[논문]재발성 아프타성 궤양 환자의 타액에서 Herpes Simplex Virus, Varicella Zoster Virus, Helicobacter pylori 그리고 Candida 검출

허웅; 윤창륙; 안종모

재발성 아프타성 궤양 환자의 타액에서 Herpes Simplex Virus, Varicella Zoster Virus, Helicobacter pylori 그리고 Candida 검출
Detection of Herpes Simplex Virus, Varicella Zoster Virus, Helicobacter Pylori and Candida in Saliva of Patients with Recurrent Aphthous Ulceration 원문보기

대한구강내과학회지 = Korean journal of oral medicine, v.30 no.3 = no.60, 2005년, pp.319 - 328

허웅 (조선대학교 치과대학 구강내과학교실.법의치과학연구소) , 윤창륙 (조선대학교 치과대학 구강내과학교실.법의치과학연구소) , 안종모 (조선대학교 치과대학 구강내과학교실.법의치과학연구소)

초록
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재발성 아프타성 궤양 환자의 타액에서 구강내 궤양성 병소를 유발할 수 있고 감염성이 비교적 높은 미생물로 알려진 Herpes Simplex virus, Varicella Zoster virus, Helicobacter pylori 그리고 Candida가 발현되는지 여부와 병소의 발생과 상관관계가 있는지를 알아보고자 조선대학교 치과병원 구강내과에 내원한 재발성 아프타성 궤양을 가진 환자 29명과 대조군 29명의 타액을 이용하여 PCR과 배양을 통해 발현율을 비교한바 다음과 같은 결과를 얻었다. 1. HSV DNA는 재발성 아프타성 궤양 환자군에서 41.4%, 대조군에서 55.2%가 발현되었으나 두 군간에 유의한 차이는 없었고(P>0.05), VZV DNA는 두 군에서 모두 나타나지 않았다. 2. H. pylori DNA는 재발성 아프타성 궤양 환자군에서 27.6%, 대조군에서 48.3%가 발현되었으나 두 군간에 유의한 차이는 없었다(P>0.05). 3. Candida는 재발성 아프타성 궤양 환자군에서 13.8%, 대조군에서 6.9%가 배양되었으나 두 군간에 유의한 차이는 없었다(P>0.05). 이상의 연구를 종합하여 보았을 때, HSV, VZV, H. pylori 그리고 Candida는 재발성 아프타성 궤양의 발생에 직접적인 역할을 한다고는 볼 수 없으므로 향후 더 많은 표본을 대상으로 다른 미생물이 병소 발생의 유발요인으로 작용하는지 연구하여야 할 것으로 사료된다.

Abstract ▼ AI-Helper

To examine whether HSV, VZV, H. pylori and Candida that are known to be microorganisms causing ulcerative disease in oral cavity and have the relatively high contigiousness are detected in saliva of patients with RAU and related to the development with RAU, PCR and culture were performed on the saliva of 29 patients with RAU and 29 control subjects who visited the Department of Oral Medicine, Dental Hospital, Chosun University. The results were obtained as follows; 1. HSV DNA was detected in 41.4% patients with RAU, and 55.2% control subjects, however, a significant difference between the two groups was not detected, (P>0.05), and VZV DNA was not detected in both groups. 2. H. pylori DNA was detected in 27.6% patients with RAU, and 48.3% control subjects, however, a significant difference between the two groups was not detected (P>0.05). 3. Candida was cultured in 13.8% patients with RAU, and 6.9% control subjects, however, a significant difference between the two groups was not detected (P>0.05). This results suggest that HSV, VZV, H. pylori and Candida can not be regarded to play a direct role in the development of RAU. Thus it is considered that in future, on a larger sample, also, it has to be examined whether other microorganisms acts as a trigger factor of the development of RAU.

주제어

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제안 방법

As the mixture for the second PCR of H. pylori genomic DNA, to the AccuPower PCR Premix (Bioneer, Daegeon, Korea) containing 10 mM Tris-HCl (pH 9.0), 40 mM KCl, 1.5 mM MgCl 2, 250 ㎛ dNTP, and 1 U Tag DNA Polymerase, 2 ㎕ of the first PCR product and 2 ul of each Primer (10 pmole/㎕) were added, and the final volume was adjusted to 20 ㎕ with 8-mop. The condition of second PCR was the same of first PCR.
For the first PCR of H. pylori genomic DNA, to the AccuPower PCR Premix (Bioneer, Daegeon, Korea) containing 10 mM Tris-HCl (pH 9.0), 40 mM KCl, 1.5 mM MgCl 2 , 250 ㎛dNTP, and 1 U Tag DNA Polymerase, 5 ㎕ of the template DNA and 2 ㎕ of each Primer (10 pmole/㎕) were added, and the final volume was adjusted to 20 ㎕ with 8-mop. The PCR was performed under conditions of 94℃ for 5 minutes, 1 cycle; 94℃ for 30 seconds, 55℃ for 30 seconds, 72℃ for 50 seconds, 30 cycles; 72℃ for 5 minutes, 1 cycle.
In this study, therefore, to find the etiology of RAU, microbiological analysis in the saliva was performed by molecular biological methods.
PCR of HSV DNA was performed by using the RealArt TM HSV 1/2 GEL PCR Kit (Artus, Hamburg, Germany). 45 ㎕ of the master mix contained in the kit was mixed with 5㎕ of the template DNA extracted from the saliva in the oral cavity, and it was used for PCR.
The concentration of genomic DNA was determined at 260 nm and 280 nm with Ultraspec 2000 (Pharmacia biotech, Cambridge, England). Purity of DNA were determined by evaluating the A260/A280 ratio.
The PCR was performed under conditions of 94℃ for 5 minutes, 1 cycle; 94℃ for 30 seconds, 55℃ for 30 seconds, 72℃ for 45 seconds, 10 cycles; 94℃ for 30seconds, 53℃ for 30 seconds, 72℃ for 45 seconds, 10 cycles; 94℃ for 30 seconds, 50℃ for 30 seconds, 72℃ for 45 seconds, 10 cycles; 72℃ for 10 minutes, 1 cycle.
5 mM MgCl 2 , 250 ㎛dNTP, and 1 U Tag DNA Polymerase, 5 ㎕ of the template DNA and 2 ㎕ of each Primer (10 pmole/㎕) were added, and the final volume was adjusted to 20 ㎕ with 8-mop. The PCR was performed under conditions of 94℃ for 5 minutes, 1 cycle; 94℃ for 30 seconds, 55℃ for 30 seconds, 72℃ for 50 seconds, 30 cycles; 72℃ for 5 minutes, 1 cycle.
45 ㎕ of the master mix contained in the kit was mixed with 5㎕ of the template DNA extracted from the saliva in the oral cavity, and it was used for PCR. The PCR was performed under conditions of 94℃ for 5 minutes, 1 cycle; 94℃ for 30 seconds, 60℃ for 30 seconds, 72 ℃ for 45 seconds, 5 cycles; 94℃ for 30 seconds, 58 ℃ for 30 seconds, 72℃ for 45 seconds, 35 cycles; 72 ℃ for 10 minutes, 1 cycle.
To 1.5% agarose gel containing Ethidium bromide (0.5 ㎍/㎖), 9 ㎕ of the PCR product, 1 ㎕ of 10 X DNA loading buffer (20% Ficoll 400; 0.1 M EDTA pH 8.0; 1% SDS; 0.25% Bromphenol Blue;0.25% Xylene cyanol) were mixed and electrophoresis was performed, and subsequently, by using a UV Transilluminator, the size of PCR products was analyzed. As the Marker, 100 bp DNA Ladder (Bioneer, Daegeon, Korea) was used.

데이터처리

For the statistical analysis of the patients group with RAU and the control group, Pearson's χ 2 test was performed using the SPSS windows 10.0 program.

성능/효과

1. HSV DNA was detected in 41.4% patients with RAU, and 55.2% control subjects, however, a significant difference between the two groups was not detected, (P＞0.05), and VZV DNA was not detected in both groups.
2. H. pylori DNA was detected in 27.6% patients with RAU, and 48.3% control subjects, however, a significant difference between the two groups was not detected (P＞0.05).
3. Candida was cultured in 13.8% patients with RAU, and 6.9% control subjects, however, a significant difference between the two groups was not detected (P＞0.05).
In this study, H. pylori was detected in saliva of 8 patients with RAU (27.6%), and 14 control subjects (48.3%), however, it was not statistically significant, and furthermore, it was isolated more in control group. Therefore, H.
In this study, HSV DNA was found to be positive in saliva of 12 patients with RAU (41.4%) and 16 control subjects (55.2%), however, a significance was not detected, and VZV DNA was not detected in both groups. Thus, it is considered that these two viruses did not mediate a direct effect on the development of RAU.
In this study, different from previous studies performed about microbiological factors in the lesion and the blood of patients by numerous investigators, using the saliva that mediates a great effect on the oral environment and has the protective function of oral mucosa, it was examined whether HSV, VZV, H. pylori, and Candida that have been shown to be able to induce ulcerative disease in oral cavity and have the relatively high contagiousness are detected in the saliva of patients with RAU, and related to the development of RAU.
The result of the culture on CHROMagar^Ⓡ Candida (CHROMagar, Paris, France) showed that 4 patients with RAU (13.8%) and 2 control subjects (6.9%) were found to be positive, however, a significant difference between the two groups was not detected (P＞0.05) (Fig. 5, Table 2).
The result of the electrophoresis on 1.5% agarose gel of the first and second PCR products to H. pylori DNA showed that 8 patients with RAU (27.6%), and 14 control subjects (48.3%) were found to be positive, however, a significant difference between the two groups was not detected (P＞0.05) (Fig. 3 and 4, Table 2).
The result of the electrophoresis on 1.5% agarose gel showed that the 148bp size HSV DNA detected in 12 patients with RAU (41.4%), and 16 control subjects (55.2%), however, a significant difference between the two groups was not detected(P＞ 0.05)(Fig. 1 and Table 2).

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표제어: PCR

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용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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