梁紅霞,余志晟*,劉如銦,張洪勛,吳 鋼

?

基于擬桿菌16S rRNA基因進(jìn)行微生物溯源的研究進(jìn)展

梁紅霞1,余志晟1*,劉如銦1,張洪勛1,吳 鋼2

(1.中國科學(xué)院大學(xué)資源與環(huán)境學(xué)院,北京 100049；2.中國科學(xué)院生態(tài)環(huán)境研究中心,北京 100085)

微生物溯源方法可利用糞便中的微生物區(qū)分來自人或動物的糞便污染.其中,擬桿菌以其豐度高?不能體外繁殖和宿主特異性強(qiáng)等優(yōu)勢被廣泛應(yīng)用于微生物溯源研究中.本文以擬桿菌16S rRNA基因?yàn)闃?biāo)記物,總結(jié)了擬桿菌及其標(biāo)記物在環(huán)境中的衰減?擬桿菌引物的敏感性和特異性以及分子生物學(xué)技術(shù)在微生物溯源中的運(yùn)用,可為糞便污染源解析提供一定的科學(xué)參考依據(jù).

微生物溯源；擬桿菌；16S rRNA基因；糞便污染

近年來,隨著我國畜牧業(yè)的快速發(fā)展,糞便的產(chǎn)生量日益增加.產(chǎn)生的糞便因管理不善或雨水沖刷等原因進(jìn)入水中,容易引發(fā)一系列健康和環(huán)境問題.一方面,糞便中的病原菌會對公眾健康造成一定威脅[1-2].研究表明糞便是水體病原菌的重要來源[3-4],水體病原菌可通過飲用水?貝類和水上娛樂活動等進(jìn)行傳播[5-6],進(jìn)而增加傳染病爆發(fā)的風(fēng)險(xiǎn);另一方面,糞便引起的水體污染難以治理.未經(jīng)處理的糞便N?P含量較高,易引起水體富營養(yǎng)化,使原有水體喪失功能[1,7].那么,如何區(qū)分人和動物的糞便污染對污染源的確定至關(guān)重要[8-10],而微生物溯源技術(shù)可實(shí)現(xiàn)快速有效區(qū)分.

微生物溯源(MST)[11-12]又稱糞便污染溯源(FST),其原理是微生物與宿主腸道環(huán)境長期適應(yīng)的過程中,逐漸形成了宿主專一性,并將這種專一性遺傳給后代,使得這類微生物均具有某種特定的標(biāo)記,通過這種特定的標(biāo)記即可判斷污染樣品和可能污染物之間是否存在聯(lián)系,從而實(shí)現(xiàn)糞便污染溯源[13-15].

在進(jìn)行糞便污染溯源時(shí),選擇合適的糞便污染指示微生物至關(guān)重要.許多研究者推薦將擬桿菌作為微生物溯源的指示菌[16-17].擬桿菌是糞便中的優(yōu)勢菌.糞便干重的1/3為細(xì)菌,擬桿菌占糞便細(xì)菌總數(shù)的30%~40%[18-19].每克人糞便中的擬桿菌細(xì)胞數(shù)約為109~1011個(gè),比腸桿菌或腸球菌高4~7個(gè)數(shù)量級[17,20-21];擬桿菌不能在體外環(huán)境中繁殖.擬桿菌進(jìn)入水體后無法繁殖,故可通過其含量反映水體受污染的程度[16-17,22];擬桿菌具有宿主特異性.擬桿菌在與宿主長期適應(yīng)的過程中,使其16S rRNA基因具有一定的宿主特異性.可利用擬桿菌16S rRNA基因判斷污染樣品和污染物之間的關(guān)系,進(jìn)而確定污染源[23-24].

目前,大量研究表明以擬桿菌16S rRNA基因?yàn)闃?biāo)記物可成功區(qū)分哺乳動物、反芻動物和禽類等的糞便污染[24-26].本文在前人研究的基礎(chǔ)上對相關(guān)內(nèi)容進(jìn)行歸納總結(jié),以期為糞便污染的預(yù)防和微生物溯源技術(shù)在我國的發(fā)展提供一定幫助.

1 擬桿菌及其標(biāo)記物的衰減

作為微生物溯源常用的指示菌,擬桿菌在宿主體外的存活時(shí)間備受關(guān)注,許多學(xué)者進(jìn)行了相關(guān)研究.大量研究表明,擬桿菌在環(huán)境中的存活時(shí)間較短,有些糞便中的擬桿菌在環(huán)境水體中能存活數(shù)小時(shí)至數(shù)天[27-28],擬桿菌標(biāo)記物則可持續(xù)數(shù)周[17,29-30],故可以很好的指示水體短期內(nèi)發(fā)生的污染[16-17,30].

影響擬桿菌存活的因素較多,溫度和捕食是主要因素,其次是光照和鹽度.另外,水體營養(yǎng)程度?溶解氧含量和擬桿菌培養(yǎng)基質(zhì)的狀態(tài)等環(huán)境因素也會對擬桿菌及其標(biāo)記物的衰減造成影響[31-33].

1.1 溫度

研究發(fā)現(xiàn)溫度越低,擬桿菌及其標(biāo)記物的存在時(shí)間越長. Kreade[34]在河水中加入人的糞便模擬污水,發(fā)現(xiàn)擬桿菌標(biāo)記物在4℃條件下可存在2周,14℃存在4~5d,而在24℃僅存在1~2d,30℃僅存在1d. Seurinck等[35]在厭氧條件下培養(yǎng)人糞便污水,發(fā)現(xiàn)擬桿菌標(biāo)記物HF183在4和12℃的條件下可持續(xù)24d,而在28℃的條件下僅持續(xù)8d; Dick等[36]、Bae等[27]和Okabe等[37]在研究了不同宿主糞便來源的擬桿菌標(biāo)記物后也得出了類似結(jié)論.

1.2 捕食

水體中的原生微生物或原生動物對擬桿菌標(biāo)記物存在一定的捕食行為,且捕食行為越強(qiáng),擬桿菌標(biāo)記物的衰減速度越快.即水中活性因素的影響越大,擬桿菌標(biāo)記物的存在時(shí)間越短. Bell等[38]將馬糞和溪水混合后模擬糞便污水,分別研究了廢水在過濾和不過濾兩種情況下,擬桿菌標(biāo)記物AllBac的衰減.研究發(fā)現(xiàn)將水樣過濾后,擬桿菌標(biāo)記物存在的時(shí)間更長. Dick等[36]研究發(fā)現(xiàn)捕食對人糞源擬桿菌標(biāo)記物的影響比人工日光?沉積物和溫度要大. Kreader[34]、Kobayashi等[39]及Ballesté等[31]的研究也一致認(rèn)為捕食是影響擬桿菌標(biāo)記物存在的主要因素.

1.3 光照

光照對擬桿菌標(biāo)記物衰減速度的影響,研究結(jié)果并不一致,但這種不一致在其他微生物研究中也存在[40-41]. Bae等[27]研究了光照對4種擬桿菌及其標(biāo)記物的影響.發(fā)現(xiàn)自然光并未明顯地影響擬桿菌和其DNA的衰減(牛糞源擬桿菌標(biāo)記物BacCow-UCD除外). Walters等[42]的發(fā)現(xiàn)與上述研究結(jié)果一致,該研究發(fā)現(xiàn)在光照和黑暗兩種情況下,淡水中人糞源擬桿菌標(biāo)記物HF134?HF183和反芻動物糞源標(biāo)記物CF193的衰減行為并無明顯差別.然而, Dick等[36]研究發(fā)現(xiàn)暴露在太陽光下會加速人糞源擬桿菌標(biāo)記物qHF183和BacHum的衰減. Walters等[29]的研究也發(fā)現(xiàn)人糞源擬桿菌標(biāo)記物BacHum的衰減速度在自然光下比黑暗中更快. Green等[43]的發(fā)現(xiàn)與Dick等[36]和Walters等[29]的結(jié)果一致.

1.4 鹽度

鹽度越高,擬桿菌標(biāo)記物的衰減速度越慢. Green等[43]分別研究了人糞源擬桿菌標(biāo)記物在海水和淡水中的衰減行為,結(jié)果表明擬桿菌標(biāo)記物在海水中的持續(xù)時(shí)間比在淡水中長3d左右. Okabe等[37]研究了不同鹽濃度的河水對通用擬桿菌、人糞源擬桿菌、牛糞源擬桿菌和豬糞源擬桿菌標(biāo)記物存在時(shí)間的影響.這4種標(biāo)記物的衰減行為一致說明鹽度越高,擬桿菌標(biāo)記物衰減速度越慢.值得說明的是此現(xiàn)象只出現(xiàn)在過濾后的水樣,而在未過濾水樣中則未出現(xiàn),可能因?yàn)樵谖催^濾水樣中捕食是主要影響因素.

2 擬桿菌引物的敏感性和特異性

2.1 擬桿菌引物

不同動物的飲食習(xí)慣、生活環(huán)境及腸道系統(tǒng)有所差異,擬桿菌在與宿主長期適應(yīng)的過程中,使得其16S rRNA基因表現(xiàn)出一定的宿主特異性[24].利用擬桿菌16S rRNA基因構(gòu)建系統(tǒng)發(fā)育樹時(shí),同種宿主糞源的擬桿菌群落表現(xiàn)出一定的相似性,不同宿主糞源的擬桿菌群落則出現(xiàn)分異,說明可根據(jù)擬桿菌標(biāo)記物的存在指示不同宿主來源的糞便污染.通常使用擬桿菌引物判斷標(biāo)記物是否存在,擬桿菌引物分為擬桿菌通用引物和特異性引物.擬桿菌通用引物適用于多種動物,擬桿菌特異性引物則只針對某一種或某一類動物.

本節(jié)總結(jié)前人利用擬桿菌16S rRNA基因所設(shè)計(jì)的擬桿菌通用引物和特異性引物(表1),其中特異性引物針對的擬桿菌宿主包括:哺乳動物(人、豬、狗、馬、北美海貍)?反芻動物(牛?麋鹿)及禽類(加拿大雁、雞、鴨)等.目前,利用擬桿菌特異性標(biāo)記物能夠很好的區(qū)分人和反芻動物的糞便污染,但也存在一些問題,如關(guān)于禽類和水鳥糞便的溯源研究較少,主要有以下原因:(1)擬桿菌在有些禽類糞便中并不是優(yōu)勢菌[7].(2)禽類動物不僅包括城市中的禽類也包括野鳥,表現(xiàn)出高度多樣的生境和飲食特征[44-45]. (3)不同的禽類會共同生活在同一片區(qū)域,無法獲得單一性糞樣[46].

2.2 引物的敏感性和特異性

擬桿菌標(biāo)記物是否適合指示某種目標(biāo)宿主的糞便污染,可用擬桿菌特異性引物的敏感性和特異性衡量.當(dāng)引物的敏感性值=100%時(shí),說明引物對應(yīng)的擬桿菌標(biāo)記物存在于所有目標(biāo)宿主糞便樣品中;特異性值=100%時(shí),說明對應(yīng)的擬桿菌標(biāo)記物在所有非目標(biāo)宿主糞便樣品中均不存在.即擬桿菌引物敏感性和特異性值越高,對應(yīng)的標(biāo)記物越適合指示該宿主糞便的污染.

引物的敏感性()和特異性()可用下式表示[47]:

R

= TP/(TP + FN) (1)

S

= TN/(TN + FP) (2)

式中: TP(真陽性)為目標(biāo)宿主顯陽性的數(shù)量; FN(假陰性)為目標(biāo)宿主顯陰性的數(shù)量; TN(真陰性)為非目標(biāo)宿主顯陰性的數(shù)量; FP(假陽性)為非目標(biāo)宿主顯陽性的數(shù)量.

為了比較不同微生物溯源標(biāo)記物的效果,世界范圍內(nèi)曾進(jìn)行過3次大規(guī)模比選[48].(1) 2003年美國實(shí)驗(yàn)室間的比較研究[49-51];(2) 2006年歐洲實(shí)驗(yàn)室間的比較研究[52];(3) 2013年美國實(shí)驗(yàn)室間的比較研究[53-54].研究表明,擬桿菌通用引物Bac32F和Bac708R所對應(yīng)的標(biāo)記物存在于各種動物糞便中,可利用該引物對擬桿菌16S rRNA基因進(jìn)行擴(kuò)增后再進(jìn)行后續(xù)實(shí)驗(yàn)[23,55-56];在多種人糞源擬桿菌標(biāo)記物中, HF183表現(xiàn)出較強(qiáng)的敏感性和特異性[57-59];反芻動物標(biāo)記物Rum-2-Bac和BacR在比選中表現(xiàn)出良好的特異性和敏感性,而且地理分布較廣[54,60]；但牛糞源擬桿菌標(biāo)記物易與其他反芻動物或馬發(fā)生交叉反應(yīng),說明牛糞源標(biāo)記物也可能存在于其他反芻動物或馬糞便中[54].

表1中的敏感性和特異性值由引物設(shè)計(jì)者實(shí)驗(yàn)得出,在一定程度上可為后續(xù)研究者在選擇引物時(shí)提供參考.但不同擬桿菌標(biāo)記物的地理分布有所差異,故不一定能重現(xiàn)這些數(shù)值. Reischer等[60]比較了人糞源標(biāo)記物(BacH、BacHum)和反芻動物糞源標(biāo)記物(BacCow、BacR和BoBac)在6個(gè)大洲的16個(gè)國家中的地理分布情況.5種標(biāo)記物敏感性值分別是77%、87%、92%、90%和82%,說明牛糞源擬桿菌標(biāo)記物地理分布最廣;特異性值分別為53%、68%、57%、84%和59%,說明這5種標(biāo)記物均存在交叉反應(yīng),即在非目標(biāo)宿主中也存在. Shanks等[61]對2種反芻動物(CF128和CF193)和5種牛(Bac2、Bac3、BoBac、CowM2和CowM3)糞源擬桿菌標(biāo)記物進(jìn)行比較,結(jié)果表明反芻動物糞源標(biāo)記物CF128和牛糞源標(biāo)記物BoBac的敏感性最好且地理分布最廣,但特異性較差,分別是76%和47.4%;牛糞源標(biāo)記物Bac2、Bac3、CowM2和CowM3的特異性值均大于98.9%,但敏感性沒有CF128和BoBac高. Tambalo等[62]對犬類糞便擬桿菌標(biāo)記物CanBac-UCD進(jìn)行了評估,只有31%的特異性,與設(shè)計(jì)者的86.15%相差較大.

表1 擬桿菌通用引物和特異性引物

續(xù)表1

目標(biāo)微生物標(biāo)記物引物/探針序列敏感性(%)特異性(%) 人和動物通用糞源擬桿菌BacPre1[23]qBac560FTTTATTGGGTTTAAAGGGAGCGTA100(16/16)- qBac725RCAATCGGAGTTCTTCGTGATATCTA AllBac[55]AllBac296FGAGAGGAAGGTCCCCCAC100(34/34)- AllBac412RCGCTACTTGGCTGGTTCAG AllBac375BhqrFAM-CCATTGACCAATATTCCTCACTGCTGCCT-BHQ-1 哺乳動物糞源擬桿菌人HF134[24]HF134FGCCGTCTACTCTTGGCC43.75(7/16)94.74(18/19) HF654RCCTGCCTCTACTGTACTC HF183[24,63]HF183FATCATGAGTTCACATGTCCG87.5(14/16)100(19/19) Bac708RCAATCGGAGTTCTTCGTG BacHum-UCD[65]BacHum-160FTGAGTTCACATGTCCGCATGA81.25(26/32)97.56(40/41) BacHum-241RCGTTACCCCGCCTACTATCTAATG BacHum-193p6-FAM-TCCGGTAGACGATGGGGATGCGTT-TAMRA Human-Bac1[23]qHS601FGTTGTGAAAGTTTGCGGCTCA100(4/4)/(與牛和豬糞交叉反應(yīng)) qBac725RCAATCGGAGTTCTTCGTGATATCTA qHS 624MGBCGTAAAATTGCAGTTGA HuBac[55]HuBac566FGGGTTTAAAGGGAGCGTAGG100(6/6)68(19/28) HuBac692RCTACACCACGAATTCCGCCT HuBac594BhqfFAM-TAAGTCAGTTGTGAAAGTTTGCGGCTC-BHQ-1 BacH[66]BacHFCTTGGCCAGCCTTCTGAAAG97.5(39/40)97.5(39/40) BacHRCCCCATCGTCTACCGAAAATAC BacH-pCFAM-TCATGATCCCATCCTG-NFQ-MGB BacH-pTFAM-TCATGATGCCATCTTG-NFQ-MGB HumM2[67]Hum2FCGTCAGGTTTGTTTCGGTATTG100(36/36)99.2(247/249) Hum2RTCATCACGTAACTTATTTATATGCATTAGC ProbeFAM-TATCGAAAATCTCACGGATTAACTCTTGTGTACGC-TAMRA HumM3[67]Hum3FGTAATTCGCGTTCTTCCTCACAT100(36/36)97.2(242/249) Hum3RGGAGGAAACAAGTATGAAGATAGAAGAATTAA ProbeFAM-AGGTCTGTCCTTCGAAATAGCGGT-TAMRA 豬Pig-Bac1[23]qPS422FCGGGTTGTAAACTGCTTTTATGAAG100(5/5)100(11/11) qBac581RCGCTCCCTTTAAACCCAATAAA Pig-Bac2[23,68]qBac41FTACAGGCTTAACACATGCAAGTCG100(10/10)54(16/30) qPS183RCTCATACGGTATTAATCCGCCTTT Pig-1-Bac[68]Pig-1-Bac32FmAACGCTAGCTACAGGCTTAAC98.55(68/69)100(54/54) Pig-1-Bac108RCGGGCTATTCCTGACTATGGG Pig-1-Bac44PFAM-ATCGAAGCTTGCTTTGATAGATGGCG-BHQ-1 Pig-2-Bac[68]Pig-2-Bac41FGCATGAATTTAGCTTGCTAAATTTGAT100(69/69)100(54/54) Pig-2-Bac163RmACCTCATACGGTATTAATCCGC Pig2Bac113VIC-TCCACGGGATAGCC-NFQ-MGB PF[63,69]PF163FGCGGATTAATACCGTATGA100(2/2)100(10/10) Bac708RCAATCGGAGTTCTTCGTG 犬類BacCan-UCD[23,65]BacCan-545F1GGAGCGCAGACGGGTTTT62.5(5/8)86.15(56/65) BacUni-690R1CAATCGGAGTTCTTCGTGATATCTA BacUni-690R2AATCGGAGTTCCTCGTGATATCTA BacUni-656p6-FAM-TGGTGTAGCGGTGAAA-TAMRA-MGB DF[63,70]DF475FCGCTTGTATGTACCGGTACG100(2/2)100(6/6) Bac708RCAATCGGAGTTCTTCGTG 馬HoF[63,69]HoF597FCCAGCCGTAAAATAGTCGG100(2/2)100(10/10) Bac708RCAATCGGAGTTCTTCGTG 北美海貍Beapo101[71]Beapol-F02AGCATTTTTCAAGCTTGCTT100(17/17)100(63/63) Beapol-R01ACTTAATGCCATCCCGTATTAA Beapol-PHEX-CAACCTACCGTTTACTCTCGG-BHQ-1

續(xù)表1

目標(biāo)微生物標(biāo)記物引物/探針序列敏感性(%)特異性(%) 反芻動物糞源擬桿菌反芻動物RUM[24,63]CF128FCCAACYTTCCCGWTACTC100(19/19)100(16/16) Bac708RCAATCGGAGTTCTTCGTG RUM[24,63]CF193FTATGAAAGCTCCGGCC100(19/19)100(16/16) Bac708RCAATCGGAGTTCTTCGTG Rum-2-Bac[56]BacB2-590FACAGCCCGCGATTGATACTGGTAA97(29/30)100(40/40) Bac708RmCAATCGGAGTTCTTCGTGAT BacB2-626PFAM-ATGAGGTGGATGGAATTCGTGGTGT-BHQ-1 BacR[72]BacR-FGCGTATCCAACCTTCCCG100(57/57)100(38/38) BacR-RCATCCCCATCCGTTACCG BacR-PFAM-CTTCCGAAAGGGAGATT-NFQ-MGB 牛Cow-Bac1[23]qCS406FGAAGGATGAAGGTTCTATGGATTGT100(7/7)100(9/9) qBac581RCGCTCCCTTTAAACCCAATAAA Cow-Bac2[23]qCS621FAACCACAGCCCGCGATT100(7/7)100(9/9) qBac725RCAATCGGAGTTCTTCGTGATATCTA Cow-Bac3[23]qBac41FTACAGGCTTAACACATGCAAGTCG100(7/7)100(9/9) qCS160RTCAACGGGCTATTCCTGAGTAAG BoBac[55]BoBac367FGAAG(G/A)CTGAACCAGCCAAGTA100(11/11)100(23/23) BoBac467RGCTTATTCATACGGTACATACAAG BoBac402PFAM-TGAAGGATGAAGGTTCTATGGATTGTAAACTT-BHQ-1 CowM2[73]CowM2FCGGCCAAATACTCCTGATCGT100(60/60)/ CowM2RGCTTGTTGCGTTCCTTGAGATAAT probeFAM-AGGCACCTATGTCCTTTACCTCATCAACTACAGACA-TAMRA CowM3[73]CowM3FCCTCTAATGGAAAATGGATGGTATCT100(60/60)/ CowM3RCCATACTTCGCCTGCTAATACCTT probeFAM-TTATGCATTGAGCATCGAGGCC-TAMRA BacCow-UCD[63,65]CF128FCCAACYTTCCCGWTACTC100(8/8)95.89(70/73) BacCow-305RGGACCGTGTCTCAGTTCCAGTG BacCow-257p6-FAM-TAGGGGTTCTGAGAGGAAGGTCCCCC-TAMRA 麋鹿EF[70]EF447FAATAACACCATCTACGTGTAGA100(2/2)80(8/10) EF990RGCCTGTCCAGTGCAATTTAA 禽類糞源擬桿菌雞/鴨Chicken/Duck-Bac[26]qCD362F-HUAATATTGGTCAATGGGCGAGAG79.59(39/49)100(78/78) qcD464R-HUCACGTAGTGTCCGTTATTCCCTTA qBac394FAM-TCCTTCACGCTACTTGG-MGB 雞Chicken-Bac[26]qC160F-HUAAGGGAGATTAATACCCGATGATG69.57(16/32)88.46(69/78) qBac265R-HUCCGTTACCCCGCCTACTAC 鴨Duck-Bac[26]qBac366F-HUTTGGTCAATGGGCGGAAG84.62(22/26)94.87(74/78) qDuck474R-HUGCACATTCCCACACGTGAGA qBac394FAM-TCCTTCACGCTACTTGG-MGB 加拿大雁CGOF1-Bac[74]CG1FGTAGGCCGTGTTTTAAGTCAGC57.43(58/101)100(291/291) CG1RAGTTCCGCCTGCCTTGTCTA ProbeFAM-CCGTGCCGTTATACTGAGACACTTGAG-BHQ-1 CGOF2-Bac[74]CG2FACTCAGGGATAGCCTTTCGA50.50(51/101)100(291/291) CG2RACCGATGAATCTTTCTTTGTCTCC ProbeFAM-AATACCTGATGCCTTTGTTTCCCTGCA-BHQ-1

注:/為未命名或數(shù)據(jù)未提供;-為數(shù)據(jù)不存在.

3 分子生物學(xué)技術(shù)在微生物溯源中的運(yùn)用

在微生物溯源研究中，PCR技術(shù)常與其他技術(shù)結(jié)合使用以實(shí)現(xiàn)對污染物的定性分析,實(shí)時(shí)熒光定量PCR技術(shù)則常用于污染物的定量分析;基因芯片最大的特點(diǎn)是通量高,可同時(shí)檢測成千上萬個(gè)樣品,成功識別污染源的同時(shí)還可對樣品中微生物多樣性進(jìn)行分析.以上方法各有優(yōu)缺點(diǎn),在實(shí)際研究工作中,可根據(jù)實(shí)驗(yàn)?zāi)康倪x擇合適的方法,還可同時(shí)使用多種方法以增加實(shí)驗(yàn)的可信度[75-77].

3.1 PCR及其聯(lián)用技術(shù)

3.1.1 PCR主要操作如下 使用擬桿菌引物進(jìn)行PCR擴(kuò)增,觀察目標(biāo)條帶是否出現(xiàn).使用擬桿菌通用引物時(shí),出現(xiàn)目標(biāo)條帶則說明樣品存在糞便污染;使用擬桿菌特異性引物時(shí),出現(xiàn)目標(biāo)條帶則說明樣品中存在特異性糞便污染.目前該方法已成功地判斷出水樣被何種動物的糞便污染[24,63].但PCR技術(shù)也存在一定的缺陷,如易產(chǎn)生非特異性擴(kuò)增,只能對污染物定性而不能定量等.

3.1.2 末端標(biāo)記限制性酶切長度多態(tài)性(T- RFLP) 同一目的基因由于堿基的插入?缺失?重排或點(diǎn)突變,故在不同的微生物間存在長度多態(tài)性[78].用帶有熒光標(biāo)記的引物擴(kuò)增DNA樣品,然后用限制性核酸內(nèi)切酶酶切擴(kuò)增產(chǎn)物,由于不同微生物的同一基因的核苷酸序列存在差異,所以同一個(gè)基因的DNA片段經(jīng)相同酶切后可能得到長度不同的限制性內(nèi)切片段,在T-RFLP檢測中表現(xiàn)為不同的熒光信號[79]. Bernhard等[24,63]根據(jù)T-RFLP圖譜區(qū)別出人和牛糞便中的擬桿菌16S rRNA基因特異性片段,并據(jù)此設(shè)計(jì)了擬桿菌通用引物和特異性引物. Dick等[69]在設(shè)計(jì)擬桿菌引物時(shí),也用到了T-RFLP技術(shù).

3.1.3 變性梯度凝膠電泳(DGGE) 利用菌株的標(biāo)記基因獲得樣品的特異性指紋圖譜,以指紋圖譜的差異表征菌株的差異,進(jìn)而分析被污染樣品與污染物之間的關(guān)系.目前, DGGE技術(shù)在以大腸桿菌為指示菌進(jìn)行微生物溯源方面研究較多[80-82],而以擬桿菌為指示菌進(jìn)行微生物溯源方面的相關(guān)研究國內(nèi)外鮮有報(bào)道.張曦等[83]利用擬桿菌特異性16S rRNA基因和大腸桿菌特異性基因phoE這兩種標(biāo)記基因,經(jīng)DGGE技術(shù)對塘壩型飲用水污染進(jìn)行溯源,研究結(jié)果顯示擬桿菌DGGE圖譜比大腸桿菌圖譜的條帶更豐富,且樣品之間的顯著性更高,表明可利用擬桿菌的DGGE圖譜表征污染水體之間的關(guān)系.

3.2 實(shí)時(shí)熒光定量PCR技術(shù)(qPCR)

qPCR技術(shù)相比常規(guī)PCR技術(shù)有以下優(yōu)點(diǎn):(1) qPCR技術(shù)可對污染物進(jìn)行定量分析,即回答污染物類型和污染物的量,而常規(guī)PCR技術(shù)只能對污染物進(jìn)行定性分析,即回答污染物類型;(2) qPCR技術(shù)的靈敏性和特異性更好.基于 qPCR技術(shù)的優(yōu)勢,其在微生物溯源中有更廣泛的運(yùn)用,主要有以下3點(diǎn):

3.2.1 區(qū)分不同類型的糞便污染并對污染物定量 Seurinck等[35]首次利用人糞源擬桿菌HF183標(biāo)記物對水中的人糞源污染進(jìn)行研究并取得很好的定量效果. Okabe等[23]選取了1種人糞源?3種牛糞源和2種豬糞源的擬桿菌標(biāo)記物進(jìn)行溯源實(shí)驗(yàn),成功地判斷出河水的糞便污染來源并測定出污染量. Jeong等[84]使用人糞源和牛糞源擬桿菌標(biāo)記物進(jìn)行TaqMan qPCR實(shí)驗(yàn),結(jié)果表明利用qPCR技術(shù)能可靠地識別和量化糞便污染,為流域水質(zhì)管理和改進(jìn)方面提供有效的信息.

3.2.2 驗(yàn)證標(biāo)記物的敏感性和特異性 Raith等[85]檢驗(yàn)了5種擬桿菌標(biāo)記物是否適用于加利福尼亞地區(qū)反芻動物的糞便污染.研究表明將牛糞源擬桿菌標(biāo)記物CowM2和反芻動物糞源擬桿菌標(biāo)記物BacR或Rum2Bac結(jié)合使用,最適合該地區(qū)反芻動物的糞便污染檢驗(yàn). Mieszkin等[68]使用兩種豬糞源擬桿菌標(biāo)記物評估了養(yǎng)豬場下游水域污染. qPCR實(shí)驗(yàn)結(jié)果表明這兩種標(biāo)記物具有良好的敏感性和特異性,可用于檢驗(yàn)水環(huán)境中豬糞便的污染. Lee等[86]使用通用?人糞源和牛糞源擬桿菌標(biāo)記物分別進(jìn)行TaqMan qPCR實(shí)驗(yàn),驗(yàn)證了這3種標(biāo)記物具有良好的特異性和敏感性.

3.2.3 發(fā)現(xiàn)糞便中的優(yōu)勢菌 Matsuki等[19,87]利用多種特異性引物進(jìn)行qPCR實(shí)驗(yàn),表明脆弱擬桿菌是人糞便中的優(yōu)勢菌.

3.3 基因芯片技術(shù)

基因芯片又稱DNA芯片或DNA微陣列,原理是采用光導(dǎo)原位合成或顯微印刷等方法將大量特定序列的探針分子密集、有序地固定于經(jīng)過相應(yīng)處理的硅片、玻片、硝酸纖維素膜等載體上,然后加入標(biāo)記的待測樣品,進(jìn)行多元雜交,通過雜交信號的強(qiáng)弱及分布,來分析目的分子的有無?數(shù)量及序列,從而獲得受檢樣品的遺傳信息[88].

基因芯片技術(shù)在微生物溯源中表現(xiàn)出一定的可靠性[89-93],主要應(yīng)用在以下兩方面:

3.3.1 評估水體污染狀況 Dubinsky等[94]采集了42個(gè)包括人、鳥、牛、馬、麋鹿和鰭足類動物的糞便排泄物,使用59316種不同的細(xì)菌16S rRNA基因探針進(jìn)行檢測.其中梭狀芽胞桿菌門和擬桿菌門中的多種科能把人、食草動物和鰭足亞目類動物3者的污染區(qū)分開,為加利福尼亞沿海水域提供污染源信息. Inoue等[95]調(diào)查加德滿都谷地的淺井地下水污染狀況時(shí),以941個(gè)病原菌為對象進(jìn)行基因芯片實(shí)驗(yàn),證明該地區(qū)的淺井地下水普遍受到糞便污染.

3.3.2 分析糞便樣品中微生物多樣性 Li等[96]在前人研究基礎(chǔ)上,利用雞、牛、家禽和豬糞便中微生物的DNA或RNA,將基因芯片、qPCR技術(shù)及二代測序技術(shù)相結(jié)合,檢測到不同糞便中含有相同病原菌且病原菌主要有兩類:和. Wang等[90]使用CY-5熒光基團(tuán)標(biāo)記人糞便中的腸道細(xì)菌的16S rRNA基因,熒光雜交結(jié)果說明糞便中主要的腸道細(xì)菌是普通擬桿菌、梭形桿菌屬、多型擬桿菌、瘤胃球菌屬、消化鏈球菌和真桿菌,與前人的研究結(jié)果一致[97-98].

基因芯片在微生物溯源研究中有許多優(yōu)點(diǎn).如:(1)通量高,可同時(shí)檢測成千上百個(gè)樣品,全面分析樣品中的致病微生物;(2)檢測速度快;(3)在樣品量很少的情況下仍能保持高敏感性.當(dāng)然,它也存在一定的缺陷.如:(1)以16S rRNA基因?yàn)榇郎y基因時(shí),16S rRNA基因數(shù)據(jù)庫不足,導(dǎo)致探針設(shè)計(jì)存在缺陷;(2)成本和操作復(fù)雜度高.總體來說,基因芯片技術(shù)是一種快速有效判定污染物來源的技術(shù),具有廣闊的應(yīng)用空間.

4 結(jié)語

近年來,利用擬桿菌16S rRNA基因?qū)S便污染進(jìn)行溯源已經(jīng)取得了很大的進(jìn)展,但仍面臨一些挑戰(zhàn),現(xiàn)做如下總結(jié)和展望:

4.1 擬桿菌標(biāo)記物可實(shí)現(xiàn)對人和反芻動物糞便污染的準(zhǔn)確溯源,但擬桿菌在一些禽類和鳥類糞便中并不是優(yōu)勢菌,故利用擬桿菌對禽類糞便污染溯源時(shí),效果并不理想.需進(jìn)一步發(fā)現(xiàn)其他標(biāo)記物以實(shí)現(xiàn)鳥類和禽類糞便污染的準(zhǔn)確溯源.

4.2 擬桿菌是嚴(yán)格厭氧菌,在水體中的存在量隨時(shí)間逐漸減少,適用于指示近期發(fā)生的污染,但無法對污染時(shí)間較長的水體進(jìn)行評估,故在實(shí)際研究中可將擬桿菌標(biāo)記物與其他標(biāo)記物,如線粒體DNA結(jié)合使用.

4.3 需盡快建立擬桿菌標(biāo)記物含量與水體污染程度之間的對應(yīng)關(guān)系,為政府問責(zé)及司法鑒定提供依據(jù).

4.4 各國實(shí)驗(yàn)室應(yīng)加強(qiáng)合作,積極開展擬桿菌的地理分布研究,在世界范圍內(nèi)確定敏感性和特異性效果最好的擬桿菌標(biāo)記物.

[1] Harwood V J, Staley C, Badgley B D, et al. Microbial source tracking markers for detection of fecal contamination in environmental waters: relationships between pathogens and human health outcomes [J]. Fems Microbiology Reviews, 2013,38(1):1-40.

[2] Samadpour M, Stewart J, Steingart K, et al. Laboratory investigation of anO157: H7outbreak associated with swimming in Battle Ground Lake, Vancouver, Washington [J]. Journal of Environmental Health, 2002,64(10):16-20.

[3] Hamilton M J, Yan T, Sadowsky M J. Development of goose- and duck-specific DNA markers to determine sources ofin waterways [J]. Applied and Environmental Microbiology, 2006,72(6):4012-4019.

[4] Santo Domingo J W, Bambic D G, Edge T A, et al. Quo vadis source tracking? Towards a strategic framework for environmental monitoring of fecal pollution [J]. Water Research, 2007,41(16):3539- 3552.

[5] Alexander L M, Heaven A, Tennant A, et al. Symptomatology of children in contact with sea water contaminated with sewage [J]. Journal of Epidemiology and Community Health, 1992,+46(4):340- 344.

[6] US Department of Health and Human Services, Center for Disease Control and Prevention. Outbreak ofparahaemolyticus infections associated with eating raw oysters-Pacific Northwest, 1997 [J]. Morbidity and Mortality Weekly Report, 1998,47(22):457-462.

[7] Lu J R, Domingo J S, Shanks O C. Identification of chicken-specific fecal microbial sequences using a metagenomic approach [J]. Water Research, 2007,41(16):3561-3574.

[8] Ahmed W, Sritharan T, Palmer A, et al. Evaluation of bovine feces- associated microbial source tracking markers and their correlations with fecal indicators and zoonotic pathogens in a Brisbane, Australia, reservoir [J]. Applied and Environmental Microbiology, 2013,79(8): 2682-2691.

[9] Lu J R, Ryu H, Vogel J, et al. Molecular detection ofspp. and fecal indicator bacteria during the northern migration of sandhill cranes () at the central platte river [J]. Applied and Environmental Microbiology, 2013,79(12):3762-3769.

[10] Mclellan S L, Eren A M. Discovering new indicators of fecal pollution [J]. Trends in Microbiology, 2014,22(12):697-706.

[11] Wiggins B A. Discriminant analysis of antibiotic resistance patterns in fecal, a method to differentiate human and animal sources of fecal pollution in natural waters [J]. Applied and Environmental Microbiology, 1996,62(11):3997-4002.

[12] Hagedorn C, Robinson S L, Filtz J R, et al. Determining sources of fecal pollution in a rural Virginia watershed with antibiotic resistance patterns in fecal[J]. Applied and Environmental Microbiology, 1999,65(12):5522-5531.

[13] Simpson J M, Santo Domingo J W, Reasoner D J. Microbial source tracking: state of the science [J]. Environmental Science and Technology, 2002,36(24):5279-5288.

[14] Scott T M, Rose J B, Jenkins T M, et al. Microbial source tracking: current methodology and future directions [J]. Applied and Environmental Microbiology, 2002,68(12):5796-5803.

[15] 馮廣達(dá),鄧名榮,郭 俊,等.廣東某農(nóng)村塘壩飲用水污染的微生物溯源 [J]. 中國環(huán)境科學(xué), 2011,31(1):96-104.

[16] Allsop K, Stickler D J. An assessment offragilis group organisms as indicators of human faecal pollution [J]. Journal of Applied Microbiology, 1985,58(1):95-99.

[17] Fiksdal L, Maki J S, Lacroix S J, et al. Survival and detection ofspp. prospective indicator bacteria [J]. Applied and Environmental Microbiology, 1985,49(1):148-150.

[18] Hayashi H, Sakamoto M, Benno Y. Phylogenetic analysis of the human gut microbiota using 16S rDNA clone libraries and strictly anaerobic culture-based methods [J]. Microbiology and Immunology, 2002,46(8):535-548.

[19] Matsuki T, Watanabe K, Fujimoto J, et al. Use of 16s rRNA gene- targeted group-specific primers for real-time PCR analysis of predominant bacteria in human feces [J]. Applied and Environmental Microbiology, 2004,70(12):7220-7228.

[20] Hold G L, Pryde S E, Russell V J, et al. Assessment of microbial diversity in human colonic samples by 16S rDNA sequence analysis [J]. Fems Microbiology Ecology, 2002,39(1):33-39.

[21] Leser T D, Amenuvor J Z, Jensen T K, et al. Culture-independent analysis of gut bacteria: the pig gastrointestinal tract microbiota revisited [J]. Applied and Environmental Microbiology, 2002,68(2): 673-690.

[22] Avelar K E, Moraes S R, Pinto L J, et al. Influence of stress conditions onfragilis survival and protein profiles [J]. Zentralblatt Für Bakteriologie, 1998,287(4):399-409.

[23] Okabe S, Okayama N, Savichtcheva O, et al. Quantification of host-specific, 16S rRNA genetic markers for assessment of fecal pollution in freshwater [J]. Applied Microbiology and Biotechnology, 2007,74(4):890-901.

[24] Bernhard A E, Field K G. A PCR assay to discriminate human and ruminant feces on the basis of host differences in-genes encoding 16S rRNA [J]. Applied and Environmental Microbiology, 2000,66(10):4571-4574.

[25] 張 楊,吳仁人,張一敏,等.珠三角河網(wǎng)地區(qū)糞便污染源解析 [J]. 中國環(huán)境科學(xué), 2017,37(9):3446-3454.

[26] Kobayashi A, Sano D, Hatori J, et al. Chicken- and duck-associated, genetic markers for detecting fecal contamination in environmental water [J]. Applied Microbiology and Biotechnology, 2013,97(16):7427-7437.

[27] Bae S, Wuertz S. Rapid decay of host-specific fecalcells in seawater as measured by quantitative PCR with propidium monoazide [J]. Water Research, 2009,43(19):4850-4859.

[28] Liang Z B, He Z L, Zhou X X, et al. High diversity and differential persistence of fecalpopulation spiked into freshwater microcosm [J]. Water Research, 2012,46(1):247-257.

[29] Walters S P, Yamahara K M, Boehm A B. Persistence of nucleic acid markers of health-relevant organisms in seawater microcosms: implications for their use in assessing risk in recreational waters [J]. Water Research, 2009,43(19):4929-4939..

[30] Tambalo D D, Fremaux B, Boa T, et al. Persistence of host-associatedgene markers and their quantitative detection in an urban and agricultural mixed prairie watershed [J]. Water Research, 2012,46(9):2891-2904.

[31] Ballesté E, Blanch A R. Persistence ofspecies populations in a river as measured by molecular and culture techniques [J]. Journal of Applied Microbiology, 2010,76(22):7608-7616.

[32] Rocha E R, Herren C D, Smalley D J, et al. The complex oxidative stress response offragilis: the role of OxyR in control of gene expression [J]. Anaerobe, 2003,9(4):165-173.

[33] Brooks Y, Aslan A, Tamrakar S, et al. Analysis of the persistence of enteric markers in sewage polluted water on a solid matrix and in liquid suspension [J]. Water Research, 2015,76:201-212.

[34] Kreader C A. Persistence of PCR-detectable Bacteroides distasonis from human feces in river water [J]. Applied and Environmental Microbiology.1998,64(10):4103-4105.

[35] Seurinck S, Defoirdt T, Verstraete W, et al. Detection and quantification of the human-specific HF183, 16S rRNA genetic marker with real-time PCR for assessment of human faecal pollution in freshwater [J]. Environmental Microbiology, 2005,7(2): 249-259.

[36] Dick L K, Stelzer E A, Bertke E E, et al. Relative decay ofmicrobial source tracking markers and cultivatedin freshwater microcosms [J]. Applied and Environmental Microbiology, 2010,76(10):3255-3262.

[37] Okabe S, Shimazu Y. Persistence of host-specific16S rRNA genetic markers in environmental waters: effects of temperature and salinity [J]. Applied Microbiology and Biotechnology, 2007,76(4):935-944.

[38] Bell A, Layton A C, Mckay L, et al. Factors influencing the persistence of fecalin stream water [J]. Journal of Environmental Quality, 2009,38(3):1224-1232.

[39] Kobayashi A, Sano D, Okabe S. Effects of temperature and predator on the persistence of host-specificgenetic markers in water [J]. Water Science and Technology A Journal of the International Association on Water Pollution Research, 2013,67(4): 838-845.

[40] Barcina I, Lebaron P, VivesRego J. Survival of allochthonous bacteria in aquatic systems: a biological approach [J]. Fems Microbiology Ecology, 1997,23(1):1–9.

[41] Rozen Y, Belkin S. Survival of enteric bacteria in seawater [J]. Fems Microbiology Reviews, 2001,25(5):513-529.

[42] Walters S P, Field K G. Survival and persistence of human and ruminant-specific faecalin freshwater microcosms [J]. Environmental Microbiology, 2009,11(6):1410–1421.

[43] Green H C, Shanks O C, Sivaganesan M, et al. Differential decay of human faecalin marine and freshwater [J]. Environmental Microbiology, 2011,13(12):3235-3249.

[44] Zhu X Y, Zhong T Y, Pandya Y, et al. 16S rRNA-based analysis of microbiota from the cecum of broiler chickens [J]. Applied and Environmental Microbiology, 2002,68(1):124-137.

[45] Xenoulis P G, Gray P L, Brightsmith D, et al. Molecular characterization of the cloacal microbiota of wild and captive parrots [J]. Veterinary Microbiology, 2010,146(3/4):320-325.

[46] Ohad S, Ben-Dor S, Prilusky J, et al. The development of a novel qPCR assay-set for identifying fecal contamination originating from domestic fowls and waterfowl in Israel [J]. Frontiers in Microbiology, 2016,7(e893378):145.

[47] Ballesté E, Bonjoch X, Belanche L A, et al. Molecular indicators used in the development of predictive models for microbial source tracking [J]. Applied and Environmental Microbiology, 2010,76(6):1789-1795.

[48] 魏源送,鄭嘉熹,王光宇,等.地表水微生物溯源技術(shù)的開發(fā)和應(yīng)用進(jìn)展 [J]. 水資源保護(hù), 2016,32(1):1-11.

[49] Field K G, Chern E C, Dick L K, et al. A comparative study of culture-independent, library-independent genotypic methods of fecal source trackin [J]. Journal of Water and Health, 2003,1(4):181-194.

[50] Harwood V J, Wiggins B, Hagedorn C, et al. Phenotypic library-based microbial source tracking methods: efficacy in the California collaborative study [J]. Journal of Water and Health, 2003,1(4):153- 166.

[51] Myoda S P, Carson C A, Fuhrmann J J, et al. Comparison of genotypic-based microbial source tracking methods requiring a host origin database [J]. Journal of Water and Health, 2003,1(4):167-180.

[52] Blanch A R, Belanche-Munoz L, Bonjoch X, et al. Integrated analysis of established and novel microbial and chemical methods for microbial source tracking [J]. Applied and Environmental Microbiology, 2006, 72(9):5915-5926.

[53] Stewart J R, Boehm A B, Dubinsky E A, et al. Recommendations following a multi-laboratory comparison of microbial source tracking methods [J]. Water Research, 2013,47(18):6829-6838.

[54] Boehm A B, Van De Werfhorst L C, Griffith J F, et al. Performance of forty-one microbial source tracking methods: A twenty-seven lab evaluation study [J]. Water Research, 2013,47(18):6812-6828.

[55] Layton A, Mckay L, Williams D, et al. Development of16S rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water [J]. Applied and Environmental Microbiology, 2006,72(6):4214-4224.

[56] Mieszkin S, Yala J F, Joubrel R, et al. Phylogenetic analysis of16S rRNA gene sequences from human and animal effluents and assessment of ruminant faecal pollution by real-time PCR [J]. Journal of Applied Microbiology, 2010,108(3):974–984.

[57] Ahmed W, Goonetilleke A, Powell D, et al. Comparison of molecular markers to detect fresh sewage in environmental waters [J]. Water Research, 2009,43(19):4908-4917.

[58] Ahmed W, Masters N, Toze S. Consistency in the host specificity and host sensitivity of the, HF183marker for sewage pollution tracking [J]. Letters in Applied Microbiology, 2012,55(4):283–289.

[59] Layton B A, Cao Y, Ebentier D L, et al. Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study [J]. Water Research, 2013,47(18):6897-6908.

[60] Reischer G H, Ebdon J E, Bauer J M, et al. Performance characteristics of qPCR assays targeting human- and ruminant- associatedfor microbial source tracking across sixteen countries on six continents [J]. Environmental Science and Technology, 2013,47(15):8548-8556.

[61] Shanks O C, White K, Kelty C A, et al. Performance assessment PCR-based assays targetinggenetic markers of bovine fecal pollution [J]. Applied and Environmental Microbiology, 2010,76(5):1359-1366.

[62] Tambalo D D, Boa T, Liljebjelke K, et al. Evaluation of two quantitative PCR assays usingand mitochondrial DNA markers for tracking dog fecal contamination in waterbodies [J]. Journal of Microbiological Methods, 2012,91(3):459-467.

[63] Bernhard A E, Field K G. Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16s ribosomal DNA genetic markers from fecal anaerobes [J]. Applied and Environmental Microbiology, 2000,66(4):1587-1594.

[64] Manz W, Amann R, Ludwig W, et al. Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylumin the natural environment [J]. Microbiology, 1996,142(Pt5):1097-1106.

[65] Kildare B J, Leutenegger C M, Mcswain B S, et al. 16S rRNA-based assays for quantitative detection of universal, human-, cow-, and dog-specific fecal: a Bayesian approach [J]. Water Research, 2007,41(16):3701-3715.

[66] Reischer G H, Kasper D C, Steinborn R, et al. A quantitative real-time PCR assay for the highly sensitive and specific detection of human faecal influence in spring water from a large alpine catchment area [J]. Letters in Applied Microbiology, 2007,44(4):351-356.

[67] Shanks O C, Kelty C A, Sivaganesan M, et al. Quantitative PCR for genetic markers of human fecal pollution [J]. Applied and Environmental Microbiology, 2009,75(17):5507-5513.

[68] Mieszkin S, Furet J P, Corthier G, et al. Estimation of pig fecal contamination in a river catchment by real-time PCR using two pig-specific16S rRNA genetic markers [J]. Applied and Environmental Microbiology, 2009,75(10):3045-3054.

[69] Dick L K, Bernhard A E, Brodeur T J, et al. Host distributions of uncultivated fecalbacteria reveal genetic markers for fecal source identification [J]. Applied and Environmental Microbiology, 2005,71(6):3184-3191.

[70] Dick L K, Simonich M T, Field K G. Microplate subtractive hybridization to enrich forgenetic markers for fecal source identification [J]. Applied and Environmental Microbiology, 2005,71(6):3179-3183.

[71] Marti R, Zhang Y, Tien Y-C, et al. Assessment of a newmarker targeting North American beaver () fecal pollution by real-time PCR [J]. Journal of Microbiological Methods, 2013,95(2):201-206.

[72] Reischer G H, Kasper D C, Steinborn R, et al. Quantitative PCR method for sensitive detection of ruminant fecal pollution in freshwater and evaluation of this method in alpine karstic regions [J]. Applied and Environmental Microbiology, 2006,72(8):5610-5614.

[73] Shanks O C, Atikovic E, Blackwood A D, et al. Quantitative PCR for detection and enumeration of genetic markers of bovine fecal pollution [J]. Applied and Environmental Microbiology, 2008,74(3):745-752.

[74] Fremaux B, Boa T, Yost C K. Quantitative real-time PCR assays for sensitive detection of Canada goose-specific fecal pollution in water sources [J]. Applied and Environmental Microbiology, 2010,76(14): 4886-4889.

[75] Roslev P, Bukh A S. State of the art molecular markers for fecal pollution source tracking in water [J]. Applied Microbiology and Biotechnology, 2011,89(5):1341-1355.

[76] Lee D Y, Lauder H, Cruwys H, et al. Development and application of an oligonucleotide microarray and real-time quantitative PCR for detection of wastewater bacterial pathogens [J]. Science of the Total Environment, 2008,398(1-3):203-211.

[77] Lee D Y, Shannon K, Beaudette L A. Detection of bacterial pathogens in municipal wastewater using an oligonucleotide microarray and real-time quantitative PCR [J]. Journal of Microbiological Methods, 2006,65(3):453-467.

[78] Suzuki M, Rappe M S, Giovannoni S J. Kinetic bias in estimates of coastal picoplankton community structure obtained by measurements of small-subunit rRNA gene PCR amplicon length heterogeneity [J]. Applied and Environmental Microbiology, 1998,64(11):4522-4529.

[79] Liu W T, Marsh T L, Cheng H, et al. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA [J]. Applied and Environmental Microbiology, 1997,63(11):4516-4522.

[80] Farnleitner A H, Kreuzinger N, Kavka G G, et al. Simultaneous detection and differentiation ofpopulations from environmental freshwaters by means of sequence variations in a fragment of the beta-D-glucuronidase gene [J]. Applied and Environmental Microbiology, 2000,66(4):1340-1346.

[81] Ram J L, Ritchie R P, Fang J, et al. Sequence-based source tracking ofbased on genetic diversity of beta-glucuronidase [J]. Journal of Environmental Quality, 2004,33(3):1024-1032.

[82] Esseili M A, Kassem I I, Sigler V. Optimization of DGGE community fingerprinting for characterizing, communities associated with fecal pollution [J]. Water Research, 2008,42(17): 4467-4476.

[83] 張 曦,朱昌雄,馮廣達(dá),等.基于擬桿菌特異性16S rRNA基因的塘壩型飲用水污染溯源研究 [J]. 農(nóng)業(yè)環(huán)境科學(xué)學(xué)報(bào), 2011,30(9): 1880-1887.

[84] Jeong J Y, Park H D, Lee K H, et al. Quantitative analysis of human- and cow-specific 16s rRNA gene markers for assessment of fecal pollution in river waters by real-time PCR [J]. Journal of Microbiology and Biotechnology, 2010,20(2):245-253.

[85] Raith M R, Kelty C A, Griffith J F, et al. Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources [J]. Water Research, 2013, 47(18):6921-6928.

[86] Lee D Y, Weir S C, Lee H, et al. Quantitative identification of fecal water pollution sources by TaqMan real-time PCR assays using16S rRNA genetic markers [J]. Applied Microbiology and Biotechnology, 2010,88(6):1373-1383.

[87] Matsuki T, Watanabe K, Fujimoto J, et al. Development of 16S rRNA-gene-targeted group-specific primers for the detection and identification of predominant bacteria in human feces [J]. Applied and Environmental Microbiology, 2002,68(11):5445-5451.

[88] Bao N, Kassegne S K. DNA Microarrays [J]. Nanoscience Nanobiotechnology and Nanobiology, 2008,109(1):433-453.

[89] Indest K J, Betts K, Furey J S. Application of oligonucleotide microarrays for bacterial source tracking of environmentalsp. Isolates [J]. International Journal of Environmental Research and Public Health, 2005,2(1):175-185.

[90] Wang R F, Beggs M L, Robertson L H, et al. Design and evaluation of oligonucleotide-microarray method for the detection of human intestinal bacteria in fecal samples [J]. Fems Microbiology Letters, 2002,213(2):175-182.

[91] Straub T M, Daly D S, Wunshel S, et al. Genotypingwith an70single-nucleotide polymorphism microarray [J]. Applied and Environmental Microbiology, 2002,68(4):1817-1826.

[92] Maynard C, Berthiaume F, Lemarchand K, et al. Waterborne pathogen detection by use of oligonucleotide-based microarrays [J]. Applied and Environmental Microbiology, 2005,71(12):8548-8557.

[93] Li X, Harwood V J, Nayak B, et al. Ultrafiltration and microarray for detection of microbial source tracking marker and pathogen genes in riverine and marine systems [J]. Applied and Environmental Microbiology, 2016,82(5):1625-1635.

[94] Dubinsky E A, Esmaili L, Hulls J R, et al. Application of phylogenetic microarray analysis to discriminate sources of fecal pollution [J]. Environmental Science and Technology, 2012,46(8):4340-4347.

[95] Inoue D, Hinoura T, Suzuki N, et al. High-throughput DNA microarray detection of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley, Nepal [J]. Current Microbiology, 2015,70(1):43-50.

[96] Li X, Harwood V J, Nayak B, et al. A novel microbial source tracking microarray for pathogen detection and fecal source identification in environmental systems [J]. Environmental Science and Technology, 2015,49(12):7319-7329.

[97] Moore W E C, Holdeman L V. Human fecal flora: the normal flora of 20Japanese-Hawaiians [J]. Applied Microbiology, 1974,27(5):961- 979.

[98] Cerniglia C E, Kotarski S. Evaluation of veterinary drug residues in food for their potential to affect human intestinal microflora [J]. Regulatory Toxicology and Pharmacology, 1999,29(3):238-261.

致謝：感謝中國科學(xué)院大學(xué)常冬冬博士對本論文的審閱和修訂.

Research progress of microbial source tracking based on16S rRNA gene.

LIANG Hong-xia1, YU Zhi-sheng1*, LIU Ru-yin1, ZHANG Hong-xun1, WU Gang2

(1.College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China；2.Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China)., 2018,38(11)：4236~4245

Microbial source tracking (MST) is a method for distinguishing fecal contamination from different animals by identifying specific fecal microbes.is widely used in MST because of its high abundance in feces, non-reproduction, and host specificity. Given thats 16S rRNA gene is a common biomarker for MST, this paper reviewed the decay ofand its biomarkers, the sensitivity and specificity of the 16S rRNA gene primers for, and the application of molecular techniques in MST. It will provide the appropriate scientific reference for the source apportionment of feces.

microbial source tracking；；16S rRNA gene；fecal pollution

X172

A

1000-6923(2018)11-4236-10

梁紅霞(1990-),女,河南鹿邑人,碩士研究生,主要從事環(huán)境微生物方向的研究.

2018-04-08

國家重點(diǎn)研發(fā)計(jì)劃(2016YFC0503601);中國科學(xué)院戰(zhàn)略性先導(dǎo)科技專項(xiàng)(B類)(XDB15010200)

*責(zé)任作者, 教授, yuzs@ucas.ac.cn