Abstract
摘要:本計畫欲利用核酸質譜儀的高敏感度特性及自行研發設計的探針做為偵測特定基因序列的方法,應用於臨床結核分枝桿菌的早期快速檢測以及多重抗藥基因突變偵測。結核桿菌為引起結核病的病原菌,結核病為世界上非常重要的慢性傳染病,根據世界衛生組織最新統計,全球2008年新增結核病人數目即超過940萬人(發生率約平均每十萬人中有139人),並有200萬人死於此疾病。全球約有三分之一的人口被結核桿菌感染,其中又以亞洲(55%)、非洲(30%)最多,大部分的人雖會因自身免疫力而不發病,但仍需面對終身可能再活化(reactivation)的潛在危險,其中約有10%被感染的人會再活化而發病。如能在受感染的潛伏期發現並接受治療,可有效減少日後發病的機會,也可以防止這些潛伏型帶原者繼續將結核菌傳染他人;發病後若即時接受適當病藥物治療,幾可完全治癒,但若不予治療或錯失治療黃金時期,約有一半的病人會在發病後三年內死亡。此外有四分之一的結核病死亡患者與愛滋病毒有關,顯示結核病與愛滋病的合併感染,更是當前結核病防治工作中的一項重大挑戰。在台灣,結核病是個案數最多的法定傳染病,結核病的防治亦為政府防疫重點之一。根據行政院衛生署統計,全台灣每年至少新增1萬5000名病例,發生率為每十萬人口63.7,死亡數為每十萬人口3.3。近年來因藥物濫用產生的多重抗藥結核分枝桿菌(MDR-TB, multidrug-resistant tuberculosis)及廣泛抗藥性結核分枝桿菌(XDR-TB, extensively drug-resistant tuberculosis)更是嚴重威脅結核病的管控。傳統鑑定結核分枝桿菌以及其抗藥性的方式主要仍以實驗室培養為主,由病人痰液進行抗酸性染色後,塗盤培養至藥物感受性試驗,耗時約為六至八週,極易錯失早期診斷早期治療之黃金時間。近年來,分子生物學的進步對於結核分枝桿菌抗藥機制更為了解,因此,也發展出以此為基礎的分子診斷技術,減短篩檢時程,然而操作便利性、特異性與敏感度仍倍受挑戰。為此,本計畫將應用核酸質譜儀技術,以SEQUENOM MassARRAY MALDI-TOF MS平台進行分枝桿菌的鑑定以及抗藥基因突變偵側。此系統具有高敏感度與高產量的優點,在操作上,從反應進行到上機分析,只需一到兩個工作天,可顯著改善傳統檢測耗時甚久的情況,大幅提升效率,加上其高敏感度特性,本計畫預計將來可應用於門診病人痰液中進行檢測,並進一步挑戰以呼出冷凝液做為檢體進行偵測,有效把握防疫先機。此外,設計聚合酶鍊鎖反應的引子搭配特異性探針進行單一核苷酸延展反應,除可大幅提高檢測病原菌的特異性外,更可以進行多基因突變整合同時偵測,本計畫將對目前已知抗藥基因突變,包括:rpoB、katG、pncA、embB、rpsL、inhA等基因共十多種突變整合於一至二個反應中同時進行分析,大幅降低反應成本。本計畫期望可以達到以下目標:專一鑑定出結核桿菌、有效偵測檢體內少量病原菌、抗藥性偵測及早預測抗藥結核分枝桿菌,期能成為目前臨床診斷與防治微生物感染疾病之有力的輔助工具,以提供結核病患更優質的照護與正確完善的診治方案。
Abstract: The specific aims of this proposal are to achieve early detection of Mycobacterium tuberculosis (MTB) and its gene mutations confer to drug-resistance in multiplex by high sensitive nucleotide mass spectrometry and our designed specific probes. Tuberculosis caused by Mycobacterium tuberculosis (MTB) infection is the leading cause of morbidity and mortality worldwide. According to recent estimates from the World Health Organization (WHO), 9.4 million new active disease cases were occurred (with incidence of 139 per 100,000 populations) with 2 million associated deaths in 2008. One third of worldwide population are suffered by MTB infection with the highest number of TB cases in Asia (55%) followed by Africa (30%). Although most people are with latent MTB infection due to self-immune defense, 10% of them will fall ill because of reactivation. Early diagnosis and treatment in the incubation period not only can effectively reduce the chance of disease but also prevent latent MTB carriers to continue to infect others. In addition, immediately received appropriate medical treatment after the onset of disease, some can be completely cured while half of patients will die with no treatment or missed the golden time of treatment within three years. A quarter of deaths is correlated to HIV infection indicates MTB and HIV co-infection is a major challenge to current MTB control. In Taiwan, TB is the largest case number of legal infectious disease and it is also one of the priorities in epidemic prevention and control. According to the statistics from the Department of Health, there are at least 15,000 new cases every year with the incidence rate of 63.7 per million population and the death rate of 3.3 per million population. Recently, MDR-TB (multidrug-resistant tuberculosis) and XDR-TB (extensively drug-resistant tuberculosis) due to drug abuse have been a serious threat to TB control. Traditional identification of MTB including its drug susceptibility remains to be based on laboratorial culture method. It takes six to eight weeks to identify MTB from acid-fast stain of patients’ sputum to plate culture and drug susceptibility test. Early diagnosis and treatment may be easily missed. In recent years, advances in molecular biology facilitates comprehended understanding the mechanism of MTB drug resistance and, therefore, as a basis to develop molecular diagnostic techniques to minimize the screening time cost. However, convenient operation, specificity and sensitivity are still much challenge. To this end, this proposal will utilize SEQUENOM MassARRAY platform based MALDI-TOF MS (matrix-assisted laser desorption inoization-time of flight mass spectrometry) to perform the identification of MTB and analyze its gene mutations related to drug resistance. This has advantages in high sensitivity and high throughput. Technically, it takes one to two days from biochemical reaction to analysis on the machine. This can significantly improve the long time duration of traditional analysis and greatly enhance the efficiency. Besides, high sensitivity facilitates this platform to be applied to sputum examination even further to respiratory condensate analysis of out-patients for effectively grasping the prevention opportunities. Furthermore, combination of primers for PCR and specific probes for single nucleotide extension reaction can not only enhance the specificity but also multiplex detect gene mutations including up to twenty residues among rpoB、katG、pncA、embB、rpsL、inhA genes. In conclusion, this proposal will anticipate to achieve followed aims: 1. Specifically identification of MTB; 2. Efficiently detection of rare pathogen in the sample; 3. Detection of gene mutations related to MTB drug resistance. This is presented on clinical diagnosis and prevention of MTB infection to powerful aid to provide better care of TB patients complete treatment with correct solution.
Keyword(s)
結核分枝桿菌
高敏感度快速檢測平台
多重抗藥基因偵測
核酸質譜儀