplified target DNA that could be directly visualized by ethid ium bromide staining starting from one to two molecules of Neisseria g enome. The third phase was designed to generate a sequence of several hundred nt directly from the amplified DNA. A number of culture-negati ve cerebrospinal fluid samples from individuals suspected of meningiti s during a vaccine trial were analyzed by this strategy to obtain more accurate information on the actual number of cases that occurred in t he study and the non-study populations. The basic HNPCR strategy descr ibed here could be applied to amplify and sequence target DNAs from an y low-copy-number biological sample.