本文選題：白色念珠菌　切入點(diǎn)：內(nèi)轉(zhuǎn)錄間隔區(qū)　出處：《藥物分析雜志》2017年06期

【摘要】：目的:建立TaqMan-小溝結(jié)合物探針實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)方法,快速定量檢定臨床標(biāo)本中的白色念珠菌并進(jìn)行抗真菌藥物敏感性分析。方法:針對白色念珠菌內(nèi)轉(zhuǎn)錄間隔區(qū)和26S核糖體核糖核酸基因設(shè)計(jì)特異性引物和探針,構(gòu)建含有上述基因的標(biāo)準(zhǔn)質(zhì)粒進(jìn)行定量分析,建立TaqMan-小溝結(jié)合物探針實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)檢測方法,評價(jià)其特異性、靈敏度、重復(fù)性和穩(wěn)定性。對1 185份臨床標(biāo)本中的白色念珠菌進(jìn)行檢測,同時(shí)進(jìn)行真菌培養(yǎng)、普通PCR、基因克隆和測序鑒定。對所有真菌分離株進(jìn)行抗真菌藥物敏感性試驗(yàn)。結(jié)果:研究結(jié)果顯示,建立的白色念珠菌TaqMan-小溝結(jié)合物探針實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)檢測方法專屬性強(qiáng),能準(zhǔn)確檢出白色念珠菌,而與其他念珠菌屬真菌、非念珠菌屬真菌、細(xì)菌、寄生蟲和病毒均無交叉反應(yīng),特異性為100%。該技術(shù)靈敏度高,能精確定量檢測白色念珠菌DNA線性范圍達(dá)11個(gè)數(shù)量級,白色念珠菌最低檢測限度為5個(gè)菌落形成單位。該方法重復(fù)性非常好,組內(nèi)和組間相對標(biāo)準(zhǔn)偏差均小于2%。測試中相關(guān)系數(shù)、斜率和效率沒有顯著變化,表明該方法穩(wěn)定性好,精密度高。將其成功應(yīng)用于臨床標(biāo)本中白色念珠菌載量的定量檢測,用普通PCR和基因克隆測序分析進(jìn)行了確證。整個(gè)檢測過程可在2 h內(nèi)完成,可以用作定量分析快速診斷方法。應(yīng)用TaqMan-小溝結(jié)合物探針實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng)從1 185份臨床標(biāo)本中檢出94份白色念珠菌陽性標(biāo)本,真菌培養(yǎng)獲得8株存活的白色念珠菌,抗真菌藥物敏感性試驗(yàn)分析結(jié)果顯示:這些白色念珠菌分離株對制霉菌素、咪康唑、克霉唑、益康唑、氟康唑、沃爾康唑均敏感,但對兩性霉素B、酮康唑、特比萘芬、氟胞嘧啶已經(jīng)產(chǎn)生了耐藥性。結(jié)論:本研究新開發(fā)評價(jià)的TaqMan-小溝結(jié)合物探針實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)椒磻?yīng),可以快速簡易,精準(zhǔn)穩(wěn)定,特異靈敏地定量檢定白色念珠菌,適用于動物源性產(chǎn)品中白色念珠菌檢測、食品藥品生物制品醫(yī)療器械安全檢查、環(huán)境監(jiān)測、流行病學(xué)調(diào)查。
[Abstract]:Objective: to establish a real-time fluorescent quantitative polymerase chain reaction method with Taq Man-small groove conjugate probe to detect Candida albicans in clinical samples and to analyze their susceptibility to antifungal drugs.Methods: specific primers and probes were designed for the internal transcription spacer and 26s ribonucleic acid gene of Candida albicans, and the standard plasmid containing the above genes was constructed for quantitative analysis.A real-time fluorescent quantitative polymerase chain reaction method for the detection of TaqMan-small groove conjugate probe was established to evaluate its specificity, sensitivity, reproducibility and stability.Candida albicans were detected in 1 185 clinical specimens, fungi culture, common PCR, gene cloning and sequencing were performed.The antimicrobial susceptibility test was carried out on all fungi isolates.Results: the results showed that the established real-time fluorescent quantitative polymerase chain reaction method for the detection of Candida albicans TaqMan-small groove conjugate probe was highly specific and could accurately detect Candida albicans, but with other Candida fungi.Noncandida fungi, bacteria, parasites and viruses did not cross reaction, the specificity was 100.The sensitivity of this technique is high, and the linear range of accurate quantitative detection of Candida albicans DNA is up to 11 orders of magnitude, and the minimum detection limit of Candida albicans is 5 colony forming units.The reproducibility of the method was very good, and the relative standard deviations within and between groups were less than 2.The correlation coefficient, slope and efficiency are not significantly changed in the test, which indicates that the method has good stability and high precision.It was successfully applied to the quantitative detection of Candida albicans in clinical specimens and confirmed by PCR and gene clone sequencing.The whole detection process can be completed within 2 hours and can be used as a rapid diagnostic method for quantitative analysis.94 Candida albicans positive specimens were detected from 1 185 clinical specimens by real time fluorescence quantitative polymerase chain reaction with TaqMan-small groove conjugate probe. Eight strains of Candida albicans were isolated from fungal culture.The results of antimicrobial susceptibility test showed that the isolates were sensitive to nystatin, miconazole, clotrimazole, econazole, fluconazole, and Wolconazole, but were sensitive to amphotericin B, ketoconazole, terbinafine, amphotericin B, ketoconazole, terbinafine, amphotericin B, ketoconazole and terbinafine.Fluorocytosine has developed resistance.Conclusion: the Taq Man-small groove conjugate probe developed in this study can be used to detect Candida albicans in a rapid, simple, accurate and sensitive manner.It is suitable for the detection of Candida albicans in animal products, safety inspection of medical instruments for food, drug and biological products, environmental monitoring and epidemiological investigation.
【作者單位】： 中國食品藥品檢定研究院;
【基金】：國家科技支撐計(jì)劃(2013BAK11B01)資助項(xiàng)目
【分類號】：R440;R519

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