PCR 방법에 의한 Shigella 속 균주와 enteroinvasive E. coli 검출방법의 확립

Abstract

Invasive Shigella species and enteroinvasive E. coli (EIEC) are enteropathogenic for humans causing dysenteries with abdominal pain and frequent stools containing blood and mucus. To identify these microorganisms, biochemical and serological methods have been used in most clicinal laboratories. However, these tests have difficulties in distinguishing each strains and can not be performed in a routine laboratory procedure on large numbers of stools. Therefore, isolates thought to be invasive Shigella species or EIEC are required to be confirmed by animal inoculation or tissue culture studies. Recent discovery of a few virulence plasmid-encoded loci from has provided the altemative approach such as hybridization assay or polymerized chain reaction (PCR) to identify these strains. In this study, the optimal thermocycler conditions was established and the sensitivity of PCR was estimated by the cell numbers as well as by the amount of DNA for each standard strains: Shigella dysenteriae (ATCC 9752) , Shigella flexneri (ATCC 9403), E. coli O29 (ATCC 43892) , and E. coli O124 (ATCC 43893). In addition, one strain of Shigella flexneri strain, two strains of Shigella sonnei and E. coli O157 strain isolated from patients stools were investigated. PCR was performed with DNA extracted from suspension of each organism diluted in the range of 108 to 102 cells or with total cellular DNA in the concentration of 101 to 10-6㎍/㎕ using primer Sh-1 and Sh-2, followed by the detection of 320bp DNA fragmen t. The most suitable reaction conditions consisted of 40 cycles of 1 min at 94℃(denaturation) , 1min at 55℃ (annealing) , and subsequently 1 min at 72℃ (extension). The sensitivity estimated by the cell number was 104 for Shigella dysenteriae (A TCC 9752), Shigella flexneri (A TCC 9403), and E. coli 029 (A TCC43892) and 105 for E. coli 0124 (ATCC 43893). The organisms isolated from the patient stools exhibited sensitivity of 106 for all Shigella flexneri strain, 107 for one Shigella sonnei strain and 108 for another Shigella sonnei strain. However, no bands were produced from the E. coli O157 strain. On the other hand, the sensitivity estimated by amount of DNA was 10-2㎍ for Shigella dysenteria (ATCC 9752) and E. coli 029 (ATCC 43892), and 10-3㎍ for Shigella flexneri (ATCC 9403) and E. coli O124 (ATCC 43893) For the organism obtained from patient specimens the sensitivity was 10-3 for one all Shigella flexneri strain, 10-1 for one Shigella sonnei strain and 101 for another Shigella sonnei strain. No bands were from the E. coli O157 strain. PCR can be used as good alternative method to identify invasive Shigella species and EIEC, since this method is sensitive, and rapid, and does not require selective enrichment or high cell numbers. In the future, more studies with clinical specimens are required to evaluate the conditions of PCR examined in this investigation.