基于擬桿菌16S rRNA基因進行微生物溯源的研究進展

梁紅霞,余志晟*,劉如銦,張洪勛,吳 鋼

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基于擬桿菌16S rRNA基因進行微生物溯源的研究進展

梁紅霞1,余志晟1*,劉如銦1,張洪勛1,吳 鋼2

(1.中國科學院大學資源與環境學院,北京 100049；2.中國科學院生態環境研究中心,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等優勢被廣泛應用于微生物溯源研究中.本文以擬桿菌16S rRNA基因為標記物,總結了擬桿菌及其標記物在環境中的衰減?擬桿菌引物的敏感性和特異性以及分子生物學技術在微生物溯源中的運用,可為糞便污染源解析提供一定的科學參考依據.

微生物溯源；擬桿菌；16S rRNA基因；糞便污染

近年來,隨著我國畜牧業的快速發展,糞便的產生量日益增加.產生的糞便因管理不善或雨水沖刷等原因進入水中,容易引發一系列健康和環境問題.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行傳播[5-6],進而增加傳染病爆發的風險;另一方面,糞便引起的水體污染難以治理.未經處理的糞便N?P含量較高,易引起水體富營養化,使原有水體喪失功能[1,7].那么,如何區分人和動物的糞便污染對污染源的確定至關重要[8-10],而微生物溯源技術可實現快速有效區分.

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境長期適應的過程中,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記,通過這種特定的標記即可判斷污染樣品和可能污染物之間是否存在聯系,從而實現糞便污染溯源[13-15].

在進行糞便污染溯源時,選擇合適的糞便污染指示微生物至關重要.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勢菌.糞便干重的1/3為細菌,擬桿菌占糞便細菌總數的30%~40%[18-19].每克人糞便中的擬桿菌細胞數約為109~1011個,比腸桿菌或腸球菌高4~7個數量級[17,20-21];擬桿菌不能在體外環境中繁殖.擬桿菌進入水體后無法繁殖,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系,進而確定污染源[23-24].

目前,大量研究表明以擬桿菌16S rRNA基因為標記物可成功區分哺乳動物、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上對相關內容進行歸納總結,以期為糞便污染的預防和微生物溯源技術在我國的發展提供一定幫助.

1 擬桿菌及其標記物的衰減

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間備受關注,許多學者進行了相關研究.大量研究表明,擬桿菌在環境中的存活時間較短,有些糞便中的擬桿菌在環境水體中能存活數小時至數天[27-28],擬桿菌標記物則可持續數周[17,29-30],故可以很好的指示水體短期內發生的污染[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,水體營養程度?溶解氧含量和擬桿菌培養基質的狀態等環境因素也會對擬桿菌及其標記物的衰減造成影響[31-33].

1.1 溫度

研究發現溫度越低,擬桿菌及其標記物的存在時間越長. Kreade[34]在河水中加入人的糞便模擬污水,發現擬桿菌標記物在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]在厭氧條件下培養人糞便污水,發現擬桿菌標記物HF183在4和12℃的條件下可持續24d,而在28℃的條件下僅持續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記物后也得出了類似結論.

1.2 捕食

水體中的原生微生物或原生動物對擬桿菌標記物存在一定的捕食行為,且捕食行為越強,擬桿菌標記物的衰減速度越快.即水中活性因素的影響越大,擬桿菌標記物的存在時間越短. 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,擬桿菌標記物AllBac的衰減.研究發現將水樣過濾后,擬桿菌標記物存在的時間更長. Dick等[36]研究發現捕食對人糞源擬桿菌標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]的研究也一致認為捕食是影響擬桿菌標記物存在的主要因素.

1.3 光照

光照對擬桿菌標記物衰減速度的影響,研究結果并不一致,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種擬桿菌及其標記物的影響.發現自然光并未明顯地影響擬桿菌和其DNA的衰減(牛糞源擬桿菌標記物BacCow-UCD除外). Walters等[42]的發現與上述研究結果一致,該研究發現在光照和黑暗兩種情況下,淡水中人糞源擬桿菌標記物HF134?HF183和反芻動物糞源標記物CF193的衰減行為并無明顯差別.然而, Dick等[36]研究發現暴露在太陽光下會加速人糞源擬桿菌標記物qHF183和BacHum的衰減. Walters等[29]的研究也發現人糞源擬桿菌標記物BacHum的衰減速度在自然光下比黑暗中更快. Green等[43]的發現與Dick等[36]和Walters等[29]的結果一致.

1.4 鹽度

鹽度越高,擬桿菌標記物的衰減速度越慢. Green等[43]分別研究了人糞源擬桿菌標記物在海水和淡水中的衰減行為,結果表明擬桿菌標記物在海水中的持續時間比在淡水中長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記物存在時間的影響.這4種標記物的衰減行為一致說明鹽度越高,擬桿菌標記物衰減速度越慢.值得說明的是此現象只出現在過濾后的水樣,而在未過濾水樣中則未出現,可能因為在未過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

不同動物的飲食習慣、生活環境及腸道系統有所差異,擬桿菌在與宿主長期適應的過程中,使得其16S rRNA基因表現出一定的宿主特異性[24].利用擬桿菌16S rRNA基因構建系統發育樹時,同種宿主糞源的擬桿菌群落表現出一定的相似性,不同宿主糞源的擬桿菌群落則出現分異,說明可根據擬桿菌標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.

本節總結前人利用擬桿菌16S rRNA基因所設計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記物能夠很好的區分人和反芻動物的糞便污染,但也存在一些問題,如關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

擬桿菌標記物是否適合指示某種目標宿主的糞便污染,可用擬桿菌特異性引物的敏感性和特異性衡量.當引物的敏感性值=100%時,說明引物對應的擬桿菌標記物存在于所有目標宿主糞便樣品中;特異性值=100%時,說明對應的擬桿菌標記物在所有非目標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,對應的標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)為目標宿主顯陽性的數量; FN(假陰性)為目標宿主顯陰性的數量; TN(真陰性)為非目標宿主顯陰性的數量; FP(假陽性)為非目標宿主顯陽性的數量.

為了比較不同微生物溯源標記物的效果,世界范圍內曾進行過3次大規模比選[48].(1) 2003年美國實驗室間的比較研究[49-51];(2) 2006年歐洲實驗室間的比較研究[52];(3) 2013年美國實驗室間的比較研究[53-54].研究表明,擬桿菌通用引物Bac32F和Bac708R所對應的標記物存在于各種動物糞便中,可利用該引物對擬桿菌16S rRNA基因進行擴增后再進行后續實驗[23,55-56];在多種人糞源擬桿菌標記物中, HF183表現出較強的敏感性和特異性[57-59];反芻動物標記物Rum-2-Bac和BacR在比選中表現出良好的特異性和敏感性,而且地理分布較廣[54,60]；但牛糞源擬桿菌標記物易與其他反芻動物或馬發生交叉反應,說明牛糞源標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計者實驗得出,在一定程度上可為后續研究者在選擇引物時提供參考.但不同擬桿菌標記物的地理分布有所差異,故不一定能重現這些數值. Reischer等[60]比較了人糞源標記物(BacH、BacHum)和反芻動物糞源標記物(BacCow、BacR和BoBac)在6個大洲的16個國家中的地理分布情況.5種標記物敏感性值分別是77%、87%、92%、90%和82%,說明牛糞源擬桿菌標記物地理分布最廣;特異性值分別為53%、68%、57%、84%和59%,說明這5種標記物均存在交叉反應,即在非目標宿主中也存在. Shanks等[61]對2種反芻動物(CF128和CF193)和5種牛(Bac2、Bac3、BoBac、CowM2和CowM3)糞源擬桿菌標記物進行比較,結果表明反芻動物糞源標記物CF128和牛糞源標記物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記物CanBac-UCD進行了評估,只有31%的特異性,與設計者的86.15%相差較大.

表1 擬桿菌通用引物和特異性引物

續表1

目標微生物標記物引物/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) 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

續表1

目標微生物標記物引物/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:/為未命名或數據未提供;-為數據不存在.

3 分子生物學技術在微生物溯源中的運用

在微生物溯源研究中，PCR技術常與其他技術結合使用以實現對污染物的定性分析,實時熒光定量PCR技術則常用于污染物的定量分析;基因芯片最大的特點是通量高,可同時檢測成千上萬個樣品,成功識別污染源的同時還可對樣品中微生物多樣性進行分析.以上方法各有優缺點,在實際研究工作中,可根據實驗目的選擇合適的方法,還可同時使用多種方法以增加實驗的可信度[75-77].

3.1 PCR及其聯用技術

3.1.1 PCR主要操作如下 使用擬桿菌引物進行PCR擴增,觀察目標條帶是否出現.使用擬桿菌通用引物時,出現目標條帶則說明樣品存在糞便污染;使用擬桿菌特異性引物時,出現目標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技術也存在一定的缺陷,如易產生非特異性擴增,只能對污染物定性而不能定量等.

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

3.1.3 變性梯度凝膠電泳(DGGE) 利用菌株的標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,進而分析被污染樣品與污染物之間的關系.目前, DGGE技術在以大腸桿菌為指示菌進行微生物溯源方面研究較多[80-82],而以擬桿菌為指示菌進行微生物溯源方面的相關研究國內外鮮有報道.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這兩種標記基因,經DGGE技術對塘壩型飲用水污染進行溯源,研究結果顯示擬桿菌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系.

3.2 實時熒光定量PCR技術(qPCR)

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

3.2.1 區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標記物對水中的人糞源污染進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記物進行溯源實驗,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記物進行TaqMan qPCR實驗,結果表明利用qPCR技術能可靠地識別和量化糞便污染,為流域水質管理和改進方面提供有效的信息.

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

3.2.3 發現糞便中的優勢菌 Matsuki等[19,87]利用多種特異性引物進行qPCR實驗,表明脆弱擬桿菌是人糞便中的優勢菌.

3.3 基因芯片技術

基因芯片又稱DNA芯片或DNA微陣列,原理是采用光導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、有序地固定于經過相應處理的硅片、玻片、硝酸纖維素膜等載體上,然后加入標記的待測樣品,進行多元雜交,通過雜交信號的強弱及分布,來分析目的分子的有無?數量及序列,從而獲得受檢樣品的遺傳信息[88].

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

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種不同的細菌16S rRNA基因探針進行檢測.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者的污染區分開,為加利福尼亞沿海水域提供污染源信息. Inoue等[95]調查加德滿都谷地的淺井地下水污染狀況時,以941個病原菌為對象進行基因芯片實驗,證明該地區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技術及二代測序技術相結合,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熒光基團標記人糞便中的腸道細菌的16S rRNA基因,熒光雜交結果說明糞便中主要的腸道細菌是普通擬桿菌、梭形桿菌屬、多型擬桿菌、瘤胃球菌屬、消化鏈球菌和真桿菌,與前人的研究結果一致[97-98].

基因芯片在微生物溯源研究中有許多優點.如:(1)通量高,可同時檢測成千上百個樣品,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.當然,它也存在一定的缺陷.如:(1)以16S rRNA基因為待測基因時,16S rRNA基因數據庫不足,導致探針設計存在缺陷;(2)成本和操作復雜度高.總體來說,基因芯片技術是一種快速有效判定污染物來源的技術,具有廣闊的應用空間.

4 結語

近年來,利用擬桿菌16S rRNA基因對糞便污染進行溯源已經取得了很大的進展,但仍面臨一些挑戰,現做如下總結和展望:

4.1 擬桿菌標記物可實現對人和反芻動物糞便污染的準確溯源,但擬桿菌在一些禽類和鳥類糞便中并不是優勢菌,故利用擬桿菌對禽類糞便污染溯源時,效果并不理想.需進一步發現其他標記物以實現鳥類和禽類糞便污染的準確溯源.

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

4.3 需盡快建立擬桿菌標記物含量與水體污染程度之間的對應關系,為政府問責及司法鑒定提供依據.

4.4 各國實驗室應加強合作,積極開展擬桿菌的地理分布研究,在世界范圍內確定敏感性和特異性效果最好的擬桿菌標記物.

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致謝：感謝中國科學院大學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,主要從事環境微生物方向的研究.

2018-04-08

國家重點研發計劃(2016YFC0503601);中國科學院戰略性先導科技專項(B類)(XDB15010200)

*責任作者, 教授, yuzs@ucas.ac.cn