利用聚合酶連鎖反應結合膠體金側流向技術自臨床檢體快速偵測結核桿菌

Use of PCR-Colloidal Gold Lateral flow Technique for Rapid Detection of Mycobacterium tuberculosis from Clinical Specimens

黃建中(Chien-Chung Huang)

指導教授 ： 賴信志 ； 何憲武

國立臺灣大學/醫學院/醫事技術學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.00177

摘要

結核病每年在全球造成兩百萬人死亡跟八百萬人的新興病例。造成結核病的病菌統稱為Mycobacterium tuberculosis complex，包含有M. tuberculosis、M. bovis、M. africanum，M. microti和M. canetti，其中在人體中造成結核病的最主要病菌為M. tuberculosis，而造成結核病的最主要部位為肺部。在臨床檢體中最主要的檢體為病人的痰檢體。傳統上檢查方法以培養方法為主，另外可利用acid-fast stain作為快速檢測方法，可是因為培養花費3~8週，花費時間太久，而acid-fast stain的敏感度跟特異性都不夠高，因此我們希望發展一個分子生物快速檢測方法，縮短檢測時間，並提高敏感度跟特異性。
IS6110是Mycobacterium tuberculosis complex中特有的DNA序列，我們針對IS6110設計primer，利用PCR方法來放大病人痰檢體中微量的細菌DNA。PCR可以有效地放大在檢體中所含有的微量DNA，提高DNA濃度而達到易於偵測的濃度，傳統上偵測PCR產物是利用電泳方法並利用EtBr染色來加以判讀，我們利用側流向(lateral flow)技術來作為一個新的偵測平台。膠體金側流向技術已普遍使用在一般的快速檢測試劑中。其優點為使用方便且成本低廉。利用biotin跟FITC標定的primer，使得PCR產物帶有biotin跟FITC。測試條上的測試區的抗體會抓住FITC，而biotin跟膠體金上的streptavidin結合。如果有PCR產物時，測試條上的測試區便會顯現一條紅線。結合PCR的高專一性跟高敏感度，以及側流向平台的快速方便使用，將可以有效地縮短檢測時間並擁有一定的檢測正確性。在臨床呼吸道檢體中，若以培養結果為標準，敏感度可達93.3%，特異性可達96.2%，若結合臨床症狀跟培養結果，敏感度為65.0%，特異性為98.2%。
某些研究中顯示在其他種類的非結核桿菌(NTM)的細菌DNA中也含有與IS6110相似的DNA片段，因此如果針對IS6110設計的PCR反應中，有可能會出現小量的偽陽性結果，另外在臨床檢查上，希望能夠更專一地檢測出是M. tuberculosis complex中何種細菌，因為M. tuberculosis是結核病中最主要的致病細菌，利用之前M. tuberculosis和M. bovis、M. bovis BCG的基因序列比對報告，得知M. tuberculosis中所特有的RD1~RD16等DNA序列，其中我們針對RD9進行primer的設計來進行PCR，在標準菌種的測試中已經得到可行的結果，然而在臨床檢體的測試中，卻發現靈敏度不足的現象，在靈敏度的測試中顯示，有關RD9的PCR靈敏度遠比IS6110 PCR低了100~1000倍，必須突破這一個限制，才能繼續應用於臨床的TB診斷。實驗顯示nest PCR lateral flow在TB診斷中是一可行之方法，但其敏感度跟特異性有更好的改進空間。

關鍵字

RD region ； IS6110 ； 聚合酶側流向技術 ； 臨床檢體 ； 結核桿菌 ； 連鎖反應

並列摘要

Tuberculosis, a predominant disease in the nineteenth century, remains one of the most life-threatening diseases at the beginning of the new millennium. It causes two million deaths per year and eight million new case of TB infection. The M. tuberculosis complex comprises M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti and M. canneti. The major pathogen for tuberculosis among M. tuberculosis complex is M. tuberculosis. The major syndrome is pulmonary tuberculosis and the most often clinical specimens are sputum. Traditional diagnosis of tuberculosis are culture and acid-fast stain. Culture need 3~8 weeks and the sensitivity of acid-fast stain is low. We want to develop a molecular diagnosis method to shorten time and has high sensitivity and specificity.
IS6110 is the specific DNA fragment exist the M. tuberculosis complex. We use PCR for IS6110 to amplify the fewer mycobacteria DNA in clinical sputum specimen. PCR method can amplify the DNA and raise the concentration of DNA for easy detection. Traditionally electrophoresis and EtBr stain is the method to detect PCR product. It is a trouble for clinical diagnosis that making a electrophoresis agarose gel and EtBr staining. We use a lateral-flow platform to detect PCR product. Advantages of lateral-flow are economic and easy to use. Use of biotin and FITC labeled primers in PCR, the amplicon contain the biotin and FITC. The anti-FITC antibody captures FITC and biotin linked with colloidal gold conjugated streptavidin. The test strip show a red line in test zone if amplicon was amplified in PCR procedure. Combine the high sensitivity and specificity of PCR and the rapid and easy of lateral-flow, we can shorten test time and have good accuracy. In clinical respiratory specimens, culture as standard, the sensitivity and specificity are 93.3% and 96.2%. The sensitivity and specificity are 65.0% and 98.2% when resolved results as standard.
In some studies report there are some IS6110 homologs exist in NTM chromosome DNA and make false positive results in IS6110 PCR. In the other way, we want to separate which species among MTB complex in clinical diagnosis. Former study reported that compare of M. tuberculosis, M. bovis and M. bovis BCG and shows there were 16 RD extra region in M. tuberculosis DNA. Another study shows RD9 region seem to be the most specific region for M. tuberculosis. We try to design primers for RD9 PCR and showed good results in reference strains PCR. But the sensitivity of RD9 PCR is very poor in clinical specimen PCR. According to the test showed that the sensitivity of RD9 PCR is 100~1000 fold lower than IS6110 PCR. In this study showed nest PCR lateral flow is work in identification of M. tuberculosis, although the sensitivity and specificity need to be approved.

並列關鍵字

IS6110 ； Mycobacterium tuberculosis ； lateral flow ； PCR ； RD region ； clinical specimen

參考文獻

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