Serotype Prediction for Frequently Isolated Serotypes of Streptococcus Pneumoniae by Heteroduplex Analysis and Modification of This Technique to Real-Time Fluorometric Nucleic Acid Detection System

Abstract

A AB BS S T TR RA AC CT T O Ob b j je ec c t ti i v ve e:: Se ve ral mo le cu lar se roty pe pre dic ti on met hods ha ve be en pub lis hed in or der to simp lify the pne u mo coc cal se roty ping. The aim of this study is to de ve lop a mo le cu lar se roty pe pre dic ti on tech ni qu e al ter na tive to ot her mo le cu lar ap pro ac hes. M Ma a t te e r ri i a al l a an nd d M Me et t h ho od ds s: : S. pne u mo ni a e se roty pes 1, 3, 5, 6A, 7F, 8, 9V, 11A, 14, 15B, 18C, 19A, 19F and 23B, that are the most fre qu ently iso la ted se roty pes from in fec ti ons in Tur key, we re inc luded in the study. S. pne u mo ni a e cpsA and cpsB ge nes that are in vol ved in the pro ces sing, re gu la ti on and ex port of the cap su lar poly sacc ha ri des, we re amp li fi ed by polymerase chain reacsion (PCR). Then, he te ro dup lex analy sis was per for med to the 1.8 kb PCR pro ducts ob ta i ned from all se roty pes with cons tant se roty pe 1. This tech ni qu e was then mo di fi ed to re al-ti me flu o ro met ric nuc le ic acid de tec ti on system. R Re e s su ul lt ts s: : The PCR pro ducts ob ta i ned from seroty pes 1, 6A, 14 and 19F sho wed only ho mo dup lex bands whi le tho se from se roty pes 3, 5, 7F, 8, 9V, 11A, 15B and 23B sho wed spe ci fic he te ro dup lex bands. Alt ho ugh se roty pes 18C and 19A sho wed a spe ci fic he te ro dup lex ban ding pat tern, they co uld not be dif fe ren ti a ted from each ot her. When furt her he te ro dup lex analy sis was per for med to seroty pes 1, 6A, 14 and 19F with se roty pe 5 as a cons tant com po nent, se roty pe 1 and 6A co uld be dif fe ren ti a ted from the ot hers. He te ro dup lex and ho mo dup lex DNA strands co uld be dis tin gu is hed by mel ting cur ve analy sis in re al-time flu o ro met ric nuc le ic acid de tec ti on system. C Co on nc c l lu u s si i o on n: : As a ra pid and cost-ef fec ti ve met hod, re al-ti me he te rodup lex analy sis may be an al ter na ti ve to ot her mo le cu lar met hods for se roty pe pre dic ti on of pne u mo coc ci. K Ke ey y W Wo or rd ds s: : Streptococcus pneumoniae; serotyping; polymerase chain reaction; heteroduplex analysis Ö ÖZ ZE ET T A Am ma aç ç: : Li te ra tür de pnö mo kok se ro tip len dir me si ni da ha kul la nış lı ha le ge tir mek için bir kaç mo le kü ler sero tip tah min yön te mi ya yın lan mış tır. Bu ça lış ma nın ama cı bu mo le kü ler yak la şım la ra al ter na tif oluş tu ra cak farklı bir mo le kü ler se ro tip tah min yön te mi ge liş tir mek tir. G Ge e r re eç ç v ve e Y Yö ön n t te em m l le er r: : Tür ki ye' de en fek si yon lar dan en sık lık la izo le edi len S. pne u mo ni a e ser tip le ri olan 1, 3, 5, 6A, 7F, 8, 9V, 11A, 14, 15B, 18C, 19A, 19F ve 23B, ça -lış ma ya da hil edil miş tir. S. pne u mo ni a e kap sül po li sak ka ri ti nin iş len me si, dü zen len me si ve ta şın ma sı ile il gi li gen ler olan cpsA ve cpsB böl ge le ri PCR ile ço ğal tıl mış tır. El de edi len 1.8 kb bo yun da ki PCR ürün le ri he te ro dupleks ana li zi ne alın mış tır. Ana liz de tüm se ro tip le re a...