TWI392740B - Method for identifying mycobacterium - Google Patents
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本發明係關於一種用於鑑定分枝桿菌(Mycobacterium spp.)之方法;特定言之,本發明係關於一種使用專一性探針鑑定分枝桿菌之方法。The present invention relates to a method for identifying Mycobacterium spp.; in particular, the present invention relates to a method for identifying mycobacteria using a specific probe.
由結核桿菌群引起之結核病(tuberculosis)全球每年約有920萬新案例,並造成約170萬人死亡,台灣每年亦有14,500至16,500個肺結核病例,故是全世界公共健康的大問題。The tuberculosis caused by Mycobacterium tuberculosis is about 9.2 million new cases each year and causes about 1.7 million deaths. Taiwan has 14,500 to 16,500 cases of tuberculosis every year, which is a major public health problem worldwide.
分枝桿菌屬包括絕對致病菌、兼性致病菌及腐生菌。目前已分離出將近100種分枝桿菌,其中跟人類疾病相關的有20多種,臨床上常見的如結核桿菌群(Mycobacterium tuberculosis comples,MTBC)、痲瘋分枝桿菌(M. leprae )、潰瘍分枝桿菌(M. ulcerans )、潰瘍腫分枝桿菌(M. abscessus )及鳥分枝桿菌群(M. avium complex)等(Murray等人,2005,Medical Microbiology,fifth ed,vol. Elsevier Mosby.)。Mycobacteria include absolute pathogenic bacteria, facultative pathogens, and saprophytic bacteria. Nearly 100 mycobacteria have been isolated, of which more than 20 are related to human diseases. Clinically common such as Mycobacterium tuberculosis comples (MTBC), M. leprae , and ulcers M. ulcerans , M. abscessus , and M. avium complex, etc. (Murray et al., 2005, Medical Microbiology, fifth ed, vol. Elsevier Mosby.) .
結核桿菌群包含以下菌種:非洲結核桿菌(Mycobacterium africanum )、牛結核桿菌BCG(M. bovis BCG)、牛結核桿菌(M. bovis )、卡柏結核桿菌(M. caprae )、坎結核桿菌(M. canettii )、邁克結核桿菌(M. microti )以及結核桿菌(M. tuberculosis )(Djelouadji等人,2008,PLoS Negl. Trop. Dis. 2:e253;Murray等人,2007,Manual of CLINICAL MICROBIOLOGY,9th ed,vol. 1),這些菌種彼此間的形態相近且皆會引發人類或動物結核病,其中以非洲結核桿菌、牛結核桿菌及結核桿菌較常造成人類肺結核病(Singleton及Sainsbury,2006,Dictionary of Microbiology and Molecular Biology,3rd ed,vol.),尤以結核桿菌為最。The Mycobacterium tuberculosis group includes the following species: Mycobacterium africanum , M. bovis BCG, M. bovis , M. caprae , and Mycobacterium tuberculosis ( M. canettii ), M. microti and M. tuberculosis (Djelouadji et al., 2008, PLoS Negl. Trop. Dis. 2: e253; Murray et al., 2007, Manual of CLINICAL MICROBIOLOGY, 9 th ed, vol. 1) These strains are similar in shape to each other and cause human or animal tuberculosis. Among them, tuberculosis, tuberculosis and tubercle bacilli in Africa cause tuberculosis in humans more often (Singleton and Sainsbury, 2006). , Dictionary of Microbiology and Molecular Biology, 3 rd ed, vol.), especially Mycobacterium tuberculosis.
正確鑑定MTBC中的菌種有助於結核病的流行病學調查,但因MTBC成員在基因上的相似性非常高(Sreevatsan等人,1997,Proc. Natl. Acad. Sci. USA 94:9869-9874),在16S rRNA基因中有99-100%的序列相似性,故此段基因的序列並不容易區分這些菌種;近年來,用來區分MTBC的目標基因包括pncA 、oxyR 、mtp40 、hupB 、gyrB 等(Liebana等人,1996,J. Clin. Microbiol. 34:933-938;Prabhakar等人,2004,J. Clin. Microbiol. 42:2724-2732;Scorpio及Zhang,1996,Nat. Med. 2:662-667;Sreevatsan等人,1996,J. Clin. Microbiol. 34:2007-2010),其中gyrB 基因已被證實是一個區分MTBC菌種的較佳基因(Chimara等人,2004,Mem. Inst. Oswaldo. Cruz. 99:745-748;Goh等人,2006,Mol. Cell. Probes. 20:182-190;Niemann等人,2000,J. Clin. Microbiol. 38:3231-3234)。Correct identification of strains in MTBC contributes to the epidemiological investigation of tuberculosis, but the genetic similarity of MTBC members is very high (Sreevatsan et al., 1997, Proc. Natl. Acad. Sci. USA 94:9869-9874 ), there is 99-100% sequence similarity in the 16S rRNA gene, so the sequence of this gene is not easy to distinguish these species; in recent years, the target genes used to distinguish MTBC include pncA , oxyR , mtp40 , hupB , gyrB Et al. (Liebana et al., 1996, J. Clin. Microbiol. 34: 933-938; Prabhakar et al., 2004, J. Clin. Microbiol. 42: 2724-2732; Scorpio and Zhang, 1996, Nat. Med. 2: 662-667; Sreevatsan et al., 1996, J. Clin. Microbiol. 34: 2007-2010), in which the gyrB gene has been shown to be a better gene for distinguishing MTBC species (Chimara et al., 2004, Mem. Inst. Oswaldo. Cruz. 99: 745-748; Goh et al., 2006, Mol. Cell. Probes. 20: 182-190; Niemann et al., 2000, J. Clin. Microbiol. 38: 3231-3234).
結核桿菌群之外的分枝桿菌,一般統稱為非結核桿菌群分枝桿菌(nontuberculous mycobacteria,NTM),大多數的NTM存於自然界的土壤或水中,少數則會造成人類或動物的伺機性感染(Field及Cowie,2006,Chest. 129:1653-1672),在免疫低下時較易感染致病,近年來臨床上NTM的分離率有逐漸提高的趨勢(Griffith,2007,Curr. Opin. Infect Dis. 20:198-203)。由於NTM在顯微鏡底下無法與結核桿菌區分,通常在培養分離後才能做菌種鑑定,故需要花費較長的鑑定時間。現今有一些結合分子技術的方法,例如:利用IS6110 限制性片段長度多樣性(IS6110 -restriction fragment length polymorphism,RFLP)(Kallenius等人,1999,J. Clin. Microbiol. 37:3872-3878)、熱休克蛋白65聚合酶連鎖反應及限制酶分析(hsp 65 PCR-restriction assay)(Telenti等人,1993,J. Clin. Microbiol. 31:175-178)、多位點可變數量串聯重複序列分析(mycobacterial interspersed repetitive-unit-variable-number tandem-repeat,MIRU-VNTR)或者間隔區寡核苷酸分型技術(spoligotyping)作分型(Aranaz等人,2004,J. Clin. Microbiol. 42:5388-5391;Cadmus等人,2006,J. Clin. Microbiol. 44:29-34;Godreuil等人,2007,J. Clin. Microbiol. 45:921-927;Kremer等人,2005,J. Clin. Microbiol. 43:314-320;Kulkarni等人,2005,Res. Microbiol. 156:588-596;Sola等人,2003,Infect. Genet. Evol. 3:125-133),但臨床上有時並未對NTM做進一步的鑑定。Mycobacteria other than Mycobacterium tuberculosis are generally referred to as nontuberculous mycobacteria (NTM). Most NTMs are found in natural soil or water, and a few can cause opportunistic infections in humans or animals. (Field and Cowie, 2006, Chest. 129: 1653-1672), which is more susceptible to infection when the immunity is low. In recent years, the clinical separation rate of NTM has gradually increased (Griffith, 2007, Curr. Opin. Infect Dis. 20:198-203). Since NTM cannot be distinguished from Mycobacterium tuberculosis under the microscope, it is usually only after culture and isolation that strain identification can be performed, so it takes a long time to identify. Today there are some methods molecule binding techniques, for example: the use of restriction fragment length diversity IS 6110 (IS 6110 -restriction fragment length polymorphism, RFLP) (Kallenius et al., 1999, J Clin Microbiol 37: ... 3872-3878) , heat shock protein 65 polymerase chain reaction and restriction enzyme assay ( hsp 65 PCR-restriction assay) (Telenti et al, 1993, J. Clin. Microbiol. 31: 175-178), multilocus variable number tandem repeats Analysis (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat, MIRU-VNTR) or spacer spoligotyping (Aranaz et al., 2004, J. Clin. Microbiol. 42: 5388-5391; Cadmus et al., 2006, J. Clin. Microbiol. 44:29-34; Godreuil et al., 2007, J. Clin. Microbiol. 45:921-927; Kremer et al., 2005, J. Clin. Microbiol. 43:314-320; Kulkarni et al., 2005, Res. Microbiol. 156:588-596; Sola et al., 2003, Infect. Genet. Evol. 3:125-133), but sometimes not clinically Further identification of NTM.
由於分枝桿菌生長緩慢,一般而言,臨床上一般需要2週至8週的時間才能鑑定分枝桿菌,耗時相當長。近年來,發展許多的分子生物檢測法及套組,例如INNO-LiPAMYCOBACTERIA(Innogenetics,Ghent,Belgium),使用分枝桿菌的rRNA operon作為目標序列(Suffys等人,2001,J. Clin. Microbiol. 39:4477-4482;Tortoli等人,2003,J. Clin. Microbiol. 41:4418-4420;Trombert-Paolantoni等人,2004,Pathol. Biol.(Paris) 52:462-468),可區分出結核桿菌群與16種非結核分枝桿菌;GenoType Mycobacterium CM/AS(Hain Lifesience GmbH,Nehren,Germany)則使利用23S rRNA基因作為其鑑定菌種之目標(Makinen等人,2006,Clin. Microbiol. Infect. 12:481-483;Richter等人,2006,J. Clin. Microbiol. 44:1769-1775),可區分出結核桿菌群與14種( Mycobacterium CM)或16種( Mycobacterium AS)非結核分枝桿菌;Gen-Probe AmplifiedMycobacterium tuberculosis direct test(Gen-Probe,Inc.,San Diego,CA,USA)則以16S rRNA為標的檢測結核桿菌群(Chedore及Jamieson,2002,Int. J. Tuberc. Lung Dis. 6:913-919;Lemaitre等人,2004,J. Clin. Microbiol. 42:4307-4309;Sloutsky等人,2004,J. Clin. Microbiol. 42:1547-1551);而AmplicorTM nucleic acid amplification test(Roche Diagnostic Systems,Inc.,Branchburg,NJ,USA)則是利用16S rRNA為目標檢測結核桿菌群(Bergmann及Woods,1996,J. Clin. Microbiol. 34:1083-1085;Michos等人,2006,Diagn. Microbiol. Infect Dis. 54:121-126)。LCx MTB assay,ABBOTT LCx probe system(Abbott Laboratories,Abbott Park,Ill.)(Rohner等人,1998,J. Clin. Microbiol. 36:3046-3047)利用連接酶連鎖反應偵測結核桿菌群;BD ProbeTec energy transfer(ET) system(DTB)(Becton Dickinson Biosciences Microbiology Products,Sparks,MD,USA)(Barrett等人,2002,J. Med. Microbiol. 51:895-898)利用IS6110 與16S rRNA基因為目標檢測結核分枝桿菌群。Due to the slow growth of mycobacteria, it is generally clinically necessary to take 2 weeks to 8 weeks to identify mycobacteria, which takes a long time. In recent years, many molecular bioassays and kits have been developed, such as INNO-LiPAMYCOBACTERIA (Innogenetics, Ghent, Belgium), using rRNA operon of mycobacteria as a target sequence (Suffys et al., 2001, J. Clin. Microbiol. 39 : 4477-4482; Tortoli et al, 2003, J. Clin. Microbiol. 41: 4418-4420; Trombert-Paolantoni et al, 2004, Pathol. Biol. (Paris) 52: 462-468), can distinguish between Mycobacterium tuberculosis Group with 16 non-tuberculous mycobacteria; GenoType Mycobacterium CM/AS (Hain Lifesience GmbH, Nehren, Germany) used the 23S rRNA gene as a target for its identification (Makinen et al., 2006, Clin. Microbiol. Infect. 12:481-483; Richter et al., 2006). , J. Clin. Microbiol. 44:1769-1775), can distinguish between Mycobacterium tuberculosis and 14 species ( Mycobacterium CM) or 16 species ( Mycobacterium AS) non-tuberculous mycobacteria; Gen-Probe Amplified Mycobacterium tuberculosis direct test (Gen-Probe, Inc., San Diego, CA, USA) for detection of Mycobacterium tuberculosis using 16S rRNA as the target (Chedore and Jamieson, 2002, Int J. Tuberc. Lung Dis. 6: 913-919; Lemaitre et al., 2004, J. Clin. Microbiol. 42: 4307-4309; Sloutsky et al., 2004, J. Clin. Microbiol. 42: 1547-1551) ; the Amplicor TM nucleic acid amplification test (Roche Diagnostic Systems, Inc., Branchburg, NJ, USA) using the detection of Mycobacterium tuberculosis 16S rRNA as the target group (Bergmann and Woods, 1996, J Clin Microbiol 34 :... 1083- 1085; Michos et al., 2006, Diagn. Microbiol. Infect Dis. 54: 121-126). LCX MTB assay, ABBOTT LCx probe system (Abbott Laboratories, Abbott Park, Ill.) (Rohner et al., 1998, J. Clin. Microbiol. 36: 3046-3047) detection of Mycobacterium tuberculosis using a ligase linkage reaction; BD ProbeTec Energy transfer (ET) system (DTB) (Becton Dickinson Biosciences Microbiology Products, Sparks, MD, USA) (Barrett et al, 2002, J. Med. Microbiol. 51: 895-898) targeting IS 6110 and 16S rRNA genes Detection of Mycobacterium tuberculosis.
目前亦有許多研究以液態培養基BACTEC MGIT 960陽性反應培養並確認抗酸性染色為陽性後,再結合分子生物方法鑑定其中的分枝桿菌。包括使用rpoB 基因雙套式PCR及限制酶分析(rpo B duplex PCR-restriction enzyme analysis)、光反應性與兩種生化反應,可區分29種NTM(Shen等人,2009,Int. J. Tuberc. Lung Dis. 13:472-479)。也有研究利用抗酸性染色為陽性之MGIT陽性反應管,進行以IS6110 為標的的PCR反應,成功鑑定其中MTBC。由痰塗片抗酸性染色陽性與陰性之檢測時間可分別縮短至6.4天與14.3天(Sun等人,2009,J. Formos. Med. Assoc. 108:119-125)。先前提到利用PCR來擴增hsp65 並配合RFLP技術鑑定分枝桿菌,該技術雖已廣為使用,但在培養過程仍要花不少時間,若直接消化經去汙染之臨床檢體,則常因菌量不足使得敏感度下降。Chang等人結合了毛細管電泳以及巢式聚合酶連鎖反應(nested-PCR)來增加hsp65 及RFLP之敏感度(Chang等人,2008,Talanta. 77:182-188)。台灣林口長庚醫院Wu等人的研究顯示以hsp65 為標的,以nested-PCR和RFLP,檢測陽性痰塗片檢體中的MTBC及部分NTM,有不錯的效果(Wu等人,2008,J. Clin. Microbiol. 46:3591-3594)。At present, there are many studies in which the liquid medium BACTEC MGIT 960 positive reaction culture and confirmation of acid-fast staining is positive, and then combined with molecular biological methods to identify the mycobacteria. Comprising rpoB gene using double-nested PCR and restriction enzyme analysis (rpo B duplex PCR-restriction enzyme analysis), a photoreactive biochemical reactions and Two, distinguishable 29 kinds of NTM (Shen et al., 2009, Int. J. Tuberc. Lung Dis. 13:472-479). It has also been studied to use the MGIT-positive reaction tube positive for acid-fast staining, and the PCR reaction with IS 6110 as the target was successfully carried out to identify MTBC. The detection time for positive and negative anti-acid staining of sputum smear was shortened to 6.4 days and 14.3 days, respectively (Sun et al., 2009, J. Formos. Med. Assoc. 108: 119-125). It was previously mentioned that PCR was used to amplify hsp65 and combined with RFLP technology to identify mycobacteria. Although this technique has been widely used, it still takes a lot of time in the culture process. If the decontaminated clinical specimen is directly digested, it is often The sensitivity is reduced due to insufficient bacteria. Chang et al. combined capillary electrophoresis and nested polymerase chain reaction (nested-PCR) to increase the sensitivity of hsp65 and RFLP (Chang et al, 2008, Talanta. 77: 182-188). Wu et al., from Chang Gung Hospital, Linkou, Taiwan, showed that with hsp65 as the target, it was good to detect MTBC and some NTM in positive smear samples by nested-PCR and RFLP (Wu et al., 2008, J. Clin). Microbiol. 46:3591-3594).
然而上述方法多有操作複雜及耗時長之缺點,且許多方法都無法鑑定出菌種,正確率與靈敏度亦不甚令人滿意,業界仍須開發可正確鑑定分枝桿菌之方法。However, the above methods have many disadvantages such as complicated operation and long time-consuming, and many methods cannot identify the species, and the accuracy and sensitivity are not satisfactory. The industry still needs to develop a method for correctly identifying mycobacteria.
本發明之目的在於提供一種鑑定分枝桿菌之方法,該方法包括使Myc2及Myc3之探針或其互補股(complementary strands)與待測樣品中之DNA進行雜合反應,如有至少一雜合反應發生,則該待測樣品包含分枝桿菌。It is an object of the present invention to provide a method for identifying mycobacteria, which comprises subjecting a probe of Myc2 and Myc3 or a complementary strand thereof to a heterozygous reaction with DNA in a sample to be tested, if at least one is heterozygous When the reaction occurs, the sample to be tested contains mycobacteria.
本發明之另一目的在於提供一種探針,其包含選自由下列探針及其互補股所組成之群:Myc2、Myc3、MTBC2、MTBC4、MbovG1、MbovGW1、Mbov1、MbovW1、Mcan1、McanW1、Mcap1、McapW1、Mmic1、MmicW1、Mtub1R、MtubW1R、Mabs1、Mavi1、Mche3、Mfor1、Mgas2、Mgor1、Mint2、Mkan1、Msi/le1、Mma/ul2、Mnon1、Mper1、Mscr1、Msme3、Mszu1、Mter1及Mxen2。Another object of the present invention is to provide a probe comprising a group selected from the group consisting of the following probes and their complementary strands: Myc2, Myc3, MTBC2, MTBC4, MbovG1, MbovGW1, Mbov1, MbovW1, Mcan1, McanW1, Mcap1 McapW1, Mmic1, MmicW1, Mtub1R, MtubW1R, Mabs1, Mavi1, Mche3, Mfor1, Mgas2, Mgor1, Mint2, Mkan1, Msi/le1, Mma/ul2, Mnon1, Mper1, Mscr1, Msme3, Mszu1, Mter1 and Mxen2.
本發明之又一目的在於提供一種用於鑑定一樣品中分枝桿菌之套組,該套組包括前述之探針。It is still another object of the present invention to provide a kit for identifying mycobacteria in a sample comprising the aforementioned probe.
根據本發明之方法鑑定的正確率相當高,縮短鑑定時間,並使鑑定之流程一致化,並容易朝向簡單化、快速化、和自動化,可藉由正確的菌種鑑定,於臨床上施以正確的抗生素治療。The correct rate of identification according to the method of the invention is quite high, the identification time is shortened, the identification process is consistent, and the simplification, rapidization, and automation are easy to be performed, and can be clinically applied by correct strain identification. Correct antibiotic treatment.
本發明之目的在於提供一種鑑定分枝桿菌之方法,該方法包括使Myc2及Myc3之探針或其互補股與待測樣品中之DNA進行雜合反應,如有至少一雜合反應發生,則該待測樣品包含分枝桿菌。The object of the present invention is to provide a method for identifying mycobacteria, which comprises subjecting a probe of Myc2 and Myc3 or a complementary strand thereof to a heterozygous reaction with DNA in a sample to be tested, if at least one heterozygous reaction occurs, The sample to be tested contains mycobacteria.
本發明所使用之探針資訊如表1所示。The probe information used in the present invention is shown in Table 1.
特異性探針命名方式是取分枝桿菌屬名(genus)的一個開頭大寫英文字母”M”加上種名(species)前三個小寫英文字母,最後再加上探針的序號,若為負股(anti-sense)探針會再加上大寫字母R。有19個探針選自rRNA基因間隔區(intergenic spacer region,ITS)中;另外12個探針用來區分MTBC內的菌種,設計自各菌種之gyrB 基因特有的單一位點核苷酸多型性(single nucleotide polymorphism,SNP),以兩個探針為一組,一個是特異性探針,命名方式如前述,另一個是除了目標菌種以外的其他MTBC菌種探針,加大寫字母W以作區別。The specific probe naming method is to take the uppercase English letter "M" of the genus genus and the first three lowercase English letters of the species, and finally add the serial number of the probe. The anti-sense probe will be preceded by the capital letter R. There are 19 probes selected from the rRNA intergenic spacer region (ITS); the other 12 probes are used to distinguish the strains in the MTBC, and the single nucleotides unique to the gyrB gene of each species are designed. Single nucleotide polymorphism (SNP), which consists of two probes, one is a specific probe, named as described above, and the other is a probe of other MTBC species other than the target species. W to make a difference.
探針設計原則為該探針所在位置序列不常發生變異及:The probe design principle is that the sequence of the probe is infrequently mutated and:
1.探針長度介於17~27個核苷酸是最理想的範圍,探針過短會造成與目標DNA結合不易,過長容易產生非特異性的雜合反應;1. The length of the probe is between 17 and 27 nucleotides, which is the most ideal range. If the probe is too short, it will cause difficulty in binding to the target DNA, and too long will cause non-specific heterozygous reaction;
2.G+C比例介於40%至60%,以降低二級結構產生的機率;2. The ratio of G+C is between 40% and 60% to reduce the probability of secondary structure generation;
3.探針T m 值(melting temperature)盡可能的設計在介於雜合反應(hybridization)時的雜合溫度(hybridization temperature)±5℃之間。例如雜合溫度若設定在50℃,則探針的T m 值應該盡量為45℃~55℃;3. The probe T m value (melting temperature) at the design temperature is between heterozygous hybrid reactor (Hybridization) of (hybridization temperature) ± 5 ℃ between possible. For example, if the hybrid temperature is set at 50 ° C, the T m value of the probe should be as much as possible 45 ° C ~ 55 ° C;
4.盡量避開可能會產生髮夾環結構(hairpin loops)、迴文結構(palindrome)或是重複鹼基(repeats)結構;4. Try to avoid hairpin loops, palindromes or repeats.
5.把差異點設計在整個探針的中間位置,並在探針之3’端加入8~10個胸腺嘧啶(thymine),以增加探針和尼龍膜(nylon membrane)的結合;5. Design the difference point in the middle of the probe and add 8-10 thymines at the 3' end of the probe to increase the binding of the probe to the nylon membrane;
設計完的探針經過BLAST搜索,以確定沒有與GenBank上其他菌種的序列相似,避免交叉反應(cross hybridization)。The designed probes were searched by BLAST to determine that they were not similar to the sequences of other strains on GenBank, avoiding cross hybridization.
本文所使用之「探針」一詞係指一包含連續至少8個核苷酸的分子,較佳為連續10至50個核苷酸,更佳為連續15至40個核苷酸,最佳為連續17至27個核苷酸。另一方面,較佳地,探針之3’端包含8至10個胸腺嘧啶。該探針可與標的DNA於雜合條件下進行雜合反應。本發明所屬技術領域中具通常知識者可決定雜合反應之條件,其中該雜合反應之較佳條件係於約40℃至約65℃中進行。As used herein, the term "probe" refers to a molecule comprising at least 8 nucleotides in succession, preferably 10 to 50 nucleotides in length, more preferably 15 to 40 nucleotides in length, optimal. It is 17 to 27 nucleotides in succession. On the other hand, preferably, the 3' end of the probe comprises 8 to 10 thymines. The probe can be hybridized with the target DNA under heterozygous conditions. Those of ordinary skill in the art to which the present invention pertains can determine the conditions of the hybridization reaction, wherein preferred conditions for the hybridization reaction are carried out at a temperature of from about 40 ° C to about 65 ° C.
本文所使用之「互補股」一詞係指可與根據本發明之探針雜合之核酸分子,較佳係可與根據本發明之探針鹼基完全互補之核酸分子。The term "complementary strand" as used herein refers to a nucleic acid molecule which can be hybridized to a probe according to the invention, preferably a nucleic acid molecule which is fully complementary to the base of the probe according to the invention.
根據本發明之探針可用以鑑定分枝桿菌,由於分枝桿菌普遍生長緩慢,用傳統的培養法需要較長的時間,且部分的分枝桿菌不易鑑定。先前研究指出,細菌的16S rRNA及23S rRNA二個基因間隔區在序列上具有種的特異性(species-specific),有時比利用16S rRNA基因定序法更能區分至種的層次。且rRNA基因間隔區序列在種內不同菌株間的變異性很小(Gurtler及Stanisich,1996,Microbiology. 142(Pt 1):3-16),因此,本發明主要利用rRNA基因間隔區作為分枝桿菌鑑定之目標。The probe according to the present invention can be used to identify mycobacteria. Since mycobacteria generally grow slowly, it takes a long time to use conventional culture methods, and some mycobacteria are difficult to identify. Previous studies have pointed out that the bacterial 16S rRNA and 23S rRNA two gene spacers are species-specific in sequence, and sometimes more distinguishable to the species level than 16S rRNA gene sequencing. Moreover, the rRNA gene spacer sequence has little variability among different strains within the species (Gurtler and Stanisich, 1996, Microbiology. 142 (Pt 1): 3-16). Therefore, the present invention mainly utilizes the rRNA gene spacer as a branch. The target of bacillus identification.
較佳地,根據本發明之方法可進一步區分結核桿菌群與非結核分枝桿菌群,其另包含使用MTBC2及MTBC4之探針或其互補股與該待測樣品中之DNA進行雜合反應,如該待測樣品中之DNA同時與MTBC2及MTBC4之探針或其互補股產生雜合反應,則該待測樣品包含結核桿菌群;如該待測樣品中之DNA未同時與MTBC2及MTBC4之探針或其互補股產生雜合反應,則該待測樣品包含非結核分枝桿菌。Preferably, according to the method of the present invention, the Mycobacterium tuberculosis group and the non-tuberculous mycobacterial group can be further distinguished, and the probe further comprising a probe using MTBC2 and MTBC4 or a complementary strand thereof is subjected to a heterozygous reaction with the DNA in the sample to be tested, If the DNA in the sample to be tested simultaneously generates a hybrid reaction with the probe of MTBC2 and MTBC4 or its complementary strand, the sample to be tested contains Mycobacterium tuberculosis; if the DNA in the sample to be tested is not simultaneously with MTBC2 and MTBC4 The probe or its complementary strands produce a heterozygous reaction, and the sample to be tested comprises non-tuberculous mycobacteria.
更佳地,根據本發明之方法可進一步針對已鑑定為結核桿菌群之待鑑定菌鑑定出其種名。由於結核桿菌群的rRNA基因間隔區具100%的相似度,故利用MTBC在gyrB 基因中有多個單一位點核苷酸多型性做該群中不同菌種的鑑定。More preferably, according to the method of the present invention, the species name of the strain to be identified which has been identified as Mycobacterium tuberculosis can be further identified. Since the rRNA gene spacer of the Mycobacterium tuberculosis group has 100% similarity, MTBC has multiple single-site nucleotide polymorphisms in the gyrB gene to identify different strains in the group.
根據本發明之方法較佳另包含使用Mtub1R及MtubW1R之探針或其互補股與待測樣品中之DNA進行雜合反應,如該待測樣品中之DNA與Mtub1R或其互補股雜合反應之強度,大於該待測樣品中之DNA與MtubW1R或其互補股雜合反應之強度,則該待測樣品包含結核桿菌(M. tuberculosis )。Preferably, the method according to the present invention further comprises using a probe of Mtub1R and MtubW1R or a complementary strand thereof to carry out a hybrid reaction with the DNA in the sample to be tested, for example, the DNA in the sample to be tested is heterozygously reacted with Mtub1R or its complementary strand. The intensity is greater than the intensity of the heterozygous reaction of the DNA in the sample to be tested with MtubW1R or its complementary strand, and the sample to be tested comprises M. tuberculosis .
根據本發明之方法較佳另包含使用MbovG1及MbovGW1之探針或其互補股與該待測樣品中之DNA進行雜合反應,如該待測樣品中之DNA與MbovG1或其互補股雜合反應之強度,大於該待測樣品中之DNA與MbovGW1或其互補股雜合反應之強度,則該待測樣品包含牛結核桿菌群(M. bovis group)。較佳地,該牛結核桿菌群包含牛結核桿菌(M. bovis )、牛結核桿菌BCG(M. bovis BCG)及卡柏結核桿菌(M. caprae )。於本發明之一較佳具體實施例中,可鑑定出牛結核桿菌群之個別菌種。根據本發明之方法,其較佳另包含使用Mbov1及MbovW1之探針或其互補股與該待測樣品中之DNA進行雜合反應,如該待測樣品中之DNA與Mbov1或其互補股雜合反應之強度,大於該待測樣品中之DNA與MbovW1或其互補股雜合反應之強度,則該待測樣品包含牛結核桿菌或牛結核桿菌BCG;根據本發明之方法,其較佳另包含使用Mcap1及McapW1之探針或其互補股與該待測樣品中之DNA進行雜合反應,如該待測樣品中之DNA與Mcap1或其互補股雜合反應之強度,大於該待測樣品中之DNA與McapW1或其互補股雜合反應之強度,則該待測樣品包含卡柏結核桿菌。Preferably, the method according to the present invention further comprises using a probe of MbovG1 and MbovGW1 or a complementary strand thereof to carry out a heterozygous reaction with the DNA in the sample to be tested, such as the heterozygous reaction of the DNA in the sample to be tested with MbovG1 or its complementary strand. The intensity is greater than the intensity of the heterozygous reaction of the DNA in the sample to be tested with MbovGW1 or its complementary strand, and the sample to be tested comprises the M. bovis group. Preferably, the Bovine tuberculosis population comprises M. bovis , M. bovis BCG and M. caprae . In a preferred embodiment of the invention, individual strains of the Mycobacterium tuberculosis population can be identified. Preferably, the method according to the present invention further comprises using a probe of Mbov1 and MbovW1 or a complementary strand thereof to carry out a hybrid reaction with the DNA in the sample to be tested, such as DNA in the sample to be tested and Mbov1 or its complementary strand. The intensity of the reaction is greater than the intensity of the heterozygous reaction of the DNA in the sample to be tested with MbovW1 or its complementary strand, and the sample to be tested comprises Bovine M. tuberculosis or Bovine M. tuberculosis BCG; according to the method of the invention, preferably The probe comprising Mcap1 and McapW1 or a complementary strand thereof is subjected to a heterozygous reaction with the DNA in the sample to be tested, and the intensity of the hybridization reaction between the DNA in the sample to be tested and Mcap1 or its complementary strand is greater than the sample to be tested. The strength of the hybridization reaction between the DNA and McapW1 or its complementary strands, the sample to be tested contains K. tuberculosis.
根據本發明之方法,其較佳另包含使用Mmic1及MmicW1之探針或其互補股與該待測樣品中之DNA進行雜合反應,如該待測樣品中之DNA與Mmic1或其互補股雜合反應之強度,大於該待測樣品中之DNA與MmicW1或其互補股雜合反應之強度,則該待測樣品包含邁克結核桿菌(M. microti )。Preferably, the method according to the present invention further comprises using a probe of Mmic1 and MmicW1 or a complementary strand thereof to carry out a hybrid reaction with the DNA in the sample to be tested, such as DNA in the sample to be tested and Mmic1 or its complementary strand. The intensity of the reaction is greater than the intensity of the heterozygous reaction of the DNA in the sample to be tested with MmicW1 or its complementary strand, and the sample to be tested comprises M. microti .
根據本發明之方法,其較佳另包含使用Mcan1及McanW1之探針或其互補股與該待測樣品中之DNA進行雜合反應,如該待測樣品中之DNA與Mcan1或其互補股雜合反應之強度,大於該待測樣品中之DNA與McanW1或其互補股雜合反應之強度,則該待測樣品包含坎結核桿菌(M. canettii )。Preferably, the method according to the present invention further comprises using a probe of Mcan1 and McanW1 or a complementary strand thereof to carry out a hybrid reaction with the DNA in the sample to be tested, such as DNA in the sample to be tested and Mcan1 or its complementary strand. The intensity of the reaction is greater than the intensity of the heterozygous reaction of the DNA in the sample to be tested with McanW1 or its complementary strand, and the sample to be tested comprises M. canettii .
根據本發明之方法,其較佳另包含使用第一群探針或其互補股及第二群探針或其互補股與該待測樣品中之DNA進行雜合反應,如該待測樣品中之DNA與第二群探針或其互補股雜合反應之強度,皆大於該待測樣品中之DNA與第一群探針或其互補股雜合反應之強度,則該待測樣品包含非洲結核桿菌(M. africanum ),其中該第一群探針包含Mtub1R、MbovG1、Mbov1、Mcap1、Mmic1及Mcan1;第二群探針包含MtubW1R、MbovGW1、MbovW1、McapW1、MmicW1及McanW1。Preferably, the method according to the present invention further comprises performing a hybrid reaction with the DNA in the sample to be tested using the first group probe or its complementary strand and the second group probe or its complementary strand, such as in the sample to be tested The intensity of the heterozygous reaction between the DNA and the second group of probes or their complementary strands is greater than the intensity of the heterozygous reaction of the DNA in the sample to be tested with the first group of probes or their complementary strands, and the sample to be tested comprises Africa. Mycobacterium tuberculosis ( M. africanum ), wherein the first group of probes comprises Mtub1R, MbovG1, Mbov1, Mcap1, Mmic1 and Mcan1; and the second group of probes comprises MtubW1R, MbovGW1, MbovW1, McapW1, MmicW1 and McanW1.
另一方面,更佳地,根據本發明之方法可進一步針對已鑑定為非結核分枝桿菌之待鑑定菌鑑定出其種名,其主要利用rRNA基因間隔區所設計之探針而進行。On the other hand, more preferably, the method according to the present invention can further identify the species name of the strain to be identified which has been identified as non-tuberculous mycobacteria, which is mainly carried out using a probe designed by the rRNA gene spacer.
根據本發明之方法,其較佳另包含使用一或多個選自Mabs1、Mavi1、Mche3、Mfor1、Mgas2、Mgor1、Mint2、Mkan1、Msi/le1、Mma/ul2、Mnon1、Mper1、Mscr1、Msme3、Mszu1、Mter1、Mxen2及其互補股之探針與該待測樣品中之DNA進行雜合反應;如該該待測樣品中之DNA與Mabs1探針或其互補股產生雜合反應,則該待測樣品包含潰瘍腫分枝桿菌(M. abscessus );如該待測樣品中之DNA與Mavi1探針或其互補股產生雜合反應,則該待測樣品包含鳥分枝桿菌(M. avium );如該待測樣品中之DNA與Mche3探針或其互補股產生雜合反應,則該待測樣品包含龜分枝桿菌(M. chelonae );如該待測樣品中之DNA與Mfor1探針或其互補股產生雜合反應,則該待測樣品包含偶然分枝桿菌(M. fortuitum );如該待測樣品中之DNA與Mgas2探針或其互補股產生雜合反應,則該待測樣品包含胃分枝桿菌(M. gastri );如該待測樣品中之DNA與Mgor1探針或其互補股產生雜合反應,則該待測樣品包含戈登分枝桿菌(M. gordonae );如該待測樣品中之DNA與Mint2探針或其互補股產生雜合反應,則該待測樣品包含胞內分枝桿菌(M. intracellulare );如該待測樣品中之DNA與Mkan1探針或其互補股產生雜合反應,則該待測樣品包含堪薩斯分枝桿菌(M. kansasii );如該待測樣品中之DNA與Msi/le1探針或其互補股產生雜合反應,則該待測樣品包含猿分枝桿菌(M. simiae )或蘭分枝桿菌(M. lentiflavum );如該待測樣品中之DNA與Mma/ul2探針或其互補股產生雜合反應,則該待測樣品包含海洋分枝桿菌(M. marinum )或潰瘍分枝桿菌(M. ulcerans );如該待測樣品中之DNA與Mnon1探針或其互補股產生雜合反應,則該待測樣品包含不產色分枝桿菌(M. nonchromogenicum );如該待測樣品中之DNA與Mper1探針或其互補股產生雜合反應,則該待測樣品包含培力分枝桿菌(M. peregrinum );如該待測樣品中之DNA與Mscr1探針或其互補股產生雜合反應,則該待測樣品包含瘰癘分枝桿菌(M. scrofulaceum );如該待測樣品中之DNA與Msme3探針或其互補股產生雜合反應,則該待測樣品包含恥垢分枝桿菌(M. smegmatis );如該待測樣品中之DNA與Mszu1探針或其互補股產生雜合反應,則該待測樣品包含斯氏分枝桿菌(M. szulgai );如該待測樣品中之DNA與Mter1探針或其互補股產生雜合反應,則該待測樣品包含土地分枝桿菌(M. terrae );及如該待測樣品中之DNA與Mxen2探針或其互補股產生雜合反應,則該待測樣品包含蟾蜍分枝桿菌(M. xenopi )。According to the method of the present invention, it is preferred to further comprise one or more selected from the group consisting of Mabs1, Mavi1, Mche3, Mfor1, Mgas2, Mgor1, Mint2, Mkan1, Msi/le1, Mma/ul2, Mnon1, Mper1, Mscr1, Msme3, Mszu1, Mter1, Mxen2 and their complementary strand probes are heterozygously reacted with the DNA in the sample to be tested; if the DNA in the sample to be tested is heterozygous with the Mabs1 probe or its complementary strand, then The test sample comprises M. abscessus ; if the DNA in the sample to be tested is heterozygous with the Mavi1 probe or its complementary strand, the sample to be tested comprises M. avium . If the DNA in the sample to be tested is heterozygous with the Mche3 probe or its complementary strand, the sample to be tested comprises M. chelonae ; such as the DNA in the sample to be tested and the Mfor1 probe Or the complementary strand thereof produces a heterozygous reaction, and the sample to be tested comprises M. fortuitum ; if the DNA in the sample to be tested is heterozygous with the Mgas2 probe or its complementary strand, the test is to be tested stomach sample comprising Mycobacterium (M. gastri); if the test sample DNA and the probe Mgor1 Unit generates complementary hybrid reactor, the Gordon test sample comprising Mycobacterium (M. gordonae); such that the test sample or the complementary DNA probe shares with Mint2 producing a hybrid reactor, the test The sample comprises M. intracellulare ; if the DNA in the sample to be tested is heterozygous with the Mkan1 probe or its complementary strand, the sample to be tested comprises M. kansasii ; If the DNA in the sample to be tested is heterozygous with the Msi/le1 probe or its complementary strand, the sample to be tested comprises M. simiae or M. lentiflavum ; The DNA in the sample to be tested is heterozygously reacted with the Mma/ul2 probe or its complementary strand, and the sample to be tested comprises M. marinum or M. ulcerans ; The DNA in the sample to be tested is heterozygously reacted with the Mnon1 probe or its complementary strand, and the sample to be tested comprises M. nonchromogenicum ; such as the DNA in the sample to be tested and the Mper1 probe or The complementary strands produce a heterozygous reaction, and the sample to be tested contains Mycobacterium phlei ( M. peregri) NUM); such that the test sample or the complementary DNA probe shares with Mscr1 producing a hybrid reactor, the test sample comprises scrofulous Mycobacterium (M. scrofulaceum); if the test sample of DNA with Msme3 probe shares producing a hybrid or its complementary reaction, the test sample contains Mycobacterium smegmatis (M. smegmatis); such that the test sample or the complementary DNA probe shares with Mszu1 produce heterozygous reactions, Then, the sample to be tested comprises M. szulgai ; if the DNA in the sample to be tested is heterozygous with the Mter1 probe or its complementary strand, the sample to be tested comprises Mycobacterium smegmatis ( M And terrae ; and if the DNA in the sample to be tested is heterozygous with the Mxen2 probe or its complementary strand, the sample to be tested comprises M. xenopi .
根據本發明之待測樣品可為包含待鑑定菌之培養物、取自病人之檢體,或是自待鑑定菌培養物或自病人檢體進一步處理以取得其中DNA資訊之樣品,其中病人之檢體較佳係為痰或血液,取得DNA資訊之方法較佳係藉由聚合酶連鎖(polymerase chain reaction)反應擴增。The sample to be tested according to the present invention may be a culture containing the bacteria to be identified, a sample taken from the patient, or a sample from the culture to be identified or further processed from the patient to obtain a DNA information therein, wherein the patient's sample The sample is preferably sputum or blood, and the method of obtaining DNA information is preferably amplified by a polymerase chain reaction.
由於本發明之方法係藉由rRNA基因間隔區或gyrB 基因設計之探針進行鑑定,故該待測樣品中之DNA較佳包含待鑑定菌之rRNA基因間隔區或gyrB 基因;更佳地,該待測樣品中之DNA包含由聚合酶連鎖反應擴增之片段。於本發明之一較佳具體實施例中,待測樣品中之DNA係使用sp1(SEQ ID NO. 36)及sp2(SEQ ID NO. 37)引子擴增待鑑定菌之rRNA基因間隔區;待測樣品中待鑑定菌之gyrB 基因較佳係藉由Gb1f(SEQ ID NO. 38)及Gb1r(SEQ ID NO. 39)引子擴增獲得。Since the method of the present invention is identified by a probe designed by the rRNA gene spacer or the gyrB gene, the DNA in the sample to be tested preferably comprises the rRNA gene spacer or the gyrB gene of the strain to be identified; more preferably, the method The DNA in the sample to be tested contains a fragment amplified by a polymerase chain reaction. In a preferred embodiment of the present invention, the DNA in the sample to be tested uses sp1 (SEQ ID NO. 36) and sp2 (SEQ ID NO. 37) primers to amplify the rRNA gene spacer region of the strain to be identified; Preferably, the gyrB gene of the strain to be identified in the sample is obtained by amplification of Gb1f (SEQ ID NO. 38) and Gb1r (SEQ ID NO. 39) primers.
於本發明之一較佳具體實施例中,可對樣品中DNA實施標記。DNA之標記方法,已為本發明所屬技術領域中具通常知識者所熟知。例如,當實施聚合酶連鎖反應擴增時,該引子係包含一標幟,較佳地,該標幟係為毛地黃素(digoxigenin),或經標記之dUTP可用於將一標記引入產物。In a preferred embodiment of the invention, the DNA in the sample can be labeled. Methods of labeling DNA are well known to those of ordinary skill in the art to which the invention pertains. For example, when performing polymerase chain reaction amplification, the primer comprises a marker, preferably the marker is digoxigenin, or the labeled dUTP can be used to introduce a marker into the product.
在本發明一較佳實施例中,該方法進一步包括一陽性對照步驟。任何用於鑑定一已知微生物之確定探針皆適用於此陽性對照步驟。In a preferred embodiment of the invention, the method further comprises a positive control step. Any identifying probe for identifying a known microorganism is suitable for this positive control step.
較佳地,根據本發明雜合反應可於一微陣列晶片(microarray)上進行,該微陣列晶片包含一基材,且該探針係塗佈於該基材上。本領域已良好地建立了基材材料及塗佈方法,其中該基材較佳係為尼龍或玻璃。Preferably, the hybridization reaction according to the present invention can be carried out on a microarray wafer comprising a substrate and the probe is applied to the substrate. Substrate materials and coating methods have been well established in the art, wherein the substrate is preferably nylon or glass.
根據本發明之探針較佳以微陣列方式佈置在該基材上,以便一次操作可鑑定多種微生物,節省大量時間、費用、空間及人力。該技術中任何常用之基因表現檢測方法皆可用於本發明,其可取代習知技術中針對不同分枝桿菌進行不同之生物化學反應來鑑定菌種。於本發明之一具體實施例中,以寡核苷酸晶片(oligonucleotide array)鑑定分枝桿菌,包括6種屬於結核桿菌群(M. tuberculosis complex)的細菌。由細菌的16S-23S rRNA基因間的內轉錄區域及gyrB 基因設計探針,將此探針以微矩陣點製機點佈於尼龍薄膜(nylon membrane)上,與標識毛地黃素的PCR產物進行雜合反應後,再以標記磷酸分解酶的抗體呈現反應結果。The probes according to the present invention are preferably arranged on the substrate in a microarray manner to allow identification of a plurality of microorganisms in a single operation, saving a lot of time, cost, space and labor. Any of the commonly used gene expression detection methods in the art can be used in the present invention, which can replace the different biochemical reactions for different mycobacteria in the prior art to identify the species. In a specific embodiment of the invention, the mycobacteria are identified by an oligonucleotide array comprising six bacteria belonging to the M. tuberculosis complex. The probe was designed from the inner transcribed region of the 16S-23S rRNA gene of the bacterium and the gyrB gene, and the probe was spotted on a nylon membrane by a micro-matrix dot machine, and the PCR product identifying the digoxigenin was used. After the hybridization reaction, the result of the reaction is exhibited by an antibody labeled with a phosphate decomposing enzyme.
晶片先測試模式菌株(type strain)以及參考菌株(reference strain),再進一步測試臨床菌株,最後直接測試臨床檢體,以評估該晶片用於鑑定(或檢測)分枝桿菌之可行性。本發明共測試目標參考菌株25種,包括MTBC 6種(15株)(表2),NTM 19種(73株)(表3),共88株。另測試目標臨床菌株,其中MTBC 80株(表2),NTM 121株,共201株(表3)。The wafer first tests the type strain and the reference strain, further tests the clinical strain, and finally directly tests the clinical specimen to evaluate the feasibility of the wafer for identifying (or detecting) mycobacteria. The present invention co-tested 25 target reference strains, including 6 MTBC (15 strains) (Table 2) and NTM 19 strains (73 strains) (Table 3), a total of 88 strains. The target clinical strains were also tested, among which MTBC 80 strains (Table 2) and NTM 121 strains, a total of 201 strains (Table 3).
根據本發明之晶片可在8小時內完成菌種鑑定,且試劑用量較少、成本較低廉、靈敏度及特異性高。故本發明之微陣列晶片可快速及正確地鑑定不同的分枝桿菌菌種,為一種有效的檢測工具。The wafer according to the present invention can complete the identification of the bacteria within 8 hours, and the reagents are used in a small amount, the cost is low, and the sensitivity and specificity are high. Therefore, the microarray wafer of the present invention can quickly and correctly identify different mycobacterial species and is an effective detection tool.
本發明之另一目的在於提供一種探針,其包含選自由下列序列所組成之群:Myc2、Myc3、MTBC2、MTBC4、MbovG1、MbovGW1、Mbov1、MbovW1、Mcan1、McanW1、Mcap1、McapW1、Mmic1、MmicW1、Mtub1R、MtubW1R、Mabs1、Mavi1、Mche3、Mfor1、Mgas2、Mgor1、Mint2、Mkan1、Msi/le1、Mma/ul2、Mnon1、Mper1、Mscr1、Msme3、Mszu1、Mter1、Mxen2及其互補股。Another object of the present invention is to provide a probe comprising a group selected from the group consisting of Myc2, Myc3, MTBC2, MTBC4, MbovG1, MbovGW1, Mbov1, MbovW1, Mcan1, McanW1, Mcap1, McapW1, Mmic1, MmicW1 Mtub1R, MtubW1R, Mabs1, Mavi1, Mche3, Mfor1, Mgas2, Mgor1, Mint2, Mkan1, Msi/le1, Mma/ul2, Mnon1, Mper1, Mscr1, Msme3, Mszu1, Mter1, Mxen2 and their complementary strands.
本發明之又一目的在於提供一種用於鑑定一樣品中分枝桿菌之套組,該套組包括前述之探針。It is still another object of the present invention to provide a kit for identifying mycobacteria in a sample comprising the aforementioned probe.
根據本發明,該套組進一步包括用於獲得rRNA基因間隔區或gryB 基因之試劑。該試劑之組成亦為本發明所屬技術領域中具通常知識者所熟知。在本發明一較佳實施例中,試劑係用於聚合酶連鎖反應。在本發明一更佳實施例中,該套組進一步包括sp1與sp2引子及/或Gb1f與Gb1r引子。在本發明另一較佳實施例中,該套組進一步包括雜合反應用試劑。According to the invention, the kit further comprises reagents for obtaining the rRNA gene spacer or the gryB gene. The composition of the reagents is also well known to those of ordinary skill in the art to which the invention pertains. In a preferred embodiment of the invention, the reagents are used in a polymerase chain reaction. In a more preferred embodiment of the invention, the kit further comprises sp1 and sp2 primers and/or Gb1f and Gb1r primers. In another preferred embodiment of the invention, the kit further comprises a reagent for hybrid reaction.
茲以下列實例予以詳細說明本發明,唯其並不意味本發明僅侷限於此等實例所揭示之內容。The invention is illustrated by the following examples, which are not intended to limit the invention to the invention.
本研究所使用的參考菌株分別從美國ATCC(American Type Culture Collection,Manassas,Virginia,USA)、台灣BCRC(Bioresources Collection and Research Center,Food Industry Research and Development Institute,Hsinchu,Taiwan)、瑞典CCUG(Culture Collection,University of Gteborg,Gteborg,Sweden)、法國CIP(Collection of the Institute Pasteur,rue du Docteur Roux,Paris,France)、日本JCM(Japan Collection of Microorganism,RIKEN BioResource Center,Saitama,Japan)、英國NCTC(National Collection of Type Cultures,Central Public Health Laboratory,London,UK)所購得。The reference strains used in this study were from American ATCC (American Type Culture Collection, Manassas, Virginia, USA), Taiwan BCRC (Bioresources Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan), and Swedish CCUG (Culture Collection). , University of G Teborg, G Teborg, Sweden), French CIP (Collection of the Institute Pasteur, rue du Docteur Roux, Paris, France), Japan JCM (Japan Collection of Microorganism, RIKEN BioResource Center, Saitama, Japan), British NCTC (National Collection of Type Cultures, Central Public Health Laboratory, London, UK).
本研究所使用的目標參考菌株總共有25種(88株)分枝桿菌,包括MTBC 6種(15株)(表2),NTM有19種(73株)(表3);另測試目標臨床菌株(clinical strains),其中MTBC 80株(表2),NTM 121株,共201株(表3)。非目標菌(nontarget species)中屬於分枝桿菌屬之細菌有37種(38株),其他細菌35種(35株)(表4)。The target reference strains used in this study have a total of 25 (88 strains) of mycobacteria, including 6 MTBC (15 strains) (Table 2), and NTM (19 strains) (Table 3); The strains (clinical strains), including MTBC 80 strain (Table 2) and NTM 121 strain, a total of 201 strains (Table 3). Among the nontarget species, there were 37 species (38 strains) belonging to the genus Mycobacterium, and 35 strains (35 strains) of other bacteria (Table 4).
所有的菌株培養均依照各個菌種中心所建議的培養基(如BBLTM 7H11 Agar以及Lwenstein-Jensen(L-J) agar slants)和培養條件進行次代培養。All strains were cultured according to the recommended medium for each strain center (eg BBL TM 7H11 Agar and L) wenstein-Jensen (LJ) agar slants) and culture conditions for secondary culture.
菌株DNA萃取方法為煮沸法(boiled method)(Millar等人,2000,J. Microbiol. Methods. 42:139-147):在微量離心管(Eppendorf)中加入滅菌水,取培養基上的菌落懸浮在滅菌水中,以乾浴槽加熱至100℃,30分鐘後,再用12,500rpm離心20分鐘,上清液含有細菌DNA,保存於-70℃。The strain DNA extraction method is a boiled method (Millar et al., 2000, J. Microbiol. Methods. 42: 139-147): sterilized water is added to a microcentrifuge tube (Eppendorf), and the colonies on the medium are suspended in the medium. The sterilized water was heated to 100 ° C in a dry bath, and after 30 minutes, it was centrifuged at 12,500 rpm for 20 minutes. The supernatant contained bacterial DNA and stored at -70 °C.
分別利用引子sp1(5'-ACCTC CTTTC TAAGG AGCAC C-3',SEQ ID NO. 36)及sp2(5'-GATGC TCGCA ACCAC TATCC A-3',SEQ ID NO. 37)來放大分枝桿菌的rRNA基因間隔區(Roth等人,2000,J. Clin. Microbiol. 38:1094-1104);引子Gb1f(5’-TGGTT AACGC GCTAT CCAC-3’,SEQ ID NO. 38)及Gb1r(5’-ACCAA CTCTC GTGCC TTAC-32,SEQ ID NO. 39)用來放大分枝桿菌的gyrB 基因,引子均由生工有限公司(MDBio Inc.,Taipei,Taiwan)合成。利用這兩對引子分別以聚合酶連鎖反應進行增幅,引子對Sp1及Sp2所增幅產物為rRNA基因間隔區。引子對Gb1f及Gb1r所增幅的產物為gyrB 基因的部份序列。以Applied Biosystems 2720 thermal cycler(Applied Biosystems,Taipei,Taiwan)進行PCR;總體積為50μl的PCR反應液含有DNA模板(約50 ng),75 mM Tris-HCl(pH 8.5),20 mM ammonium sulfate,1.5 mM MgCl2 ,0.8 mM deoxyribonucleoside triphosphates(0.2 mM each),1 M(each) primer,及1.25 U ofHot Start polymerase(Promega,Madison,WI,USA)。PCR反應為初始變性(initial denaturation,94℃,3 mins),40個循環的變性(denaturation,94℃,1 min),黏合(annealing,59℃,1 min)和延伸(extension,72℃,1.5 mins)反應,最後為7分鐘的延伸反應(extension,72℃,7 mins)(Gurtleret al. ,2006)分別增幅產物之後,以2%的瓊脂(agarose,,OH,USA)進行電泳產物分析。若PCR產物要和晶片進行雜合反應時,則各引子對[(sp1,sp2)及(Gb1f,Gb1r)]中之一引子或二引子之5’端標記毛地黃素。Amplification of mycobacteria by primers sp1 (5'-ACCTC CTTTC TAAGG AGCAC C-3', SEQ ID NO. 36) and sp2 (5'-GATGC TCGCA ACCAC TATCC A-3', SEQ ID NO. 37, respectively) rRNA gene spacer (Roth et al., 2000, J. Clin. Microbiol. 38: 1094-1104); primer Gb1f (5'-TGGTT AACGC GCTAT CCAC-3', SEQ ID NO. 38) and Gb1r (5'- ACCAA CTCTC GTGCC TTAC-32, SEQ ID NO. 39) was used to amplify the gyrB gene of mycobacteria, and the primers were all synthesized by MD Bio Inc., Taipei, Taiwan. These two pairs of primers were used to increase the concentration by polymerase chain reaction, and the amplified products of the primer pair Sp1 and Sp2 were rRNA gene spacers. The product of the amplification of Gb1f and Gb1r by the primer is a partial sequence of the gyrB gene. PCR was performed with an Applied Biosystems 2720 thermal cycler (Applied Biosystems, Taipei, Taiwan); a total volume of 50 μl of PCR reaction solution containing a DNA template (about 50 ng), 75 mM Tris-HCl (pH 8.5), 20 mM ammonium sulfate, 1.5 mM MgCl 2 , 0.8 mM deoxyribonucleoside triphosphates (0.2 mM each), 1 M (each) primer, and 1.25 U of Hot Start polymerase (Promega, Madison, WI, USA). The PCR reaction was initial denaturation (94 ° C, 3 mins), 40 cycles of denaturation (denaturation, 94 ° C, 1 min), adhesion (annealing, 59 ° C, 1 min) and extension (extension, 72 ° C, 1.5 Mins) reaction, and finally a 7-minute extension reaction (extension, 72 ° C, 7 mins) (Gurtler et al. , 2006), respectively, after amplifying the product, with 2% agar (agarose, , OH, USA) for electrophoresis product analysis. If the PCR product is to be heterozygously reacted with the wafer, each primer is labeled with digoxigenin at one of the [(sp1, sp2) and (Gb1f, Gb1r)] primers or the 5' end of the second primer.
將參考菌株定序,加上GenBank資料庫上大量搜集分枝桿菌的rRNA基因間隔區及gyrB 基因序列,利用序列比對軟體Vector NTI(Invitrogen Corporation,Carlsbad,CA,USA)進行序列比對(sequence alignment)之後,找出種與種之間(interspecies)的差異性序列,以及同種不同菌株間(intraspecies)的相似性序列來設計探針。本研究之晶片共有32個探針,包含1個分枝桿菌正對照組探針(mycobacteria positive control,代號為PC)及13個座標標記探針(position maker,代號M)以及31個菌種特異性探針(圖1)。正對照組探針的選定採用盡可能蒐集到的分枝桿菌rRNA基因間隔區中的高度保留區域(conserved region);座標標記探針選用已標記毛地黃素之沒有菌種特異性之寡核苷酸探針。The reference strain was sequenced, and the rRNA gene spacer and the gyrB gene sequence of the mycobacteria were collected on the GenBank database, and the sequence alignment (sequence) was performed using the sequence vector software NTI (Invitrogen Corporation, Carlsbad, CA, USA). After alignment, the differential sequences between species and species, as well as sequences of similarities between different species, were designed to design probes. The wafers in this study consisted of 32 probes, including one mycobacteria positive control (code-named PC) and 13 place marker probes (code M) and 31 species-specific probes. Sex probe (Figure 1). The positive control group was selected using the highly conserved region of the mycobacterial rRNA gene spacer collected as much as possible; the coordinate-labeled probe was selected from the labeled digoxigenin-free oligonucleotide-specific oligonucleotide. Probe.
在MTBC中,極罕見的菌種坎結核桿菌(M. canettii )在全世界的菌種中心均沒有供應,故以人工合成的坎結核桿菌gyrB 基因的序列,來測試探針是否能鑑定坎結核桿菌。從GenBank收集到的MTBC菌種中發現,坎結核桿菌和結核桿菌之gyrB 基因序列間僅有單一個核苷酸差異(SNP),故選殖結核桿菌H37Rv的gyrB 片段,再利用II site-directed mutagenesis kit(Stratagene Products,Hwy,USA),在結核桿菌H37Rv菌株的gyrB 序列上製造一個跟坎結核桿菌相同的單一個核苷酸差異,即可模擬坎結核桿菌之gyrB PCR產物,經定序確認正確無誤後,以套組(Geneaid plasmid midi kit,Taiwan)抽取細菌質體,收集模擬的片段並保存於-80℃,以供日後使用。由於MTBC中所有菌種rRNA基因間隔區序列的相似度為100%,在測試坎結核桿菌時,是以模擬之坎結核桿菌gyrB 和結核桿菌H37Rv之rRNA基因間隔區的PCR產物進行雜合反應。In MTBC, the extremely rare species of M. canettii is not available in the center of the world. Therefore, the sequence of the artificially synthesized gyrB gene of Mycobacterium tuberculosis can be used to test whether the probe can identify the tuberculosis. Bacillus. From the MTBC strain collected by GenBank, it was found that there was only one nucleotide difference (SNP) between the gyrB gene sequences of Mycobacterium tuberculosis and Mycobacterium tuberculosis, so the gyrB fragment of Mycobacterium tuberculosis H37Rv was selected and reused. II site-directed mutagenesis kit (Stratagene Products, Hwy, USA), which can produce a gyrB PCR product of Mycobacterium tuberculosis by making a single nucleotide difference in the gyrB sequence of Mycobacterium tuberculosis H37Rv strain. After confirming the correctness of the sequence, the bacterial plastids were taken with a set (Geneaid plasmid midi kit, Taiwan), and the simulated fragments were collected and stored at -80 °C for later use. Since the similarity of the rRNA gene spacer sequences of all the strains in the MTBC was 100%, in the test of Mycobacterium tuberculosis, the hybridization reaction was carried out by PCR products of the rRNA gene spacers of the simulated Mycobacterium tuberculosis gyrB and Mycobacterium tuberculosis H37Rv.
分別將探針與染劑(tracking dye)以體積比1:1(vol/vol)混合於96孔圓底的微量盤(ELISA plate,Becton Drive,NJ,USA),探針最終濃度除正負對照組及座標探針之外,其他皆為10μM;正對照組(positive control,PC)濃度為5 μM,座標探針濃度為2.5 μM,負對照組(negative control,NC)則是由無菌水和染劑等體積混合而成。The probe and the tracking dye were mixed in a 96-well round bottom microplate (ELISA plate, Becton Drive, NJ, USA) at a volume ratio of 1:1 (vol/vol), and the final concentration of the probe was divided by positive and negative. The group and the coordinate probe were all 10 μM; the positive control (PC) concentration was 5 μM, the coordinate probe concentration was 2.5 μM, and the negative control (NC) was from sterile water and The dye is mixed in equal volume.
將配置完成的微量盤置於EzspotTM arrayer SR-A300(悅生科技,台北,台灣),利用直徑為400 μm的實心針頭(solid pin)把各個探針點在尼龍膜(positively charged Nylon membrane,Roche,Mannheim,Germany)上,二點之間的中心距離為800 μm。此晶片上共有49點(7×7 dots),大小為6.8 mm×6.8 mm。晶片製作完成後,以shortwave UV(Stratalinker 1800;Stratagen,La Jolla,CA,USA)3 W/cm2 能量之紫外光照射30秒,促使探針固定在尼龍膜上,接著儲存在陰暗乾燥處備用。微陣列晶片探針位置示於圖1。The configuration of the microplate was placed Ezspot TM arrayer SR-A300 (Wyatt Health Technology, Taipei, Taiwan), using a needle having a diameter of 400 μm of solid (solid pin) points in the respective probe nylon membrane (positively charged Nylon membrane, On Roche, Mannheim, Germany, the center distance between the two points is 800 μm. There are 49 points (7 × 7 dots) on the wafer and the size is 6.8 mm × 6.8 mm. After the wafer was fabricated, it was irradiated with ultraviolet light of 3 W/cm 2 of shortwave UV (Stratalinker 1800; Stratagen, La Jolla, CA, USA) for 30 seconds to promote the probe on the nylon membrane, and then stored in a dark dry place. . The microarray wafer probe locations are shown in Figure 1.
於製作好的分枝桿菌晶片中,分別加入已標記毛地黃素的PCR產物,進行雜合反應。雜合反應進行的主要步驟如下:The PCR product of the labeled digoxigenin was separately added to the prepared mycobacterial wafer to carry out a heterozygous reaction. The main steps of the heterozygous reaction are as follows:
1. 將製作好的晶片標記之後,利用0.5×的SSC緩衝液[1×SSC緩衝液為0.15 M NaCl和0.015 M sodium citrate)-0.1% sodium dodecyl sulfate(SDS,Boehringer Mannheim,Mannheim,Germany)]清洗晶片,以震盪器震盪4次,每次3分鐘,以洗去晶片上的追蹤染劑、未結合在尼龍膜上的探針及其他雜質1. After labeling the prepared wafer, use 0.5× SSC buffer [1×SSC buffer for 0.15 M NaCl and 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS, Boehringer Mannheim, Mannheim, Germany)] The wafer was cleaned and shaken 4 times for 3 minutes each time to wash away trace dye on the wafer, probes not bonded to the nylon membrane, and other impurities.
2. 加入雜合緩衝液[hybridization solution,5×SSC,1%(w/v) blocking reagent(Roche Diagnostics,Indianapolis,IN,USA),0.1% N-laurylsarcosine and 0.02% SDS],在室溫下震盪2小時進行前雜合反應(prehybridization)2. Add hybridization solution [hybridization solution, 5 × SSC, 1% (w / v) blocking reagent (Roche Diagnostics, Indianapolis, IN, USA), 0.1% N-laurylsarcosine and 0.02% SDS], at room temperature Prehybridization before shaking for 2 hours
3. 把每一晶片依序放入24孔的細胞培養盤(24-well cell culture plate)每一孔中,每孔加入300 μl的雜合緩衝液3. Place each wafer sequentially into each well of a 24-well cell culture plate, adding 300 μl of hybrid buffer to each well.
4. 將帶有毛地黃素的PCR產物加熱至95℃,5分鐘,用以解旋(denature)成單股狀態,並立刻置於冰上4. Heat the PCR product with digoxigenin to 95 ° C for 5 minutes to denature into a single strand and immediately place on ice.
5. 在冰浴上,分別將各個單股狀態的PCR產物取10 μl加入含有雜合緩衝液的晶片中,在雜合反應箱(hybridization oven,Firstek Scientific,Taipei,Taiwan)中(50℃,60 rpm)進行1.5小時的雜合反應5. On the ice bath, 10 μl of each single-stranded PCR product was added to the wafer containing the hybrid buffer in a hybridization oven (Firstek Scientific, Taipei, Taiwan) (50 ° C, Hybrid reaction at 1.5 rpm for 1.5 hours
6. 用0.25×SSC緩衝液作為清洗緩衝液(wash buffer),清洗4次,每次5分鐘,洗掉未結合的PCR產物和雜合緩衝液6. Wash the unbound PCR product and hybrid buffer with 0.25 x SSC buffer as wash buffer and wash 4 times for 5 minutes each time.
7. 移除清洗緩衝液,加入1×填塞緩衝液[blocking buffer,10% blocking reagent(Roche Diagnostics)以maleic acid buffer(0.1 M maleic acid,0.15 M NaCl,pH 7.5)稀釋10倍],室溫震盪1小時7. Remove the washing buffer, add 1× stuffing buffer [blocking buffer, 10% blocking reagent (Roche Diagnostics) diluted 10 times with maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, pH 7.5)], room temperature Shock for 1 hour
8. 加入稀釋2500倍(以1×blocking buffer稀釋)標記有鹼性磷酸酶(alkaline phosphatase,AP)之抗毛地黃素抗體(anti-digoxigenin-AP Fab fragments,Roche),室溫震盪反應1小時8. Add 2500 times diluted (diluted with 1×blocking buffer) with anti-digoxigenin-AP Fab fragments (Roche) labeled with alkaline phosphatase (AP), room temperature shaking reaction 1 hour
9. 移除抗體緩衝液,接著以緩衝液清洗[0.3%(v/v) Tween 20 in 1×maleic acid buffer]洗去未結合的抗體,室溫下清洗3次,每次15分鐘9. Remove the antibody buffer, then wash the unbound antibody with buffer [0.3% (v/v) Tween 20 in 1 × maleic acid buffer] and wash 3 times at room temperature for 15 minutes each time.
10. 加入偵測緩衝液(detection buffer,0.1 M Tris-HCl,0.15 M NaCl,pH 9.5)室溫震盪5分鐘後,去除偵測緩衝液10. Add detection buffer (0.1 M Tris-HCl, 0.15 M NaCl, pH 9.5) for 5 minutes at room temperature, remove the detection buffer.
11. 以偵測緩衝液將鹼性磷酸酶受質[stock solution of nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate(NBT/BCIP),Roche]稀釋50倍,每個晶片上加40μl,37℃避光,進行25分鐘至45分鐘的呈色反應,反應進行中不要搖晃。11. Dilute the alkaline phosphatase substrate (stock solution of nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP), Roche) 50 times with the detection buffer. 40 μl, protected from light at 37 ° C, for a color reaction from 25 minutes to 45 minutes, do not shake during the reaction.
12. 用純水清洗晶片4次,每次5分鐘,以洗掉NBT/BCIP終止呈色反應12. Wash the wafer 4 times with pure water for 5 minutes each time to wash off NBT/BCIP to terminate the color reaction.
13. 放入烘箱中烘乾後,即可用肉眼判讀結果13. After drying in an oven, you can read the results with the naked eye.
晶片上有座標標記,可辨別上下左右,座標標記將晶片分隔成兩大區域。晶片上半部區域的探針用來鑑定MTBC及其內不同菌種,及兩個負對照組(negative control,NC);下半部區域的探針用來鑑定NTM,及一個正對照組(positive control,PC)探針,該探針可以和分枝桿菌屬細菌反應。The wafer has coordinate marks on the top and bottom, and the coordinate marks divide the wafer into two large areas. Probes in the upper half of the wafer were used to identify MTBC and its different strains, and two negative control (NC); probes in the lower half were used to identify NTM, and a positive control group ( A positive control, PC) probe that reacts with mycobacteria.
晶片反應結果必須NC點沒有雜合反應發生,而PC點有雜合反應才能進行結果判讀。若鑑定MTBC的二個探針均有反應,表示檢體中有MTBC存在,至於是MTBC中的那一種,將於下文中詳細說明。NTM的鑑定則看是那一個NTM的探針發生雜合反應。The result of the wafer reaction must have no heterozygous reaction at the NC point, and the PC point has a heterozygous reaction to perform the result interpretation. If both probes for the identification of MTBC are reactive, it indicates that MTBC is present in the sample. As for the MTBC, it will be described in detail below. The identification of NTM is based on the heterozygous reaction of the NTM probe.
以晶片鑑定結果若種名和菌種中心的種名不符合,或和傳統(或其他分生方法)所鑑定之菌名不一致時,則定序此菌種之rRNA基因間隔區、16S rRNA基因、或gyrB 序列,作為第三種參考方法。純化之PCR產物進行定序,使用核酸定序儀PRISM 3100 Sequencer(Applied Biosystems,Taipei,Taiwan),及BigDye Terminator cycle sequencing kit(version 3.7;Applied Biosystems)定序。If the species name of the wafer does not match the species name of the center of the strain, or if it is inconsistent with the name of the species identified by the traditional (or other meristem method), the rRNA gene spacer, 16S rRNA gene, or The gyrB sequence serves as a third reference method. The purified PCR products were sequenced and sequenced using a nucleic acid sequencer PRISM 3100 Sequencer (Applied Biosystems, Taipei, Taiwan), and a BigDye Terminator cycle sequencing kit (version 3.7; Applied Biosystems).
靈敏度(sensitivity):欲鑑定的目標菌株得到正確鑑定的百分比[sensitivity=(no. of target strains correctly identified/total no. of target strains tested)×100%]Sensitivity: the percentage of the target strain to be identified that was correctly identified [sensitivity=(no. of target strains correctly identified/total no. of target strains tested) × 100%]
特異性(specificity):非目標菌株,沒有得到鑑定的百分比[specificity=(no. of nontarget strains not identified/total no. of nontarget strains tested)×100%]Specificity: the percentage of non-target strains that have not been identified [specificity=(no. of nontarget strains not identified/total no. of nontarget strains tested) × 100%]
偵測極限(detection limit):待測菌株能被晶片檢測到的最低genomic DNA量。Detection limit: The minimum amount of genomic DNA that the strain to be tested can be detected by the wafer.
此探針為分枝桿菌菌屬探針,由分枝桿菌屬(Mycobacterium spp.)之rRNA基因間隔區序列中的共同序列所設計,探針代號為Myc2及Myc3,由於探針Myc2無法涵蓋所有的分枝桿菌,針對無法涵蓋的菌種另設計Myc3,再將兩者混合在一起,作為分枝桿菌菌屬探針,代號為PC。本研究中,當此探針發生雜合反應,表示待測菌種為分枝桿菌屬細菌。This probe is a Mycobacterium spp. probe designed by the common sequence in the rRNA gene spacer sequence of Mycobacterium spp. The probes are Myc2 and Myc3, since the probe Myc2 cannot cover all Mycobacteria, Myc3 was designed for the species that could not be covered, and then the two were mixed together as a probe of mycobacteria, codenamed PC. In this study, when the probe is heterozygous, it indicates that the strain to be tested is a Mycobacterium.
結核桿菌群的特異性探針共分為兩部份,一部分是針對MTBC所設計的菌群特異性探針,另一部分是針對MTBC中的各個菌種設計的菌種探針。The specific probes of the Mycobacterium tuberculosis group are divided into two parts, one is a flora-specific probe designed for MTBC, and the other is a strain probe designed for each strain in MTBC.
MTBC菌群的探針有兩個,代號分別為MTBC2和MTBC4,探針長度分別為18個及19個核苷酸,由MTBC的rRNA基因間隔區上共有的序列所設計,其序列與NTM菌種有很大的差異;凡屬於結核桿菌群的菌種,包括非洲結核桿菌,牛結核桿菌群,坎結核桿菌,卡柏結核桿菌,邁克結核桿菌,和結核桿菌均會和此二探針產生雜合反應;結核桿菌群以外的菌種,皆不會和此二探針產生反應。There are two probes in the MTBC flora, codenamed MTBC2 and MTBC4, respectively. The length of the probe is 18 and 19 nucleotides, respectively. The sequence is shared by the sequence shared by the rRNA gene spacer of MTBC. There are great differences; all strains belonging to the Mycobacterium tuberculosis group, including Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, and Mycobacterium tuberculosis Hybrid reaction; no species other than Mycobacterium tuberculosis will react with the two probes.
MTBC菌種的特異性探針共有12個,探針代號Mtub1R及MtubW1R用來鑑定結核桿菌,若菌株為結核桿菌則Mtub1R的雜合訊號會比MtubW1R強;MbovG1及MbovGW1用來鑑定牛結核桿菌群,包括牛結核桿菌、牛結核桿菌BCG、及卡柏結核桿菌和MbovG1探針的雜合訊號比和MbovGW1的雜合訊號強,但無法區分出牛結核桿菌群內的菌種。Mbov1及MbovW1用來鑑定牛結核桿菌,包括牛結核桿菌以及牛結核桿菌BCG和Mbov1的雜合訊號都比和MbovW1的雜合訊號強;Mcap1及McapW1為卡柏結核桿菌的探針,僅卡柏結核桿菌才會呈現Mcap1比McapW1強的雜合訊號;Mmic1及MmicW1為邁克結核桿菌的探針,僅邁克結核桿菌才會呈現Mmic1比MmicW1強的雜合訊號;Mcan1及McanW1為坎結核桿菌的探針,由於坎結核桿菌和結核桿菌在gyrB 基因上序列較相似,故坎結核桿菌的雜合反應會呈現Mtub1R比MtubW1R強的雜合訊號,以及Mcan1比McanW1強的反應訊號。There are 12 specific probes for MTBC strains. The probes Mtub1R and MtubW1R are used to identify Mycobacterium tuberculosis. If the strain is Mycobacterium tuberculosis, the hybrid signal of Mtub1R will be stronger than MtubW1R. MbovG1 and MbovGW1 are used to identify Mycobacterium tuberculosis. The heterozygous signal ratio of Mycobacterium tuberculosis, Mycobacterium tuberculosis BCG, and Mycobacterium tuberculosis BCG and MbovG1 probes is stronger than that of MbovGW1, but the strains in the Mycobacterium tuberculosis group cannot be distinguished. Mbov1 and MbovW1 were used to identify Mycobacterium tuberculosis, including Mycobacterium tuberculosis and Mycobacterium tuberculosis BCG and Mbov1. The hybrid signal was stronger than the MbovW1 hybrid signal; Mcap1 and McapW1 were probes for Mycobacterium tuberculosis, only Kapo Mycobacterium tuberculosis will show a strong hybrid signal of Mcap1 than McapW1; Mmic1 and MmicW1 are probes of Mycobacterium tuberculosis, only M. tuberculosis will show a strong hybrid signal of Mmic1 than MmicW1; Mcan1 and McanW1 are the probes of Mycobacterium tuberculosis Because the sequence of the Mycobacterium tuberculosis and Mycobacterium tuberculosis are similar in the gyrB gene, the heterozygous reaction of M. tuberculosis will show a strong hybrid signal of Mtub1R than MtubW1R, and a stronger reaction signal of Mcan1 than McanW1.
MTBC菌種的鑑定,首先需要菌群探針MTBC2及MTBC4同時有反應的情況下(即待測菌種屬於MTBC),才進一步鑑定是該群內的那一菌種,以雜合圖譜型式(hybridization pattern)做為菌種鑑定依據。以結核桿菌為例,它的圖譜型式為:MTBC2及MTBC4同時有反應,Mtub1R探針的反應比MtubW1R強以外,且剩下10個鑑定MTBC中菌種的探針,均同時證明其不是MTBC中的其他菌種(即探針代號中有”W”字母的探針,其反應比同一組中代號沒有”W”的探針反應強)。卡柏結核桿菌以前被放在牛結核桿菌群內,現被命名為卡柏結核桿菌(Aranaz等人,2003,J. Syst. Evol. Microbiol. 53:1785-1789),它的探針反應圖譜為MbovG1比MbovGW1反應強(即屬於牛結核桿菌群),且Mcap1比McapW1反應強(即它是卡柏結核桿菌)。The identification of MTBC strains first requires the microbial probes MTBC2 and MTBC4 to react at the same time (ie, the tested species belong to MTBC), and then further identify the species in the group, in a heterozygous pattern ( Hybridization pattern) is used as the basis for identification of strains. Take Mycobacterium tuberculosis as an example, its pattern type is: MTBC2 and MTBC4 react at the same time, Mtub1R probe is stronger than MtubW1R, and there are 10 probes for identifying strains in MTBC, which are also proved to be not in MTBC. Other strains (ie probes with the "W" letter in the probe design have a stronger reaction than probes with the "W" code in the same set). Tuberculosis bacillus was previously placed in the group of Mycobacterium tuberculosis and is now named K. tuberculosis (Aranaz et al., 2003, J. Syst. Evol. Microbiol. 53: 1785-1789), its probe reaction map MbovG1 is more reactive than MbovGW1 (ie belongs to the population of Mycobacterium bovis), and Mcap1 is more reactive than McapW1 (ie it is K. tuberculosis).
針對非結核分枝桿菌所設計之菌種探針共有17個,其中有兩個探針為群探針(complex probe),共可鑑定19種NTM目標菌種,分別為潰瘍腫分枝桿菌、鳥分枝桿菌、龜分枝桿菌、偶然分枝桿菌、胃分枝桿菌、戈登分枝桿菌、胞內分枝桿菌、堪薩斯分枝桿菌、蘭分枝桿菌、海洋分枝桿菌、不產色分枝桿菌、培力分枝桿菌、瘰癘分枝桿菌、猿分枝桿菌、恥垢分枝桿菌、斯氏分枝桿菌、土地分枝桿菌、潰瘍分枝桿菌、及蟾蜍分枝桿菌,其中Mma/ul2用來鑑定海洋分枝桿菌或潰瘍分枝桿菌,而Msi/le1用來鑑定猿分枝桿菌或蘭分枝桿菌。There are 17 species of probes designed for non-tuberculous mycobacteria. Two of them are complex probes. A total of 19 NTM target strains can be identified, respectively, which are Mycobacterium ulcerans. Mycobacterium avium, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium tuberculosis, Mycobacterium gordonii, Mycobacterium intracellulare, Mycobacterium kansii, Mycobacterium marinum, Mycobacterium marinum, non-chromosome Mycobacteria, Mycobacterium phlei, Mycobacterium phlei, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium smegmatis, Mycobacterium phlei, Mycobacterium ulcerans, and Mycobacterium phlei, among which Mma/ul2 was used to identify Mycobacterium marinum or Mycobacterium ulcerans, and Msi/le1 was used to identify Mycobacterium phlei or Mycobacterium lanatus.
雜合反應結果必須正對照組(PC)有反應,接著看那一探針有反應,例如PC和Mche3二個探針有反應,菌株鑑定為龜分枝桿菌。唯一例外是胞內分枝桿菌會和鑑定鳥分枝桿菌的探針(Mavi1)有輕微交叉反應,所以Mint2(鑑定胞內分枝桿菌的探針)和Mavi1同時有反應時,菌株鑑定為胞內分枝桿菌,但Mint2的反應較Mavi1強很多。若PC及Mavi1有雜合反應則鑑定為鳥分枝桿菌,這是由於鳥分枝桿菌和胞內分枝桿菌同屬於鳥分枝桿菌群(Mycobacterium avium complex),兩者在生化反應及rRNA基因間隔區序列上非常相似。臨床上不易區分出二菌種,通常只鑑定到鳥分枝桿菌群。The result of the heterozygous reaction must be positive in the control group (PC), and then the probe is reacted. For example, two probes of PC and Mche3 are reacted, and the strain is identified as Mycobacterium marinum. The only exception is that M. intracellulare has a slight cross-reactivity with the probe for the identification of M. avium (Mavi1), so when Mint2 (a probe for identifying M. intracellulare) and Mavi1 react simultaneously, the strain is identified as a cell. Mycobacterium, but the reaction of Mint2 is much stronger than Mavi1. If PC and Mavi1 have a heterozygous reaction, they are identified as Mycobacterium avium. This is because Mycobacterium avium and Mycobacterium avium belong to Mycobacterium avium complex, both in biochemical reaction and rRNA gene. The spacer sequences are very similar in sequence. It is difficult to distinguish the two strains clinically, and only the Mycobacterium avium group is usually identified.
晶片的測試結果如圖2及圖3所示,共測試了88株參考菌株和1個gyrB 模擬序列,包括MTBC 15株及NTM 73株。另測試目標臨床菌株,其中MTBC共80株(表2),NTM 121株,共201株(表3)。The test results of the wafer are shown in Fig. 2 and Fig. 3. A total of 88 reference strains and one gyrB simulation sequence, including MTBC 15 strain and NTM 73 strain, were tested. The target clinical strains were also tested, including 80 MTBCs (Table 2) and NTM 121 strains (201) (Table 3).
所有目標菌株289株,其中有二株(M. kansasii KMUH0903-4及M. kansasii KMUH0903-26)晶片鑑定錯誤,所以晶片靈敏度為99.3%(287/289)(表2和表3)。所測試的非目標菌(nontarget species)73株中(表4),其中有一株鑑定錯誤(M. goodii CCUG 52054被鑑定為M. smegmatis ),所以晶片特異性為98.6%(72/73)。All of the target strains were 289, and two of them ( M. kansasii KMUH0903-4 and M. kansasii KMUH0903-26) were misidentified, so the wafer sensitivity was 99.3% (287/289) (Table 2 and Table 3). Among the 73 strains of nontarget species tested (Table 4), one of them was identified incorrectly ( M. goodii CCUG 52054 was identified as M. smegmatis ), so the wafer specificity was 98.6% (72/73).
以結核桿菌H37Rv,潰瘍腫分枝桿菌CCUG 20993T 和偶然分枝桿菌JCM 6387作為測試菌株。DNA濃度均由10 ng/μl做10倍序列稀釋。結核桿菌H37Rv的偵測極限為10 fg/μl(即50 fg/每一測試),約相當於12個細菌的DNA含量[(一個E. coli 細胞含有4 fg DNA(Kubitschek and Friedman,1971)]。潰瘍腫分枝桿菌CCUG 20993T 和偶然分枝桿菌JCM 6387的檢測極限均為100 fg/μl(即500 fg/每一測試),約相當於120個細菌的DNA含量。Mycobacterium tuberculosis H37Rv, Mycobacterium tuberculosis CCUG 20993 T and Mycobacterium tuberculosis JCM 6387 were used as test strains. The DNA concentration was serially diluted by 10 ng/μl. The detection limit of Mycobacterium tuberculosis H37Rv is 10 fg/μl (ie 50 fg/each test), which is equivalent to the DNA content of 12 bacteria [(an E. coli cell contains 4 fg DNA (Kubitschek and Friedman, 1971)] The detection limit of Mycobacterium tuberculosis CCUG 20993 T and Occasional Mycobacterium JCM 6387 was 100 fg/μl (ie 500 fg per test), which corresponds to a DNA content of 120 bacteria.
上述實施例僅為說明本發明之原理及其功效,並非限制本發明。因此習於此技術之人士對上述實施例進行修改及變化仍不脫本發明之精神。本發明之權利範圍應如後述之申請專利範圍所列。The above embodiments are merely illustrative of the principles and effects of the invention and are not intended to limit the invention. Therefore, those skilled in the art can make modifications and changes to the above embodiments without departing from the spirit of the invention. The scope of the invention should be as set forth in the appended claims.
圖1顯示本發明微陣列晶片探針位置示意圖。Figure 1 shows a schematic representation of the position of a microarray wafer probe of the present invention.
圖2顯示結核桿菌群之目標菌之雜合反應圖:Figure 2 shows the heterozygous reaction diagram of the target bacteria of the Mycobacterium tuberculosis group:
(1)非洲結核桿菌ATCC 25420(1) Mycobacterium tuberculosis ATCC 25420
(2)非洲結核桿菌ATCC 35711(2) Mycobacterium tuberculosis ATCC 35711
(3)牛結核桿菌ATCC 27291(3) Mycobacterium tuberculosis ATCC 27291
(4)牛結核桿菌ATCC 35734(4) Mycobacterium tuberculosis ATCC 35734
(5)牛結核桿菌BCG ATCC 35731(5) Mycobacterium tuberculosis BCG ATCC 35731
(6)牛結核桿菌BCG ATCC 35735(6) Bovine Mycobacterium tuberculosis BCG ATCC 35735
(7)牛結核桿菌BCG ATCC 35737(7) Mycobacterium tuberculosis BCG ATCC 35737
(8)卡柏結核桿菌ATCC BAA-824(8) Mycobacterium tuberculosis ATCC BAA-824
(9)坎結核桿菌(9) Mycobacterium tuberculosis
(10)邁克結核桿菌ATCC 19422(10) Mycobacterium tuberculosis ATCC 19422
(11)邁克結核桿菌ATCC 35782(11) Mycobacterium tuberculosis ATCC 35782
(12)邁克結核桿菌NCTC 8337(12) Mycobacterium tuberculosis NCTC 8337
(13)結核桿菌H37Rv(13) Mycobacterium tuberculosis H37Rv
(14)結核桿菌ATCC 25177(14) Mycobacterium tuberculosis ATCC 25177
(15)結核桿菌ATCC 35818(15) Mycobacterium tuberculosis ATCC 35818
(16)結核桿菌ATCC 35828(16) Mycobacterium tuberculosis ATCC 35828
圖3顯示非結核分枝桿菌目標菌(1-19)與非目標菌(20-32)之雜合反應圖Figure 3 shows the heterozygous reaction diagram of non-tuberculous mycobacteria target bacteria (1-19) and non-target bacteria (20-32).
(1)潰瘍腫結核桿菌CCUG 20993(1) ulcerative tuberculosis CCUG 20993
(2)鳥分枝桿菌BCRC 15441(2) Mycobacterium avium BCRC 15441
(3)龜分枝桿菌CCUG 35749(3) Mycobacterium tuberculosis CCUG 35749
(4)偶然分枝桿菌BCRC 15320(4) Occasional Mycobacterium BCRC 15320
(5)胃分枝桿菌CCUG 29062(5) Mycobacterium tuberculosis CCUG 29062
(6)戈登分枝桿菌CCUG 21801(6) Mycobacterium gordon CCUG 21801
(7)胞內分枝桿菌CCUG 28005(7) Mycobacterium intracellulare CCUG 28005
(8)堪薩斯分枝桿菌CCUG 27785(8) Kansas Mycobacterium CCUG 27785
(9)蘭分枝桿菌CCUG 47901(9) Mycobacterium lanatus CCUG 47901
(10)海洋分枝桿菌ATCC 25039(10) Mycobacterium marinum ATCC 25039
(11)不產色分枝桿菌CCUG 28009(11) Mycobacterium faecalis CCUG 28009
(12)培力分枝桿菌CCUG 27976(12) Mycobacterium phlei CCUG 27976
(13)瘰癘分枝桿菌CCUG 29045(13) Mycobacterium tuberculosis CCUG 29045
(14)猿分枝桿菌CCUG 29114(14) Mycobacterium tuberculosis CCUG 29114
(15)恥垢分枝桿菌ATCC 19420(15) Mycobacterium smegmatis ATCC 19420
(16)斯氏分枝桿菌NCTC 10829(16) Mycobacterium tuberculosis NCTC 10829
(17)土地分枝桿菌CCUG 42429(17) Mycobacterium phlei CCUG 42429
(18)潰瘍分枝桿菌ATCC 25899(18) Mycobacterium ulcerans ATCC 25899
(19)蟾蜍分枝桿菌ATCC 19973(19) Mycobacterium phlei ATCC 19973
(20)分枝桿菌M. agri CCUG 37673(20) Mycobacterium M. agri CCUG 37673
(21)分枝桿菌M. alvei CCUG 37585(21) Mycobacterium M. alvei CCUG 37585
(22)分枝桿菌M. arupense CCUG 39146(22) Mycobacterium M. arupense CCUG 39146
(23)分枝桿菌M. asiaticum MB 031(23) Mycobacterium M. asiaticum MB 031
(24)分枝桿菌M. chitae CCUG 39504(24) Mycobacterium M. chitae CCUG 39504
(25)分枝桿菌M. chlorophenolicum BCRC 13726(25) Mycobacterium M. chlorophenolicum BCRC 13726
(26)分枝桿菌M. confluentis CCUG 37513(26) Mycobacterium M. confluentis CCUG 37513
(27)分枝桿菌M. diernhoferi BCRC 16395(27) Mycobacterium M. diernhoferi BCRC 16395
(28)分枝桿菌M. gadium CCUG 37515(28) Mycobacterium M. gadium CCUG 37515
(29)分枝桿菌M. pulveris CCUG 37668(29) Mycobacterium M. pulveris CCUG 37668
(30)分枝桿菌M. fallax CCUG 37584(30) Mycobacterium M. fallax CCUG 37584
(31)分枝桿菌M. flavescens MB 017(31) Mycobacterium M. flavescens MB 017
(32)分枝桿菌M. genavense MB 691(32) Mycobacterium M. genavense MB 691
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Non-Patent Citations (2)
Title |
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Heekyung Park, et al.,"Detection and genotyping of Mycobacterium species from clinical isolates and specimens by oligonucleotide array.", J. Clin. Microbiol., Apr. 2005, Vol.43, No.4, page1782-1788 * |
M. Fukushima, et al., "Detection and identification of Mycobacterium species isolates by DNA microarray." J. Clin. Microbiol., June 2003, Vol.41, No.6, page1782-178 * |
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US11174520B2 (en) | 2018-02-27 | 2021-11-16 | Delta Electronics, Inc. | Method for detecting presence or absence of Mycobacterium and kit thereof |
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