PCR method for identifiying a serotype of
The present invention relates to a method of identifying a serotype of Klebsiella pneumoniae , in particular to a method using specific polymerase chain reaction (PCR) primer sets designed according to a fragment of a capsular polysaccharide synthesis (cps) region to identify a K57 or a NTUH-N1 serotype and its application. NTUH-N1 is a novel serotype which differs from the previously reported 77 serotypes. This PCR-based cps genotyping method not only solves the problems of insufficient specificity and sensitivity caused by conventional immune method, but can be applied in clinical diagnosis with the advantages of rapidity and low cost. In addition, the rate of unidentifiable strains can also be reduced by this method.
1. A method of identifying a serotype of Klebsiella pneumoniae , the method comprising:
(1) extracting DNA from a sample of Klebsiella pneumoniae for use as a DNA template;
(2) performing polymerase chain reaction (PCR) using the DNA template obtained from Step (1), and pair sets of PCR primers comprising a primer set selected from the group consisting of a first primer set, a second primer set, a third primer set, a fourth primer set, a fifth primer set, a sixth primer set, a seventh primer set and a eighth primer set; and
(3) identifying a K57 serotype of Klebsiella pneumoniae if PCR products are found from the first or the second primer sets, or identifying a NTUH-N1 serotype of Klebsiella pneumoniae if PCR products are found from the third, the fourth, the fifth, the sixth, the seventh or the eighth primer set;
wherein the first primer set is consisting of SEQ ID NO:21 and SEQ ID NO:22, the second primer set is consisting of SEQ ID NO:23 and SEQ ID NO:24, the third primer set is consisting of SEQ ID NO:25 and SEQ ID NO:26, the fourth primer set is consisting of SEQ ID NO:27 and SEQ ID NO:28, the fifth primer set is consisting of SEQ ID NO:29 and SEQ ID NO:30, the sixth primer set is consisting of SEQ ID NO:31 and SEQ ID NO:27, the seventh primer set is consisting of SEQ ID NO:32 and SEQ ID NO:33, and the eighth primer set is consisting of SEQ ID NO:34 and SEQ ID NO:35.
2. The method as claimed in claim 1 , wherein a Klebsiella pneumoniae A1517 (BCRC No. 910412) is identified with the method of claim 1 as the NTUH-N1 serotype.
3. A primer set of identifying a NTUH-N1 serotype of Klebsiella pneumoniae comprising DNA of a NTUH-N1 serotype of Klebsiella pneumoniae for use as a template to obtain to a fragment of a capsular polysaccharide synthesis (cps) region of the NTUH-N1 serotype of Klebsiella pneumoniae through PCR, wherein the primer set is selected from the group consisting of a third primer set, a fourth primer set, a fifth primer set, a sixth primer set, a seventh primer set and a eighth primer set, wherein the third primer set is consisting of SEQ ID NO:25 and SEQ ID NO:26, the fourth primer set is consisting of SEQ ID NO:27 and SEQ ID NO:28, the fifth primer set is consisting of SEQ ID NO:29 and SEQ ID NO:30, the sixth primer set is consisting of SEQ ID NO:31 and SEQ ID NO:27, the seventh primer set is consisting of SEQ ID NO:32 and SEQ ID NO:33, and the eighth primer set is consisting of SEQ ID NO:34 and SEQ ID NO:35.