孟相秋，袁超文，劉文鑫，關(guān)瑋琨，杜元策，李丹丹，趙姝靜，唐 杰，師東方

大腸桿菌腸毒素基因多重PCR檢測方法的建立

孟相秋，袁超文，劉文鑫，關(guān)瑋琨，杜元策，李丹丹，趙姝靜，唐 杰，師東方

目的 建立一種快速檢測大腸桿菌耐熱腸毒素(heat-stable enterotoxin, STa, STb)和不耐熱腸毒素(heat-labile enterotoxin, LT-Ⅰ, LT-Ⅱ)基因的多重PCR方法。方法 參照文獻(xiàn)合成四對可擴增產(chǎn)腸毒素大腸桿菌(EnterotoxigenicEscherichiacoli, ETEC)耐熱腸毒素基因(estA、estB)和不耐熱腸毒素基因(elt-Ⅰ、elt-Ⅱ)的特異性引物，通過反應(yīng)條件的優(yōu)化，敏感性、特異性試驗和臨床樣品檢測，建立檢測大腸桿菌腸毒素的多重PCR方法。結(jié)果 用所建立的多重PCR方法可特異性擴增出estA(229 bp)、estB(480 bp)、elt-Ⅰ(605 bp)和elt-Ⅱ(300 bp)基因片段，最低檢出量分別為2.55×101CFU/μL、2×101CFU/μL、2×101CFU/μL和2.47×103CFU/μL。從22株大腸桿菌分離株中檢測到estA基因(2/22)，elt-Ⅱ基因(3/22)，未檢測到estB和elt-Ⅰ基因，檢測結(jié)果與常規(guī)PCR檢測結(jié)果一致。結(jié)論 建立了檢測大腸桿菌腸毒素基因(estA、estB、elt-Ⅰ和elt-Ⅱ)的多重PCR方法，該方法具有良好的特異性和敏感性，能夠滿足對細(xì)菌培養(yǎng)物的檢測要求。

產(chǎn)腸毒素大腸桿菌；耐熱腸毒素；不耐熱腸毒素；多重PCR

產(chǎn)腸毒素大腸桿菌(EnterotoxigenicEscherichiacoli, ETEC)是引起嬰幼兒、旅游者、幼齡動物腹瀉的主要病原體之一，可單獨或同時產(chǎn)生耐熱腸毒素(heat-stable enterotoxin, ST)和不耐熱腸毒素(heat-labile enterotoxin, LT)[1-3]。ST為18或19個氨基酸的小肽，有很強的毒素活性，其編碼基因位于質(zhì)粒上[4-6]。根據(jù)其生物學(xué)特性和致病性的不同，ST分為STa和STb[7]，STa溶于甲醇，可引起乳鼠腸液大量分泌，STb不溶于甲醇，在乳鼠腸內(nèi)無活性，可引起新生仔豬和斷奶仔豬腸積水[8]。LT可分為LT-I和LT-II[9]，LT- I首先發(fā)現(xiàn)于引起豬腹瀉的大腸桿菌中，其結(jié)構(gòu)基因位于質(zhì)粒上[10]，能明顯導(dǎo)致豬結(jié)扎腸段的液體潴留[11]，后來也從引起人腹瀉的ETEC中分離和鑒定[12-14]。LT-II不能被抗霍亂弧菌毒素抗體(cholera toxin, CT)和抗LT-I的抗體中和[15-16]，其結(jié)構(gòu)基因位于染色體上[15]。產(chǎn)LT-II的ETEC已從人、動物和食品中分離鑒定[17-18]。

常規(guī)微生物學(xué)檢測不能區(qū)分ETEC和其他大腸桿菌，但PCR技術(shù)的廣泛應(yīng)用為ETEC的檢測提供了新的方法。文獻(xiàn)報道顯示，國內(nèi)外已建立了多個檢測estA/estB/elt-Ⅰ、estA/estB/elt-Ⅰ/stx2e、elt-Ⅰ/estA、f4/f5/estA等毒素基因的多重PCR方法[19-24]。隨著對LT-Ⅱ研究的深入，其對人及家畜的致病性已明確[25-26]，因此，在對ETEC毒素基因檢測時，應(yīng)將elt-Ⅱ也作為檢測目標(biāo)。目前尚未見同時檢測estA、estB、elt-Ⅰ和elt-Ⅱ的多重PCR檢測方法。本研究旨在建立一種同時檢測ETEC產(chǎn)生的這4種腸毒素基因的多重PCR方法，為ETEC分離鑒定、毒素基因檢測，病原流行病學(xué)調(diào)查提供準(zhǔn)確、快速的檢測手段。

1 材料和方法

1.1 菌株 大腸桿菌參考菌株C83903(K88+, LT-Ⅰ+, STb+, EAST1+)，C83920(K99+, F41+, STa+)購自中國獸醫(yī)藥品監(jiān)察所；大腸桿菌DN1502(LT-Ⅱ+, F17+)、DN0402、DN0501、DN0602、DN1202、DN1802、DN1803、DN48A、DN50B、DN54A、DN65B、DN67A、DN69A、DN71A、DN72C、DN74B、DN79B、DN83B、DN83C、DN89B、DN104A、DN105C、DN1265由東北農(nóng)業(yè)大學(xué)動物醫(yī)學(xué)學(xué)院傳染病學(xué)教研室(以下簡稱本實驗室)從犢牛腹瀉糞便中分離鑒定并保存；綠膿桿菌30C由本實驗室從貂肺臟分離鑒定并保存；豬丹毒桿菌CVCC124、巴氏桿菌CVCC430、豬霍亂沙門氏菌CVCC503由東北農(nóng)業(yè)大學(xué)動物醫(yī)學(xué)學(xué)院藥理與毒理學(xué)教研室張秀英教授提供。

1.2 主要試劑 Trans 2k Plus Ⅱ DNA Marker、10×loading buffer、EasyTaq DNA聚合酶、10×EasyTaq buffer(含Mg2+)、dNTPs購自全式金生物技術(shù)有限公司。

1.3 引物 參照文獻(xiàn)和初步篩選后確定引物序列，并由上海生工生物工程技術(shù)服務(wù)有限公司合成。引物序列及擴增片段長度見表1。

1.4 DNA模板的制備 各菌株分別接種于麥康凱瓊脂平板，37 ℃培養(yǎng)18～24 h，然后挑取單個菌落接種于LB液體培養(yǎng)基中，37 ℃ 200 r/min 振蕩培養(yǎng)16～18 h。取1 mL培養(yǎng)物，經(jīng)10 000×g離心5 min，去上清，加入100 μL滅菌去離子水重懸，100 ℃煮沸10 min后立即置冰浴中冷卻5 min，10 000×g離心5 min，取上清于-20 ℃保存?zhèn)溆谩?/p>

1.5 反應(yīng)條件的優(yōu)化 PCR擴增體系25 μL，以C83903、C83920和DN1502 DNA為模板，分別對影響PCR擴增的引物濃度、退火溫度以及循環(huán)數(shù)等因素進(jìn)行優(yōu)化。擴增產(chǎn)物用1.5%瓊脂糖凝膠電泳分析。

1.6 特異性試驗 以優(yōu)化后的條件分別對大腸桿菌C83903、C83920、DN1502、DN69A和DN67A，豬霍亂沙門氏菌CVCC503、豬丹毒桿菌CVCC124、巴氏桿菌CVCC430、綠膿桿菌30C進(jìn)行多重PCR擴增，以大腸桿菌JM109為空白對照，檢測該方法的特異性。

1.7 敏感性試驗 將大腸桿菌C83903、C83920和DN1502接種于LB液體培養(yǎng)基，37 ℃ 200 r/min振蕩培養(yǎng)12 h，各取新鮮菌液200 μL用滅菌生理鹽水連續(xù)10倍系列稀釋6個梯度(10-1～10-6)，然后分別取各稀釋度的菌液200 μL均勻混于LB固體培養(yǎng)基中，37 ℃培養(yǎng)18 h，進(jìn)行菌落計數(shù)。同時對各稀釋度的菌液進(jìn)行多重PCR擴增，檢測該方法的敏感性。

1.8 臨床樣本檢測 用建立的多重PCR方法對本實驗室分離到的22株大腸桿菌進(jìn)行檢測，并與常規(guī)PCR[28-29]檢測結(jié)果進(jìn)行比較分析，擴增的陽性產(chǎn)物送上海生工生物工程技術(shù)服務(wù)有限公司進(jìn)行序列測定。

2 結(jié) 果

2.1 多重PCR反應(yīng)條件 通過對影響PCR反應(yīng)的引物濃度、退火溫度和循環(huán)次數(shù)等因素的優(yōu)化，最終確定該多重PCR體系為25 μL，其中10×EasyTaq Buffer (Mg2+plus) 2.5 μL，2.5 mmol/L dNTPs 2 μL，5 U/μL EasyTaq DNA 聚合酶0.5 μL，10 μmol/Lelt-Ⅱ上下游引物各1.5 μL，10 μmol/LestA、estB、elt-Ⅰ上下游引物各0.2 μL，模板2 μL，用滅菌去離子水補足至 25 μL。優(yōu)化后的反應(yīng)條件為95 ℃預(yù)變性5 min；94 ℃變性30 s，48 ℃退火30 s，72 ℃延伸45 s，共30個循環(huán)；最后72 ℃延伸10 min。

2.2 特異性試驗 用優(yōu)化后的多重PCR方法對大腸桿菌C83903、C83920、DN1502進(jìn)行檢測，均擴增到與預(yù)期大小一致的條帶；陰性對照菌株未擴增到任何條帶(見圖1)。

2.3 敏感性試驗 大腸桿菌C83920、C83903和DN1502在稀釋度為10-6時每200 μL菌液菌落計數(shù)結(jié)果分別為510 CFU、400 CFU、494 CFU；多重PCR針對estA、estB/elt-Ⅰ和elt-Ⅱ可檢測到的菌液最高稀釋倍數(shù)分別為10﹣5、10﹣5、10﹣3，最低檢出量分別為2.55×101CFU、2×101CFU和2.47×103CFU(見圖 2)。

M: Trans 2k Plus Ⅱ DNA marker; N: JM109; P: ETEC C83920 (K99+, F41+, STa+), ETEC C83903 (K88+, LT-Ⅰ+, STb+, EAST1+) and DN1502 (F17+, LT-Ⅱ+) multiplex PCR.

1: ETEC C83903 (K88+, LT-Ⅰ+, STb+, EAST1+); 2: ETEC C83920 (K99+, F41+, STa+); 3: DN1502 (F17+, LT-Ⅱ+);4:P.aeruginosa30C; 5:PasteurellaCVCC430; 6: SwineerysipelasCVCC124; 7:S.choleraesuisCVCC503; 8: DN69A;9: DN67A.

圖1 多重PCR特異性試驗

Fig.1 Specificity test for the multiplex PCR

M: Trans 2k Plus Ⅱ DNA marker; N: JM109; A. 1-7: C83903 (LT-Ⅰ+, STb+) strain 100-106; B. 1-7: C83920 (STa+) strain 100-106;C. 1-5: DN1502 (LT-Ⅱ+) strain 100-104.

圖2 敏感性試驗結(jié)果

Fig.2 Results of sensitivity test for multiplex PCR

2.4 大腸桿菌分離株的多重PCR檢測 采用所建立的多重PCR方法對實驗室分離保存的22株大腸桿菌進(jìn)行檢測，結(jié)果顯示，2株為estA陽性，3株為elt-Ⅱ陽性，未檢測到estB和elt-Ⅰ陽性菌株(見圖3)，檢測結(jié)果與常規(guī)PCR檢測結(jié)果一致。陽性PCR產(chǎn)物測序結(jié)果證明，5株陽性菌株的PCR產(chǎn)物序列分別與GenBank所收錄的V00612.1(estA)、JQ031709.1(elt-Ⅱ)序列一致。

3 討 論

國內(nèi)外研究者針對ETEC菌毛(F4、F5、F6、F18、F41)和毒素(STa、STb、LT-I、Stx2e、EAST1)基因建立了多個多重PCR檢測方法[19-24,27]，這些方法在ETEC毒力因子的檢測方面發(fā)揮了重要作用。1983年Lee等首次從大腸桿菌SA53株中發(fā)現(xiàn)新型LT(后被命名為LT-II)， Seriwatana 等從236株分離自牛、肉類食品和人腹瀉糞便樣品中的大腸桿菌中檢測出41株(17%)LT-II陽性菌株[17]，袁超文等從犢牛腹瀉糞便中也分離到了產(chǎn)LT-II的ETEC[18]，可見攜帶LT-II基因的ETEC較為普遍。目前已檢測出3種LT-II變異體(LT-IIa、LT-IIb和LT-IIc)[30]。隨著對產(chǎn)LT-II ETEC的深入研究，可能會發(fā)現(xiàn)該腸毒素與STa、STb、LT-I一樣，在人、畜腹瀉的致病過程中起著重要的作用。因此，對ETEC毒力基因的檢測應(yīng)包括LT-II。LT-IIa、LT-IIb和LT-IIc A亞單位編碼基因的同源性高達(dá)70%以上，B亞單位的編碼基因同源性較低，僅為50%左右[31]，本研究使用的引物位于LT-II A亞單位基因序列的高度保守區(qū)，可擴增LT-IIa、LT-IIb和LT-IIc 3個亞型A亞單位基因中的目的片段。研究結(jié)果證明，用所建立的多重PCR方法可同時對STa、STb、LT-I和LT-II基因進(jìn)行檢測，除陽性參考菌株外，其他菌株均無陽性擴增產(chǎn)物，特異性良好。該方法主要用于ETEC的腸毒素基因檢測與鑒定，可顯著提高樣品的檢測效率。LT-II基因的最低檢出量為2.47×103CFU，而經(jīng)37 ℃ 200 r/min振蕩培養(yǎng)12 h的ETEC可達(dá)2.47×109CFU/mL，完全可以滿足檢測要求。用該方法對22份犢牛腹瀉糞便樣本的檢測結(jié)果與常規(guī)PCR檢測結(jié)果完全一致，也進(jìn)一步證明了該方法的準(zhǔn)確性。該方法為快速檢測ETEC腸毒素基因提供了有效技術(shù)手段。

M: Trans 2k Plus Ⅱ DNA marker; N: JM109; P: C83920 (K99+, F41+, STa+), C83903 (K88+, LT-Ⅰ+, STb+, EAST1+) and DN1502 (F17+, LT-Ⅱ+) multiplex PCR.

1: DN0402; 2: DN0501; 3: DN1202; 4: DN72C; 5: DN1803; 6: DN0602; 7: DN48A; 8: DN50B; 9: DN54A; 10: DN74B; 11: DN65B; 12: DN67A; 13: DN69A; 14: DN1265; 15: DN71A; 16: DN79B; 17: DN83B; 18: DN1802; 19: DN83C; 20: DN89B; 21: DN104A;22: DN105C.

圖3 大腸桿菌分離株多重PCR檢測結(jié)果

Fig.3 Assay results ofE.coliisolated strains by multiplex PCR

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Multiplex PCR detection on enterotoxin genes in enterotoxigenicEscherichiacoli

MENG Xiang-qiu,YUAN Chao-wen,LIU Wen-xin,GUAN Wei-kun,DU Yuan-ce,LI Dan-dan,ZHAO Shu-jing,TANG-Jie,SHI Dong-fang

(DepartmentofPreventiveVeterinaryMedicine,CollegeofVeterinaryMedicine,NortheastAgriculturalUniversity,Harbin150030,China)

To develop a rapid and specific multiplex PCR method for detection of enterotoxins genes (estA,estB,elt-Ⅰandelt-Ⅱ) in ETEC, four pairs of primers were synthesized according to the conserved sequences ofestA,estB,elt-Ⅰ andelt-Ⅱ genes. The assay was tested for its optimal reaction conditions, specificity, sensitivity, detection of clinical sample, and then the multiplex PCR method were developed. Results indicated that this method had a high specificity in detecting enterotoxins genes (229 bp/estA, 480 bp/estB, 605 bp/elt-Ⅰ, and 300 bp/elt-Ⅱ), and the detection limit were 2.55×101CFU/μL, 2×101CFU/μL, 2×101CFU/μL, and 2.47×103CFU/μL, respectively. Using this multiplex PCR, we detected 22E.colistrains isolated from fecal swabs of calves with diarrhea, and found 2estAgene positive strains, 3elt-Ⅱgene positive strains, while no isolated strain was detected to carry theestBorelt-Ⅰ genes, and the assay result was in agreement with that of conventional PCR. This multiplex PCR method has high specificity and sensitivity, and it can satisfy the detection of bacterial cultures.

enterotoxigenicE.coli; heat-stable enterotoxin; heat-labile enterotoxin; multiplex PCR

Shi Dong-fang, Email:shidf@neau.edu.cn

國家科技支撐計劃項目(2012BAD12B03、2012BAD12B05)；黑龍江省科技攻關(guān)計劃項目(GC12B303)。

師東方，Email: shidf@neau.edu.cn

東北農(nóng)業(yè)大學(xué)動物醫(yī)學(xué)學(xué)院傳染病教研室，哈爾濱 150030

10.3969/cjz.j.issn.1002-2694.2015.01.002

R378.2

A

1002-2694(2015)01-0006-05

2014-06-14；

2014-10-22

Funded by grants from the National Science and Technology Support Project (Nos. 2012BAD12B03 and 2012BAD12B05), and the Science and Technology Planning Project of Heilongjiang Province (No. GC12B303)