Development of type-specific PCR for typing Pneumocystis carinii f sp hominis based on nucleotide sequence variations of internal transcribed spacer regions of rRNA genes

The nucleotide sequence variations in the internal transcribed spacer region I (ITS1) and region 2 (ITS2) of rRNA genes were found to be useful for typing Pneumocystis carinii isolates that infect humans. Two types of ITS1 (A and B) and three types of ITS2 (a, b, and c) sequences have been found, and P. carinii isolates are classified based on sequence types of ITS1 and ITS2 as Ax or Ex (where x may be a, b, or c). Type determination has been achieved by sequencing the ITS regions or by reacting the ITS regions amplified by PCR with type-specific oligonucleotide (TSO) probes. However, TSO typing alone does not work on a specimen from an individual who is infected by more than one strain of P. carinii where different ITS1 types are present in the same specimen. In this study, type-specific PCR assays were developed to supplement TSO typing. Type-specific PCR primers were made so that they differ at their 3' ends by the two nucleotides which distinguish type A from type B of ITS1 plus an additional ''A'' residue at the extreme 3' ends of the primers. These two primers were paired separately with a general primer which anneals to a region downstream from ITS2 to specifically amplify Ax or Ex. The amplified products were then reacted separately with ITS2-specific probes 2-a, 2-b, and 2-c to identify their types.