TW200533757A

TW200533757A - Method for identifying Acinetobacter species

Info

Publication number: TW200533757A
Application number: TW93109358A
Authority: TW
Inventors: Chang-Quan Zhang; Xian-Zhang Zhang; Yu-Fang Wei; Ai-Hui Huang; Zhong-Dao Tang
Original assignee: Univ Nat Cheng Kung
Priority date: 2004-04-05
Filing date: 2004-04-05
Publication date: 2005-10-16

Abstract

The present invention provides a method for identifying Acinetobacter species, and the principle of before said method is mainly based on the species specificity derived from the intergenic spacer region (ITS) between 16S-23S ribosomal DNA in Acinetobacter, so that sequence comparison is used for identifying Acinetobacter species.

Description

200533757 玖、發明說明：【發明所屬之技術領域】本發明之技術係利用細菌核糖體DNA之内轉錄區 (ITS)具有種（species)特異性之特質，藉此可用以幾定不動桿菌屬之菌種。【先前技術】 (stHctly (oxidase 不動桿菌 (Acinetobacter) 為嗜氧性 aerobic)、非運動性（nonmotile)、氧化酶陰性 negative)之葡萄糖非發酵性革蘭氏陰性桿菌，簡稱”非發酵菌，，（nonfermenters)，廣泛分佈於自然環境[8，19, 2〇]，主要是水和土壤中’亦常存在人的皮膚、呼吸道、消化道和泌展生殖道中’屬伺機性致病菌（opportunistic pathogen)，常造成院内感染（nosocomial infection)。不動桿菌有時會以球桿菌或球卤存在’在抹片上與奈瑟氏菌（τΥϋαζ’α)頗為相似，因在體液與固體培養基皆以雙球菌型態為主，有時亦形成桿菌。自腦膜炎或敗血症病人中分離到的不動桿菌，常被誤認為細臈炎奈瑟氏菌，由女性泌展生殖器所培養出的不動桿菌也易被誤以為是奈瑟氏淋病雙球菌（Whwk g⑽。然而，奈瑟氏菌能產生氧化酶（oxidase)，但不動桿菌並不具有此酵素[7]。表一係顯示目前已知之不動桿菌屬基因種，此結果係依DNA-DNA 雜合法分類所得到。表依DNA-DNA雜合法分類之不動桿菌屬基因種種名基因種 200533757 1 酉曰酉夂!弓不動桿菌（▲⑽r⑶仏⑽) 2 鮑氏不動桿菌hwmawz/z·) 3 UN 4 /谷血不動桿菌 5 瓊恩氏不動桿菌 6 UN 7 約氏不動得菌（Hnet〇bacterjohnsonii) 8 (= 8TU) 魯氏不動桿菌（土 9 (= 8TU) UN 10 UN 11 UN 12 抗無始不動择菌 0cinet〇bacter radioresistens) 13TU UN 14TU (=13BJ) UN 15TU UN 14BJ UN 15BJ UN 16BJ UN 17BJ UN Acinetobacter venetianus Acinetobacter ursingii Acinetobacter schindleri β基因種（Genospecies)之數字編號 1-12, 13TU- 15TU，13BJ- 17BJ 分別根據 Bouvet & Grimont [4]，Tjernberg & Ursing (TU) [21]，Bouvet & Jeanjean (BJ) [6]的研究報告；genospecies 8, 9 及 14TU 相等於 8TU 及 13BJ[6, 14,21]· 醋酸釣不動桿菌-鮑氏不動桿菌複合群 (Acinetobacter calcoaceticus-Acinetobacter baumannii complex) ? Acb200533757 发明 Description of the invention: [Technical field to which the invention belongs] The technology of the present invention utilizes species-specific traits of the internal transcription region (ITS) of bacterial ribosomal DNA, whereby it can be used for several species of Acinetobacter Bacteria. [Prior art] (StHctly (oxine acinetobacter is aerobic), non-motile (nonmotile, oxidase-negative) glucose non-fermentative gram-negative bacteria, referred to as "non-fermentative bacteria," (Nonfermenters), widely distributed in the natural environment [8, 19, 20], mainly in water and soil 'also often present in human skin, respiratory tract, digestive tract and genital tract' are opportunistic pathogen), often causing nosocomial infection. Acinetobacter sometimes exists as cocci or globular halogens' on smears and is quite similar to Neisseria (τΥϋαζ'α), because it is used in both body fluids and solid media. Diplococci are predominant in form and sometimes form bacilli. Acinetobacter isolated from meningitis or septicemia patients are often mistaken for Neisseria pneumoniae. Acinetobacter cultured by female genitalia is also It is easy to be mistaken for Neisseria gonorrhoeae (Whwk g⑽. However, Neisseria can produce oxidase, but Acinetobacter does not have this enzyme [7]. Table 1 Shows the currently known Acinetobacter gene species, this result is based on DNA-DNA heterogeneity classification. Table shows DNA-DNA heterogeneity classification of Acinetobacter gene species name gene species 200533757 1 (▲ ⑽r⑶ 仏 ⑽) 2 Acinetobacter bauhinii hwmawz / z ·) 3 UN 4 / Acinetobacter cerealis 5 Acinetobacter jones 6 UN 7 Hnetobacterjohnsonii 8 (= 8TU) Acinetobacter (soil 9 (= 8TU) UN 10 UN 11 UN 12 resistance 0cinet〇bacter radioresistens) 13TU UN 14TU (= 13BJ) UN 15TU UN 14BJ UN 15BJ UN 16BJ UN 17BJ UN Acinetobacter venetianus Acinetobacter schindleri Numbers of beta gene species (Genospecies) are 1-12, 13TU-15TU, 13BJ-17BJ according to Bouvet & Grimont [4], Tjernberg & Ursing (TU) [21], Bouvet & Jeanjean (BJ) [6 ] Research report; genospecies 8, 9 and 14TU are equal to 8TU and 13BJ [6, 14, 21] · Acinetobacter calcoaceticus-Acinetobacter baumann ii complex)? Acb

複合群，是由基因型不同但表現型非常相似的菌種所組成， 7 200533757 主要為4個已分類的基因種··基因種丨（醋酸約不動桿菌）、美因種2(鮑氏不動桿菌）、基因種3、基因種13TU[14]，以及2種分別為基因型接近13TU (ci〇se t0 13TU)之基因種（編號 10095號及10169號菌株）及基因型介於丨和3之間（編號 5804號及10090號菌株），也被歸納為复合群a[i2]二複合群的存在非常廣泛，無論是從大自然或醫院的環境中、健康人身上及受傷的組織常可分離出此複合群菌株。醋酸約不動桿菌屬於環境菌種[5, 9, u，12, 1δ]，一般不會引起人類致病，而其餘3個屬於複合群的基因種則 ^為重要的卩元内致病卤[12] ’會引起各種器官系統的感染，主要是對住院病人造成伺機性感染，然而，在一般的社區中偶爾也會造成感染，尤其是針對較年老者、慢性酒精中毒的人。其在臨床上感染主要的表徵可分為下列幾種[2, 3, 10, 22] :(1)呼吸道感染（reSpirat〇ry infection) ;( 2 )菌血症（bacteremia) ;( 3)尿道感染（urinary tract infection) ;( 4) 腦膜炎（meningitis);以及其他各種感染（other miscellaneous infections) 〇近年來，臨床常見的非發酵菌分離率逐漸增加，不動桿菌屬的分離率在非發酵菌中僅次於綠膿桿菌（h⑼而所⑽似其中複合群在臨床上較其他不動桿菌屬更為常見，是造成院内感染群突發 (outbreak)的重要病原菌[3]，因此對於院内感染的狀況、環境與病人的監控，及儘早提供醫師用藥資料，正確且快速的鑑定出致病原是必要的。傳統鑑定不動桿菌之方法係經由光學顯微鏡觀察革蘭氏染色呈陰性、菌體外觀呈球桿狀、嗜氧性、氧化酶陰性，可 200533757 初步鑑定至屬（genus)的階段，若要鑑定至種的階段則可依據 Bouvet d α/· [4-6]，Gerner-Smidt 以 α/· [12-14]， Vaneechoutte d α/· [23]，Nemec W α/· [17, 18]的研究報告所列之在不同溫度的生長試驗、綿羊血培養基的溶血反應、明膠的水解、從葡萄糖氧化產酸，及各種不同碳源的利用來加以區分，也有利用以聚合酶連鎖反應（PCR)為基礎的分子生物鑑定方法[17]。然而，前述傳統方法與市面上商業化套組的鑑定方法均無法有效的區分複合群中的各個菌種’而分子生物學的方法，如DNA-DNA雜合反應 (hybridization)，核糖體鑑定分型（ribotyping)雖可用來區分複合群，然而這些方法過於煩瑣且耗時，因此如何開發一簡單、有效且迅速的鑑定方法，是非常重要的工作。【發明内容】有鑑於傳統鑑定技術之缺失，本發明提供一簡單、有效且迅速的不動桿菌菌種之鑑定方法，其原理基於i6s_23s 核糖體DNA ( Hbosomal DNA，伽A )之内轉錄區⑽等❿ spacer region，ITS)序列在不動桿菌菌種間之特里性，本發明係利用前述之特性’架構臨床常見的葡萄糖非發酵菌⑽ 序列資料庫，並進而彻該資料庫來鑑定㈣桿菌菌種。本發明之利用ITS序列財不動桿菌菌種之方法係可利用聚合酶連鎖反應⑽）放大待測菌株之舰後，並盘不動桿菌之序列資料庫進行相似性之比對，其實施㈣如下：（a)自待測菌株萃取崎；⑴利用適當之引子對以增幅萃取飄中16S_23S核糖體舰之内轉錄區（its) 序列；⑴定出前述步驟(b)產物之難序列；⑷將步驟⑷ 之DNA序列與不動桿菌ITS序列f料庫進行排列分析，得 200533757 到序列相似值；以及（e)藉由比對相似值以判斷待測菌株之基因種。前述之方法較佳係用於鑑定不動桿菌屬之複合群菌種。再者’本發明之利用ITS序列鑑定不動桿菌菌種之方法亦可利用待測菌株之ITS序列與多種不動桿菌ITS序列之 DNA探針進行DNA-DNA雜合反應後，再根據雜合的結果判別待测菌株之基因種，其實施步驟如下：（a)取得多種含有不動桿菌菌種ITS序列之DNA探針，其中前述DNA探針係為單股DNA序列且可於適當環境下與其互補股進行雜 a ’（ b )自待測菌株萃取DNA ; ( c )利用適當之引子以增巾田萃取DNA中16S-23S核糖體DNA之ITS序列；（d)將步驟（Ο之DNA與步驟（a)之DNA探針於適當環境下共同培養一段時間；以及（e)偵測DNA雜合結果，以判斷待測菌株之基因種。由於ITS序列具有種特異性，因此當基因種大目同’其ITS序列相似度高，產生雜合的機率大，反之，基 :種不同’其ITS序列相似度低，產生雜合的而可以藉以判定待測菌株之基因種。本發明之具體實施態樣之一桿菌菌種！ T S序列之D N A探針固種含有不動定不動桿㈣種針固者於基材上，形成—可鐘群菌t發明之方法較佳係用於鐘定不動桿菌叙W複合 L貫施方式】本發明可提供—簡單、有致且迅速散残桿 ^本發明之__實施態樣，係利合囷株之職後，再定序並與不_之^== 200533757 行相似性之比對，包括下列步驟：（a )自待測菌株萃取 DNA ; (b)彻適當之引子以增幅萃取DNA中16S-23S核糖體DNA之1TS序列；（c)定出前述步驟（b)產物之DNa 序列，（d)將步驟（c)之DNA序列與不動桿菌之ITS序列資料庫進行排列分析，得到序列相似值；以及（e)藉由比對相似值以判斷待測菌株之基因種。前述步驟（c)係可利用各種熟悉本技術之人士常用之 DNA定序方式及儀器，或是委託專門之DNA定序單位來達成。前述步驟（d) ITS序列資料庫可藉由收集實驗菌株 16S-23S核糖體DNA之ITS序列而作成，前述實驗菌株係包含不動桿菌模式菌株以及參考菌株。16S_23S核糖體DNA 之ITS序列可藉由收集公開資料庫上的資訊來擴充，例如： GenBank上的ITS序列資料，以及藉由自行定序模式菌株或參考菌株之ITS序列而達成。前述ITS序列排列分析係可利用各種熟悉本技術人士常用之DNA排列分析程式及儀器，例如，但不限於··巨分子序列分析 GCG (Genomic Computer Group, Wisconsin Package 10.3版）之PileUp程式。前述之引子，係包括SEQ ID NO:l之核苷酸序列，其互補股、衍生物或簡併序列，其中SEQ ID NO: 1之序列為： GTCGT AACAA GGTAG CCGTA 〇前述之引子，係包括SEQ ID NO:2之核苷酸序列，其互補股、衍生物或簡併序列，其中SEQ ID NO:2之序列為： GGGTT YCCCC RTTCR GAAAT，前述 Y 為胞嘧啶（Cytosine， C )或胸腺嘧啶（Thymine，T ) ; R為腺嘌呤（Adenine，A ) 200533757 或鳥糞σ票吟（Guanine ，G )。前述”衍生物”係指於本發明之SEq ID Ν〇··ΐ、SEQ ID NO:2、前述序列互補股之核苷酸序列之3,端或5,端修飾其他核普酸序列，使其仍和原序列具有7〇%或以上相似性之核苷酸序列。前述”簡併序列”係指本發明中SEq ID N〇:i或SEQ ID NO:2序列中部分（30%或以下）核苷酸為其他核苷酸所取代，或上述兩序列互補股之核苷酸序列中部分（3〇%或以下）為其他核苷酸所取代。本發明之另一實施態樣係利用待測菌株之DNA與多種不動桿菌ITS序列之DNA探針進行DNA_DNA雜合反應後，再根據雜合的結果判別待測菌株之基因種，其實施步驟如下：（a)取得多種含有不動桿菌菌種ITS序列之DNA探針，其中前述DNA探針係為單股DNA序列且可於適當環境下與其互補股進行雜合；（b)自待測菌株萃取DNA ; ( c) 利用適當之引子以增幅萃取DNA尹16S-23S核糠體DNA之 ITS序列；（d)將步驟（c)之DNA與步驟（a)之DNA探針於適當環境下雜合（hybridization)—段時間；以及（e)摘測DNA雜合結果，以判斷待測菌株之基因種。前述步驟（a)之DNA探針可藉由收集實驗菌株16S_23S 核糖體DNA之ITS序列而作成，前述實驗菌株係包含不動才干囟模式株以及參考囟株。16S-23S核糖體DNA之ITS 序列可藉由收集公開資料庫上的資訊來擴充，例如： GenBank上的ITS序列資料，以及藉由自行定序模式菌株或參考菌株之ITS序列而達成。前述步驟（a )之DNA探針係可進一步固著於晶片上。 12 200533757 前述步驟（C)係可使用聚合酶連鎖反應進行增幅。前述步驟（c)之引子，係包括SEQ ID NO: 1之核苷酸序列，其互補股、衍生物或簡併序列，其中SEQIDNOzl 之序列為·· GTCGT AACAA GGTAG CCGTA 〇前述步驟（c)之引子，係包括SEQ ID NO:2之核苷酸序列，其互補股、衍生物或簡併序列，其中SEQIDNO:2之序列為： GGGTTYCCCCRTTCRGAAAT。其中 Y 為胞嘧啶（Cytosine， C )或胸腺17密 17定（Thymine，T ) ; R 為腺嗓呤（Adenine，A ) 或鳥糞嘌呤（Guanine ，G )。前述”衍生物”係指於本發明之SEQ ID NO:l、SEQ ID NO:2、前述序列互補股之核苷酸序列之3’端或5’端修飾其他核苷酸序列，使其仍和原序列具有70%或以上相似性之核苷酸序列。前述”簡併序列”係指本發明中SEQ ID ΝΟ··1或SEQ ID ΝΟ:2序列中部分（30%或以下）核苷酸為其他核苷酸所取代，或上述兩序列互補股之核苷酸序列中部分（30%或以下）為其他核苷酸所取代。實施例一、菌株之收集實驗菌株係向各國菌種中心，包括：台灣生物資源保存及研究中心（Bioresources Collection and Research Center，BCRC， Hsichu，Taiwan)、美國標準菌種中心（American Type Culture Collection，ATCC，USA)、瑞典大學菌種保存中心（Culture 13 200533757The complex group is composed of strains with different genotypes but very similar phenotypes. 7 200533757 Mainly 4 classified gene types · Gene types 丨 (Acinetobacter acetate), Main 2 (Bauer's fixed Bacillus), gene 3, gene 13TU [14], and 2 gene types (strains 10095 and 10169) with genotypes close to 13TU (ci0se t0 13TU), and genotypes between 丨 and 3 (Strains No. 5804 and No. 10090) can also be classified as complex group a [i2]. The existence of the second complex group is very widespread, whether it is from nature or hospital environment, healthy people and injured tissues. This complex group strain was isolated. Acinetobacter acetate is an environmental species [5, 9, u, 12, 1δ], which generally does not cause human disease, while the remaining three gene species belonging to the complex group are important pathogenic halogens [ 12] 'It can cause infections of various organ systems, mainly opportunistic infections in hospitalized patients. However, it also occasionally causes infections in the general community, especially for older people and people with chronic alcoholism. The main clinical manifestations of infection can be divided into the following types [2, 3, 10, 22]: (1) reSpiratorary infection; (2) bacteremia; (3) urethra Infection (urinary tract infection); (4) meningitis; and other miscellaneous infections. 〇 In recent years, the clinical isolation rate of non-fermentative bacteria has gradually increased. It is second only to Pseudomonas aeruginosa (h, and it seems that the complex group is more common clinically than other Acinetobacter genus, which is an important pathogen causing outbreak of nosocomial infection group [3]. Condition, environment and patient monitoring, as well as providing physician information as soon as possible, it is necessary to accurately and quickly identify the pathogen. The traditional method of identifying Acinetobacter is to observe Gram staining by optical microscope, and the appearance of the bacteria is negative. Club-shaped, oxophilic, and oxidase-negative, can be initially identified to the genus stage (200533757), and to the species stage can be identified according to Bouvet d α / · [4-6], Gerner-Smi dt listed in the research reports of α / · [12-14], Vaneechoutte d α / · [23], Nemec W α / · [17, 18] at different temperatures, the hemolytic response of sheep blood culture medium, The hydrolysis of gelatin, the oxidation of glucose to produce acid, and the use of a variety of different carbon sources are also useful for molecular biological identification methods based on polymerase chain reaction (PCR). [17] However, the aforementioned traditional methods are on the market None of the commercial sets of identification methods can effectively distinguish the individual species in the complex group. However, molecular biological methods such as DNA-DNA hybridization (ribryping) can be used to distinguish Complex groups, however, these methods are too cumbersome and time-consuming, so how to develop a simple, effective and rapid identification method is very important work. [Summary of the Invention] In view of the lack of traditional identification technology, the present invention provides a simple, effective And the rapid identification method of Acinetobacter species is based on the principle of the i6s_23s ribosomal DNA (Hbosomal DNA (Gamma A)) within the transcription region (such as the spacer region, ITS) sequence in Terry among the Acinetobacter species, the present invention using the characteristics of the system 'architecture common clinical glucose nonfermenters ⑽ sequence database, the database thus thoroughly identified (iv) mycobacterial species. The method for utilizing Acinetobacter strains of the ITS sequence according to the present invention can utilize polymerase chain reaction ⑽) after magnifying the ship of the test strain, and compare the sequence database of Acinetobacter for similarity, the implementation is as follows: (A) Extracting saki from the strain to be tested; 萃取 Using appropriate primer pairs to extract the internal transcribed region (its) sequence of the 16S_23S ribosome in the wind; ⑴ Determine the difficult sequence of the product of step (b); ⑷ step The sequence analysis of the DNA sequence of 分析 and the ITS sequence f library of Acinetobacter spp. Obtained the sequence similarity value of 200533757; and (e) judging the genetic species of the strain to be tested by comparing the similarity values. The aforementioned method is preferably used for identifying a complex bacterial species of the genus Acinetobacter. Furthermore, the method for identifying Acinetobacter strains using the ITS sequence of the present invention can also use the ITS sequence of the tested strain and DNA probes of various Acinetobacter ITS sequences to perform a DNA-DNA hybrid reaction, and then according to the result of the hybridization To identify the genetic species of the strain to be tested, the implementation steps are as follows: (a) Obtain a variety of DNA probes containing ITS sequences of Acinetobacter species, wherein the aforementioned DNA probes are single-stranded DNA sequences and can be complementary to them under appropriate circumstances. (A) Extracting DNA from the strain to be tested; (c) Extracting the ITS sequence of 16S-23S ribosomal DNA from DNA using appropriate primers; (d) DNA of step (0 and step ( a) The DNA probes are co-cultured for a period of time in an appropriate environment; and (e) The DNA hybridization results are detected to determine the genetic species of the tested strain. Because the ITS sequence has species specificity, when the genetic species are the same 'Its ITS sequences have a high degree of similarity and a high probability of heterozygosity, and conversely, the basis: species are different.' Its ITS sequences have a low degree of similarity and a heterogeneous type, which can be used to determine the genetic species of the strain to be tested. DETAILED DESCRIPTION OF THE INVENTION Bacillus sp The solid DNA probe of the TS sequence contains immobilized rods and immobilized needles on the substrate to form a method which can be invented by the group of bacteria. The method is preferably used for the application of A. bacterium ] The present invention can provide-simple, conscientious and rapid dispersal ^ __ implementation of the present invention, after the position of Lihe Bazhu, then sequenced and the ratio of similarity with the non__ ^ == 200533757 Yes, it includes the following steps: (a) extracting DNA from the strain to be tested; (b) extracting the 1TS sequence of 16S-23S ribosomal DNA in DNA with appropriate primers; (c) determining the product of step (b) above DNa sequence, (d) arrange and analyze the DNA sequence in step (c) and the ITS sequence database of Acinetobacter to obtain sequence similarity values; and (e) judge the genetic species of the strain to be tested by comparing the similarity values. Step (c) can be achieved by using various DNA sequencing methods and instruments commonly used by those skilled in the art, or by entrusting a special DNA sequencing unit. The aforementioned step (d) ITS sequence database can be collected by experimental strain 16S -23S ribosomal DNA ITS sequence, the above experimental strain Contains Acinetobacter model strains and reference strains. The ITS sequence of 16S_23S ribosomal DNA can be expanded by collecting information from public databases, such as ITS sequence data on GenBank, and by self-sequencing model strains or reference strains. ITS sequence analysis can be achieved. The aforementioned ITS sequence analysis can use various DNA sequence analysis programs and instruments familiar to those skilled in the art, such as, but not limited to, the macromolecular sequence analysis GCG (Genomic Computer Group, Wisconsin Package version 10.3) PileUp program. The aforementioned primers include the nucleotide sequence of SEQ ID NO: 1, its complementary strand, derivative or degenerate sequence, wherein the sequence of SEQ ID NO: 1 is: GTCGT AACAA GGTAG CCGTA 〇 The aforementioned primer includes SEQ The nucleotide sequence of ID NO: 2, its complementary strand, derivative or degenerate sequence, wherein the sequence of SEQ ID NO: 2 is: GGGTT YCCCC RTTCR GAAAT, the aforementioned Y is cytosine (Cytosine, C) or thymine ( Thymine, T); R is adenine (Adenine, A) 200533757 or Guano (Guanine, G). The aforementioned “derivative” refers to the 3, 5 or 5 end of the nucleotide sequence of the SEq ID NO .., SEQ ID NO: 2 of the aforementioned complementary strand of the present invention, so that other nucleotide sequences are modified so that It still has a nucleotide sequence that is 70% or more similar to the original sequence. The aforementioned “degenerate sequence” refers to the substitution of some nucleotides (30% or less) in the sequence of SEq ID No: i or SEQ ID NO: 2 in the present invention by other nucleotides, or the complementary strands of the above two sequences. Part of the nucleotide sequence (30% or less) is replaced by other nucleotides. Another embodiment of the present invention uses the DNA of the strain to be tested and DNA probes of various Acinetobacter ITS sequences for DNA_DNA hybridization, and then determines the genetic species of the strain to be tested based on the results of the hybridization. The implementation steps are as follows: : (A) Obtain a variety of DNA probes containing ITS sequences of Acinetobacter species, wherein the aforementioned DNA probes are single-stranded DNA sequences and can be hybridized with their complementary strands under appropriate circumstances; (b) extracted from the test strain DNA; (c) using appropriate primers to amplify the ITS sequence of DNA Yin 16S-23S nuclear bran DNA; (d) hybridize the DNA of step (c) with the DNA probe of step (a) under appropriate circumstances (Hybridization) —for a period of time; and (e) extracting DNA hybridization results to determine the genetic species of the strain to be tested. The DNA probe of the aforementioned step (a) can be prepared by collecting the ITS sequence of the 16S_23S ribosomal DNA of the experimental strain, and the aforementioned experimental strain includes a fixed-capacity model strain and a reference strain. The ITS sequence of 16S-23S ribosomal DNA can be expanded by collecting information from public databases, such as ITS sequence data on GenBank, and by self-sequencing ITS sequences of model strains or reference strains. The DNA probe of the aforementioned step (a) can be further fixed on the wafer. 12 200533757 The aforementioned step (C) can be amplified using a polymerase chain reaction. The primer of the aforementioned step (c) includes the nucleotide sequence of SEQ ID NO: 1, the complementary strand, derivative or degenerate sequence thereof, wherein the sequence of SEQ ID NOzl is GTCGT AACAA GGTAG CCGTA 〇 of the aforementioned step (c) The primer includes the nucleotide sequence of SEQ ID NO: 2, its complementary strand, derivative or degenerate sequence, wherein the sequence of SEQIDNO: 2 is: GGGTTYCCCCRTTCRGAAAT. Wherein Y is Cytosine (C) or Thymine (Tymine, T); R is Adenine (A) or Guanine (G). The aforementioned "derivative" refers to the modification of other nucleotide sequences at the 3 'end or 5' end of the nucleotide sequence of the complementary strand of the aforementioned sequences of SEQ ID NO: 1, SEQ ID NO: 2, and the present invention A nucleotide sequence that is 70% or more similar to the original sequence. The aforementioned "degenerate sequence" refers to a part (30% or less) of nucleotides in the SEQ ID NO ·· 1 or SEQ ID NO: 2 sequence in the present invention being replaced by other nucleotides, or the complementary strands of the two sequences above. Part of the nucleotide sequence (30% or less) is replaced by other nucleotides. Example 1. Collection of Strains Strains were collected from various strain centers in various countries, including: Taiwan Bioresources Collection and Research Center (BCRC, Hsichu, Taiwan), American Type Culture Collection (American Type Culture Collection, ATCC, USA), University of Sweden Strain Collection Center (Culture 13 200533757

Collection，University of G6teborg，Sweden，CCUG)、比利時菌種保存中心（Laboratorium voor Microbiologie Gent，LMG，Belgium)、荷蘭菌種保存中心（The Netherlands Culture Collection of Bacteria, NCCB，The Netherlands)購買臨床常見葡萄糖非發酵菌菌種之模式菌株（type strain)及參考菌株（referencestrain)，包含不動桿菌屬(表2)及非不動桿菌屬之其他葡萄糖非發酵菌（表3) ’共計42種⑽菌株）。臨床分離株由成大醫院病理部細菌室所提供。Collection, University of G6teborg, Sweden, CCUG), Laboratorium voor Microbiologie Gent, LMG, Belgium, The Netherlands Culture Collection of Bacteria, NCCB, The Netherlands Type strains and reference strains of fermentative bacteria strains include Acinetobacter (Table 2) and other glucose non-fermenting bacteria of non-Acinetobacter genus (Table 3) 'a total of 42 species of tadpole strains). The clinical isolates were provided by the bacteria department of the Department of Pathology, Chengdu University Hospital.

14 200533757 表2本研究所採用之實驗菌株（不動桿菌屬)14 200533757 Table 2 Experimental strains used in this study (Acinetobacter)

菌種/基因種菌株編號 A. calcoaceticus /\ BCRC 11562, LMG 992, LMG 1046T A. baumannii /2 BCRC 10591' BCRC 15884, BCRC 15886, LMG 984 UN/3 BCRC 15420, CCUG 26384, LMG 1035 A. haemolyticus /4 BCRC 14852' BCRC 15887, LMG 997, LMG 1001 A.junii/5 BCRC 14854T UN/6 BCRC 15421 A. johnsonii /7 BCRC 14853T，BCRC 15888, LMG 1002 A. Iwoffii/S BCRC 14855' NCCB 83020 UN/9 LMG 985, LMG1017, LMG 1300 UN/10 BCRC 15423, LMG 10600 UN/11 BCRC 15424, LMG 10603 A. radioresistens!\2 BCRC 15425T，CCUG 26388, CCUG 34434 UN/13TU BCRC 15417 UN/14TU (= UN/13BJ) LMG 1235, LMG 10627 UN/15TU CCUG 26390 UN/14BJ CCUG 14816, CCUG 34435 UN/15BJ CCUG 34436 UN/16BJ BCRC 15883 UN/17BJ CCUG 34437 A. venetianus CCUG 45561T A. ursingii LMG 19575T A. schindleri LMG 19576T 模式菌株（type strain) 15 200533757 J$$0 00 ossslcrauad'UHCyaua^^CNlu^ua^rosluHuaH^irnz.olu^us Ss6n:>aua^s6u 3>IuPQ\fn68cnliTauarir-9u:yauffl 6 寸 ssluDius \965013>133;5二5331\^〇〇26 寸uulv Ho<N8 寸 i 3Ήυρα^60 寸 i cyaua 9SSIu>I3au(N8H 3>I3S\88SUUU1V 59313(^330:17313^0519313^38^0917013^303/63013^3(¾ HCN06uu>Iuar^寸 WJuulv HocslosariISuulv 910913Ή38Γ卜寸 e寸 l 3Ήυ〒οι6ειυΉυρ3οί06πυΉυΗ\8(ΝοιιυΉυρα<0601 cyauH^OSIu^ua ^06πυΉυ8 "寸寸 OSSuulv Kr-oornsuulv 卜寸寸 60ιυΉυ8τΓης8ζ,(Ν3υ1ν Ηοοζ,90ιυΉυ3 066(n3u1v 0£8 寸 I cyaua \6Ϊ8 寸 ιυΉυρανο卜寸卜IU31Virrs 寸卜 Iυυινοοε 寸卜 I 331V ^>90ιυΉυΗ^ς6ί:ί:υ31ν/ς(Νπ3υ1νΊ7ς(Νπυυ1ν U 寸6 寸uulv \r-686(Nuulv 卜668nusPQ 906πυΉυρα\80(Νπ cyauCQ οε90ιυΉ303\6(Ν90Ιυδραοο^ιυΉυΗ 08ιΗυΉοίΏ\6ε83ιυΉυ8 ^〇〇28013>!03/59$£ 3U1V ^ooseeuulvoo'loeluulvoo^Koouulv 91 ϋιιμιάοιινηί svuoulotldculou(vls slllqomlonad svuoluosulvldM su3PVJ3i:nd VJPUVM.(vlls llu^uldaluoJSJVp^ zszlnls .J νρμηά .J sdudMllvolvopndsd Vuoopu3m .J sususdonlf S3u33llvulvPH asoulMrudaSOUOmopndsPL, smsopo S3PCM^S' SUVUOAOPloaVIJJPQ lunDl}d3sosulu3wu sdusMOJOPUl ulnT3iDvqo3s/calo lbooq aovdfvo νχωριοψιΛηρρ slantmao vjpmvnuvug 的 uvplxoso%x .dsqns euvp 一 XOSO^X.^ slao<vVJ-dsqns s 二 VU3VJ S3u3slaujy (u*sJls3dxo^«^^: 200533757 二、建構核糖體DNA之内轉辞氐序列資料庫挑取適當實驗菌株之菌落以萃取DNA,可採用傳統方法得到較純的產物，或使用操作較為簡單快速的商業化套組 (commercial kit)，若為臨床檢體貝q採用更快速的沸騰法，僅需 15分鐘即可完成。Strain / Genetic strain number A. calcoaceticus / \ BCRC 11562, LMG 992, LMG 1046T A. baumannii / 2 BCRC 10591 'BCRC 15884, BCRC 15886, LMG 984 UN / 3 BCRC 15420, CCUG 26384, LMG 1035 A. haemolyticus / 4 BCRC 14852 'BCRC 15887, LMG 997, LMG 1001 A.junii / 5 BCRC 14854T UN / 6 BCRC 15421 A. johnsonii / 7 BCRC 14853T, BCRC 15888, LMG 1002 A. Iwoffii / S BCRC 14855' NCCB 83020 UN / 9 LMG 985, LMG1017, LMG 1300 UN / 10 BCRC 15423, LMG 10600 UN / 11 BCRC 15424, LMG 10603 A. radioresistens! \ 2 BCRC 15425T, CCUG 26388, CCUG 34434 UN / 13TU BCRC 15417 UN / 14TU (= UN / 13BJ) LMG 1235, LMG 10627 UN / 15TU CCUG 26390 UN / 14BJ CCUG 14816, CCUG 34435 UN / 15BJ CCUG 34436 UN / 16BJ BCRC 15883 UN / 17BJ CCUG 34437 A. venetianus CCUG 45561T A. ursingii LMG 19575T A. schindleri LMG 19576T Type strain 15 200533757 J $$ 0 00 ossslcrauad'UHCyaua ^^ CNlu ^ ua ^ rosluHuaH ^ irnz.olu ^ us Ss6n: > aua ^ s6u 3 > IuPQ \ fn68cnliTauarir-9u: yauffl 6 inch ssluDius \ 965013 133; 5 2 5331 \ ^ 〇〇26 inch uulv Ho &N; inch 8 υρα ^ 60 inch i cyaua 9SSIu > I3au (N8H 3 > I3S \ 88SUUU1V 59313 (^ 330: 17313 ^ 0519313 ^ 38 ^ 0917013 ^ 303/63013 ^ 3 (¾ HCN06uu > Iuar ^ inchWJuulv HocslosariISuulbu 91091338 inch) 〒οι6ειυΉυρ3οί06πυΉυΗ \ 8 (ΝοιιυΉυρα < 0601 cyauH ^ OSIu ^ ua ^ 06πυΉυ8 " begins OSSuulv Kr-oornsuulv Bu begins 60ιυΉυ8τΓης8ζ, (Ν3υ1ν Ηοοζ, 90ιυΉυ3 066 (n3u1v 0 £ 8 inch I cyaua \ 6Ϊ8 inch ιυΉυρανο Bu inch Bu IU31Virrs inch Bu Iυυινοοε Inch Bu I 331V ^ > 90ιυΉυΗ ^ ς6ί: ί: υ31ν / ς (Νπ3υ1νΊ7ς (Νπυυ1ν U Inch 6 Inch uulv \ r-686 (Nuulv RUB668nu6P8 8838 Η Η6αC90QNεεαC90) ^ 〇〇28013 > 03/59 $ £ 3U1V ^ ooseeuulvoo'loeluulvoo ^ Koouulv 91 ϋιιμιάοιινηί svuoulotldculou (vls slllqomlonad svuoluosulvldM su3PVJ3i:! nd VJPUVM (vlls llu ^ uldaluoJSJVp ^ zszlnls .J νρμηά .J sdudMllvolvopndsd Vuoopu3m .J sususdonlf S3u33llvulvPH asoulMrudaSOUOmopndsPL,. smsopo S3PCM ^ S 'SUVUOAOPloaVIJJPQ lunDl} d3sosulu3wu sdusMOJOPUl ulnT3iDvqo3s / calo lbooq aovdfvo νχωριοψιΛηρρ slantmao vjpmvnuvug of uvplxoso% x euvp a XOSO ^ X. ^ slao & lt .dsqns; vVJ-dsqns s two VU3VJ S3u3slaujy (u * sJls3dxo ^ «^^: 200533757 II. Construct an internal translation database of ribosomal DNA. Pick out the colonies of appropriate experimental strains to extract DNA. You can use traditional methods to obtain pure products, or use a commercial kit that is simpler and faster to operate. A faster boiling method for clinical specimens can be completed in just 15 minutes.

接著，利用一對細囷通用引子（universal primer)進行聚合酶連鎖反應（PCR)，其中正向引子係對應於五•的16SrDNA 位置上1493-1512,命名為1512F(SEQIDΝΟ:1)，其可與反向引子對應於五· Cb/z·的23S rDNA位置上之108-130，命名為6R (SEQ IDNO:2)共同放大本實驗所有的菌種，如表4所示。表4本研究所使用通用引子對之序列及其相對位置引子序列'5’—3’）引子所在位置6 1512F GTCGT AACAA GGTAG CCGTA 16S rRNA，1493-1512 6R GGGTT YCCCC RTTCR GAAAT 23S rRNA, 130-108Next, a pair of universal primers was used for polymerase chain reaction (PCR). The forward primers corresponded to the 5S 16SrDNA position 1493-1512 and were named 1512F (SEQ ID NO: 1). The reverse primers corresponded to 108-130 on the 23S rDNA position of 5 · Cb / z · and named 6R (SEQ IDNO: 2). All strains in this experiment were amplified, as shown in Table 4. Table 4 Sequences of universal primer pairs and their relative positions used in this study Primer sequence '5’-3') Primer position 6 1512F GTCGT AACAA GGTAG CCGTA 16S rRNA, 1493-1512 6R GGGTT YCCCC RTTCR GAAAT 23S rRNA, 130-108

flY:為C或T，R:為A或G cW的核苷酸編號 PCR放大ITS序列反應進行如下，在50微升（μΐ)的 PCR反應溶液中含2微升之模板DNA (約100 ng)，5微升卩〇1131^61*(10乂），3微升去氧核糖核苷三磷酸（(1]^?)混合物（每種dNTP 2.5毫莫耳濃度（mM))，35微升滅菌蒸鶴水（sterile-distilled water)，1512F 引子及 6R 引子（最終濃度20微莫耳濃度（μΜ))各2.5微升，0.5微升DNA聚 17 200533757 合酶（2·0單位/微升之ProZyme II)，最後於反應液上添加5〇微升礦物油，以循環加熱器開始反應。循環加熱器之反應條件為：94°C 2分鐘；94°C 1分鐘，62。(： 1分鐘，72°C 1分鐘（30個循環）；72QC7分鐘（1個循環）。當PCR所放大的片段並非單一長度產物時，若不能以切取膠體上含有DNA之條帶（band)部分或以注射針頭抽取再做二次PCR放大而得到單一長度產物時，則利用分子選殖的方式將不同長度PCR產物個別篩選出來。本發明係使用 TOPO TA Cloning Kit (Invitrogen，USA)，利用 7叫聚合酶同時具有的末端連接酶（ligase)的功能，在每條pcR 擴增產物（目標DNA片段）的3’端自動添加一個3f-腺嗓呤 (3f_A)突出端（overhangs)，Topo TA cloning 提供一個線性含 3’_胸腺嘧啶（3’-T)突出端的拓樸異構酶（TopoiSomerase I) 載體（vector)，利用載體上之拓樸異構酶高效率的連接特性，可以把含3f-A突出端之PCR產物快速連接到3，-T端載體上，之後再藉由轉形作用（transformation)將其送入五. co"中，選殖接入載體中的目標DNA片段便隨著五· co/z•的複製而複製，接著再經由載體的特異性引子對（M13 primers)進行PCR反應，放大出所選殖之目標DNA片段。最後再以目標DNA片段的通用引子對將目標DNA放大出來，經電泳確認產物後即可進行定序。經PCR放大16S - 23S核糖體DNA(rDNA)之内轉錄區 (ITS)，如第一圖所示，ITS序列具有種特異性（species-specific) ，可作為區分菌種之標準， ITS 序列可能包含一至多個高保留性轉移RNA (transfer RNA; tRNA)基因序列，在革蘭氏陰性細菌中通常有兩個tRNA基因[15]，分別為攜 200533757 帶有丙胺酸（alanine; ala)與異亮胺酸（isoleucine; ile)之 tRNA基因，此二tRNA基因的存在會將ITS序列分為三個部份，本發明所針對之ITS序列包含此tRNA基因序列。 i 、# 歹1j 1^匕 # DNA序列比對則採用巨分子序列分析gcG (Genomic Computer Group，Wisconsin Package 10·3 版）程式，做並列排序（alignment)及相似值計算（similaritysc〇re)，確認每一囷種具有特異性ITS序列，並收集GenBank上已發表之常見非發酵菌的ITS序列，架構成臨床常見非發酵菌之ITS序列資料庫’作為鑑定不動桿菌菌種之依據。同種菌株序列比對同一菌種之不同菌株ITS序列以GCG之PileUp程式進行序列排列分析’以母一菌種的模式菌株（type strain)為基準，若該菌種無模式菌株，則任選一參考菌株當代表，比對不同菌株間的相似度，結果如表5。複合群之4個基因種（1，2，3，13TU)種内具有很高的相似值（> 〇 98)，唯基因種13TU僅有一株，雖無法確認與其他株之相似度，但其與GenBank的序列（U60281)相符，因此可確認此菌株之序列正確無誤。複合群菌穑之ITS比對本發明定出複合群中之各基因種之ITS序列，利用PileUp做序列排序分析，所得到的相似值如表6，結果顯示不同的基因種之間的相似值介於〇.86〜〇.92之間，亦即不同基因種之間具有至少8%以上的差異性，此差異性已足以用來區分個別的基因種。菌種之序列比對 19 200533757 將ye複合群菌種與其他不動桿菌屬菌種各取一個代表性基因種之最短ITS序列利用PileUp進行多序列排序分析，並將其所得之相似值列於表7,由表7中可以看出複合群與其他的基因種相似值在0.78以下，具有很大的差異性。非不動桿菌屬之葡萄糖非發酵菌序列比對將本實驗與GenBank上所收集的臨床常見非發酵菌之 ITS序列，利用PileUp做序列排序分析比對不同菌種之間的差異度。原則上由GenBank所收集的序列若菌株編號 (strain no.)與本實驗相同，則以本實驗之序列為主。表8 為非發酵菌不同菌種之ITS序列比對結果，由於不同菌屬的序列差異性較大，因此在表8中不動桿菌菌屬僅以鮑氏不動桿菌之序列代表。不同菌種間相似度值介於0.36〜0.96，大部分的相似度值在0.77以下，若用來區分不同的非發酵菌種，應具有相當大的潛力。flY: C or T, R: A or G cW nucleotide number PCR amplification ITS sequence reaction is performed as follows, 2 microliters of template DNA (about 100 ng) in 50 microliters (μΐ) of the PCR reaction solution ), 5 μl 卩 1311 ^ 61 * (10 (), 3 μl deoxyribonucleoside triphosphate ((1) ^?) Mixture (2.5 millimoles (mM) per dNTP), 35 μl Liters of sterile-distilled water, 1512F primer and 6R primer (final concentration 20 micromolar concentration (μΜ)) 2.5 microliters each, 0.5 microliter DNA poly 17 200533757 synthase (2.0 units / microliter ProZyme II), and finally added 50 microliters of mineral oil to the reaction solution to start the reaction with a circulating heater. The reaction conditions of the circulating heater are: 94 ° C for 2 minutes; 94 ° C for 1 minute, 62. (: 1 Minutes, 72 ° C for 1 minute (30 cycles); 72QC for 7 minutes (1 cycle). When the PCR amplified fragment is not a single-length product, if it is not possible to cut out the band portion of the colloid containing DNA or to When a single length product is obtained by extracting the injection needle and then performing secondary PCR amplification, the molecular selection method is used to individually screen PCR products of different lengths. Here, the present invention uses the TOPO TA Cloning Kit (Invitrogen, USA) and utilizes the function of 7-terminal polymerase, which also has a terminal ligase, to automatically generate the 3 'end of each pcR amplification product (target DNA fragment). Add a 3f-adenine (3f_A) overhangs. Topo TA cloning provides a linear TopoiSomerase I vector with 3'_thymine (3'-T) overhangs. Utilizing the efficient ligation characteristics of the topoisomerase on the vector, the PCR product containing the 3f-A overhang can be quickly ligated to the 3, -T end vector, and then sent by transformation In Co., the target DNA fragment in the selective accession vector is replicated with the replication of co./z. Then the PCR reaction is carried out through the specific primer pairs (M13 primers) of the vector, and amplified. The selected target DNA fragment is amplified. Finally, the target DNA fragment is amplified by the universal primer pair, and the product can be sequenced after confirming the product by electrophoresis. The internal transcription of 16S-23S ribosomal DNA (rDNA) is amplified by PCR. Area (ITS), as shown in the first figure The ITS sequence is species-specific and can be used as a criterion for distinguishing strains. The ITS sequence may contain one or more high-retention transfer RNA (tRNA) gene sequences, which are usually found in gram-negative bacteria. There are two tRNA genes [15], which are 200533757 tRNA genes with alanine (ala) and isoleucine (ile), respectively. The presence of these two tRNA genes will divide the ITS sequence into three In part, the ITS sequence targeted by the present invention comprises this tRNA gene sequence. i 、 # 歹 1j 1 ^ 匕 # DNA sequence alignment uses a macromolecular sequence analysis gcG (Genomic Computer Group, Wisconsin Package version 10.3) program to do side-by-side alignment (alignment) and similarity calculation (similarityscore), Confirm that each species has specific ITS sequences, and collect the ITS sequences of common non-fermenting bacteria that have been published on GenBank, and construct the ITS sequence database of clinical non-fermenting bacteria as a basis for identifying Acinetobacter species. Sequence alignment of different strains of the same species. ITS sequences of different strains of the same species are sequenced and analyzed based on the GCG's PileUp program. 'Based on the type strain of the parent strain. If the strain does not have a model strain, choose one. The reference strain is used as a representative, and the similarity between different strains is compared. The results are shown in Table 5. The four gene species (1,2,3,13TU) of the complex group have a high similarity value (> 〇98). There is only one strain of the gene gene 13TU, although the similarity with other strains cannot be confirmed, but It matches the sequence of GenBank (U60281), so it can be confirmed that the sequence of this strain is correct. ITS comparison of bacterial complexes in the complex group The ITS sequence of each gene species in the complex group is determined by the present invention, and Sequence Up analysis is performed using PileUp. The similarity values obtained are shown in Table 6. The results show that the similarity values between different gene species are introduced. There is a difference of at least 8% between 0.86 and 0.92, that is, between different gene species, and this difference is sufficient to distinguish individual gene species. Sequence comparison of strains 19 200533757 Take the shortest ITS sequence of a representative gene species from each of the ye complex group strain and other Acinetobacter species, and use PileUp to perform multi-sequence sequencing analysis, and list the similarity values obtained in the table. 7. It can be seen from Table 7 that the similarity value between the composite group and other gene species is below 0.78, which is very different. Sequence comparison of non-Agrobacterium saccharomyces non-fermentative bacteria. This experiment and the ITS sequences of common non-fermentative clinical bacteria collected on GenBank were used to sequence analysis using PileUp to compare the differences between different species. In principle, if the strain number (strain no.) Of the sequence collected by GenBank is the same as that of this experiment, the sequence of this experiment is the main one. Table 8 shows the results of ITS sequence comparison of different strains of non-fermenting bacteria. Because the sequences of different genus are quite different, the Acinetobacter genus in Table 8 is only represented by the sequence of Acinetobacter baumannii. The similarity value between different strains ranges from 0.36 to 0.96, and most of the similarity values are below 0.77. If used to distinguish different non-fermentative strains, it should have considerable potential.

20 200533757 _表5不多桿菌屬不同菌株之ITS序包£目似度值菌種/基因種菌株編號 ITS長度(bp)相似值b Gen Bank登錄號 A. calcoaceticus /1 LMG 1046T LMG 1046T BCRC 11562 LMG 992 A. baumannii/2 BCRC 10591T BCRC 10591T BCRC 15884 BCRC 15886 LMG 984 UN/3 LMG 1035 LMG 1035 BCRC 15420 CCUG 26384 A. haemolyticus !Aa BCRC 14852T BCRC 15887 LMG 997 LMG 1001 A. johnsonii /T BCRC 14853T BCRC 15888 LMG 1002 A. lwoffii/%a BCRC 14855T NCCB 83020 UN/10 BCRC 15423 LMG 10600 UN/11 BCRC 15424 LMG 10603 A. radioresistens!\2 BCRC 15425T CCUG 26388 CCUG 34434 UN/13TU BCRC 15417 BCRC 15417 UN/14TU (= 13BJ) LMG 1235 LMG 10627 UN/14B/ CCUG 34435 CCUG 14816 8 7 7 8 3 2 3 3 6 6 6 6 )7)9)7)7)79199 4004 301 60606060606162616161636361707070 ?8^9)0)9)0)0)0900 ·99·ο· 9 0 0 0 9 0 0 ο ο 10·1·1·1·0·1*1· U60278 U60279 U60280 963433 2025879 οι 251199 3331062 23 666655 6666666 66 949499 0.0.0. 94 8 8 0. 謂 9 9 0.20 200533757 _Table 5 ITS sequence of different strains of Polybacillus. Similarity value Strain / Genetic strain number ITS length (bp) Similar value b Gen Bank accession number A. calcoaceticus / 1 LMG 1046T LMG 1046T BCRC 11562 LMG 992 A. baumannii / 2 BCRC 10591T BCRC 10591T BCRC 15884 BCRC 15886 LMG 984 UN / 3 LMG 1035 LMG 1035 BCRC 15420 CCUG 26384 A. haemolyticus! Aa BCRC 14852T BCRC 15887 LMG 997 LMG 1001 A. johnsonii / T BCRC 14853T BCRC 15888 LMG 1002 A. lwoffii /% a BCRC 14855T NCCB 83020 UN / 10 BCRC 15423 LMG 10600 UN / 11 BCRC 15424 LMG 10603 A. radioresistens! \ 2 BCRC 15425T CCUG 26388 CCUG 34434 UN / 13TU BCRC 15417 BCRC 15417 UN / 14TU (= 13BJ) LMG 1235 LMG 10627 UN / 14B / CCUG 34435 CCUG 14816 8 7 7 8 3 2 3 3 6 6 6) 7) 9) 7) 7) 79199 4004 301 60606060606162616161636361707070 8) 9) 0) 0) 0900 · 99 · ο · 9 0 0 0 9 0 0 ο ο 10 · 1 · 1 · 1 · 0 · 1 * 1 · U60278 U60279 U60280 963433 2025879 οι 251199 3331062 23 666655 6666666 66 949499 0.0.0. 94 8 8 0. It means 9 9 0.

8 8 0. 094 U60281 α有多個ITS片段，長度不同，以最短長度進行比較"以列在每一菌種之第一個菌株為相似值之計算基準；^亥登錄號（accession no·)之菌株編號已轉換成與本實驗相同的對應編號，如U60278之菌株編號為ATCC 23055T (= LMG 1046τ) ; Τ 模式菌株；UN，未命名. 21 200533757 表6 複合群不同基因種間ITS序列相似度值基因種a 菌株編號 ITS長度 (b.p·) 相似值 1 2 3 13TU 1 LMG 1046τ 638 0.86 0.89 0.91 2 BCRC 10591τ 607 0.86 0.91 0.92 3 LMG 1035 619 0.89 0.91 0.89 13TU BCRC 15417 615 0.91 0.92 0.89 β基因種：1.醋酸鈣不動桿菌；2.鮑氏不動桿菌；未命名之基因種3及13TU; 7 模式菌株係購自台灣生物資源保存及研究中心（BCRC)與比利時菌種保存中心（LMG)。 200533757 13 0<Nos【 0018 8 0. 094 U60281 α There are multiple ITS fragments with different lengths, and the shortest length is used for comparison. "The first strain listed in each species is used as the basis for calculation of similar values; ^ Access number (accession no. The strain number of) has been converted to the same corresponding number as this experiment, for example, the strain number of U60278 is ATCC 23055T (= LMG 1046τ); T model strain; UN, unnamed. 21 200533757 Table 6 ITS sequences of different genes in the complex group Similarity value gene species a Strain number ITS length (bp) Similarity value 1 2 3 13TU 1 LMG 1046τ 638 0.86 0.89 0.91 2 BCRC 10591τ 607 0.86 0.91 0.92 3 LMG 1035 619 0.89 0.91 0.89 13TU BCRC 15417 615 0.91 0.92 0.89 β gene Species: 1. Acinetobacter calcium acetate; 2. Acinetobacter baumannii; unnamed gene species 3 and 13TU; 7 model strains were purchased from the Taiwan Biological Resources Conservation and Research Center (BCRC) and the Belgian Strain Conservation Center (LMG) . 200533757 13 0 < Nos 【001

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France)確認該分離株屬於鲍氏不動桿菌或醋酸鈣不動桿菌，之後再將每一分離株測定ITS序列，並送入所建立的ITS序列資料庫進行比對，如此便可得到一個與其最相近的菌種名稱及相似度值，之後再與之前利用商業化套組（Αρι 20 NE)鑑定之結果進行比較，結果如表9所示。臨床分離株複合群共收集有82株，以API 20 Ne梦定社果63株為鮑氏不動桿菌，其餘19株為醋酸鈣不動桿菌。作細France) confirmed that the isolate belonged to Acinetobacter baumannii or Acinetobacter calcium acetate, and then determined the ITS sequence of each isolate and sent it to the established ITS sequence database for comparison. The strain name and similarity value were compared with the previous identification results using a commercial kit (Αρι 20 NE). The results are shown in Table 9. A total of 82 strains of clinical isolates were collected. 63 strains of API 20 Ne Mengding were Acinetobacter baumannii and the remaining 19 strains were Acinetobacter calcium acetate. Make fine

—、、'工 I 丁 S 序列分析63株鮑氏不動桿菌中僅有47株為正確鑑定，2株^ + _ 桿菌基因種3，12株為基因種13TU; 2株無法分基因種但與基因種3 序列較接近，其相似值分別為0.93及0.96。19株醋酸舞不動桿菌中，以ITS序列分析沒有一株是醋酸鈣不動桿菌，16株為不動桿菌基因種3，1株為基因種10，1株為基因種13TU，1株為基因種 15TU。其中基因種3、10、13TU和15TU之結果並由核糖體DNA φ 剪切分析（ribosomal DNA restriction analysis，ARDRA) [23]加以 25 200533757 基因種15TU無法分基因種 2 (3.2%) 0 0 1 (5.3%) ITS序列分析基因種13TU 12 (19%) 1 (5.3%) 基因種10 0 1 (5.3%) 基因種3 2 (3.2%) 16 (84.2%) 鮑氏不動桿菌 47 (74.6%) 0 C \ \ < tn m Os VO r-H 以 API 20 NE 鑑定結果 m m 竑 If 眾 τ ι u ι001 δ 寸(Noo碱潆 200533757 本發明成功地利用核醣體DNA之ITS序列架構資料庫，並用來區分生化特性相似的複合群菌種，若利用常見的商業化套組API 20 NE分析，多半被鑑定為鮑氏不動桿菌或醋酸鈣不動桿菌。然而正如表九所示，經過本發明之鑑定後，發現API 20 NE 鑑定為同基因種的不動桿菌其實仍有相當程度的誤差。本發明之方法不僅提供一更正確的判別基因種的方式，且於菌種培養後約需1天時間就能區分種名，相較於傳統的生化鑑定方法來的更為簡易、迅速。雖然本發明已以實施例揭露如上，然其並非用以限定本發明，任何熟悉此技藝者，在不脫離本發明之精神和範圍内，當可鲁作各種之更動與潤飾，因此，本發明之保護範圍，當視後附之申請專利範圍所界定者為準。【圖式簡單說明】第一圖係為16S-23S核糖體DNA之ITS序列示意圖。-,, 'Gong I D S sequence analysis Only 47 of the 63 strains of Acinetobacter baumannii were correctly identified, 2 strains were ^ + _ 3 gene strains, 12 strains were genetic species 13TU; 2 strains could not be genetically classified but were related to The sequences of gene 3 are relatively close, and their similarity values are 0.93 and 0.96. Of the 19 strains of Acinetobacter acetate, no ITS sequence analysis was Acinetobacter calcium acetate, 16 strains were Acinetobacter gene species 3, and 1 strain was gene. Species 10, 1 gene was 13TU gene gene, 1 gene was 15TU gene gene. The results of gene species 3, 10, 13TU and 15TU were determined by ribosomal DNA φ shear analysis (ARDRA) [23] plus 25 200533757 gene species 15TU could not be divided into gene species 2 (3.2%) 0 0 1 (5.3%) ITS sequence analysis Gene 13TU 12 (19%) 1 (5.3%) Gene 10 1 (5.3%) Gene 3 2 (3.2%) 16 (84.2%) Acinetobacter baumannii 47 (74.6% ) 0 C \ \ < tn m Os VO rH with API 20 NE identification result mm 竑 If zhong τ ι u ι 001 δ inch (Noo base 潆 200533757) The present invention successfully uses the ITS sequence architecture database of ribosomal DNA, and is used to Differentiating complex populations with similar biochemical characteristics, most of them are identified as Acinetobacter baumannii or Acinetobacter calcium acetate if they are analyzed by the common commercial set API 20 NE. However, as shown in Table 9, after the identification of the present invention, It was found that API 20 NE was not identified as a homogeneous species of Acinetobacter. In fact, there is still a considerable degree of error. The method of the present invention not only provides a more accurate method for identifying genetic species, but also takes about one day after the culture of the species. Differentiate species names compared to traditional biochemical references The method is simpler and faster. Although the present invention has been disclosed in the above examples, it is not intended to limit the present invention. Anyone skilled in the art can make various changes without departing from the spirit and scope of the present invention. Changes and retouching, therefore, the scope of protection of the present invention shall be determined by the scope of the attached patent application. [Brief description of the drawings] The first diagram is a schematic diagram of the ITS sequence of 16S-23S ribosomal DNA.

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[23] Vaneechoutte, M.? L. Dijkshoom, I. Tjemberg, A. Elaichouni, P. de vos, G. Claeys, and G. Verschraegen. (1995). Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis. J. Clin. Microbiol. 33: 11-15.[23] Vaneechoutte, M.? L. Dijkshoom, I. Tjemberg, A. Elaichouni, P. de vos, G. Claeys, and G. Verschraegen. (1995). Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis. J. Clin. Microbiol. 33: 11-15.

[24] Vaneechoutte, Μ·, I. Tjemberg，F· Baldi，M. Pepi，R· Fani, E. R· Sullivan, J. van der Toorn, and L. Dijkshoom. (1999). 0\\-&Qgvdi0mg Acinetobacter strain RAG-1 and strains described as 'Acinetobacter venetianus sp. nov.f belong to the same genomic species. Res. Microbiol. 150: 69-73.[24] Vaneechoutte, M., I. Tjemberg, F. Baldi, M. Pepi, R. Fani, E. R. Sullivan, J. van der Toorn, and L. Dijkshoom. (1999). 0 \\-& Qgvdi0mg Acinetobacter strain RAG-1 and strains described as' Acinetobacter venetianus sp. Nov.f belong to the same genomic species. Res. Microbiol. 150: 69-73.

30 200533757 04p0072序列表序列表 <110> 國立成功大學 <120> 鑑定不動桿菌菌種之方法 <130> 04p0072 <160> 2 <170> Patentln version 3.2 <210> <211> <212> <213> <220> 1 20 DNA 人工序列 <223>弓[子 <400> 1 gtcgtaacaa ggtagccgta 20 <210> 2 <211> <212> <213> 20 DNA 人工序列 <220> <223> <400> 引子 2 gggttycccc rttcrgaaat 20 第1頁30 200533757 04p0072 sequence list sequence list < 110 > National Success University < 120 > Method for identifying Acinetobacter strains < 130 > 04p0072 < 160 > 2 < 170 > Patentln version 3.2 < 210 > < 211 > < 212 > < 213 > < 220 > 1 20 DNA artificial sequence < 223 > bow [sub < 400 > 1 gtcgtaacaa ggtagccgta 20 < 210 > 2 < 211 > < 212 > < 213 > 20 DNA artificial sequence < 220 > < 223 > < 400 > Primer 2 gggttycccc rttcrgaaat 20 Page 1

Claims

200533757 The scope of patent application: 1. A method for identifying Acinetobacter strains, including the following steps: (a) extracting DNA from the test strain; (b) using appropriate primers to extract 16S-23S ribosomes from DNA in an amplified manner Internal transcription region (ITS) sequence of DNA; (c) Determine the DNA sequence of the product of step (b); (d) Align and analyze the DNA sequence of step (c) with the ITS sequence database of Acinetobacter species to obtain Sequence similarity; and (e) judging the genetic species of the test strain by aligning the similarity. 2. The method as described in item 1 of the scope of patent application, wherein the aforementioned primers include the nucleotide sequence shown in φ SEQ ID NO: 1, its complementary strand, derivative, or degenerate sequence 歹 | J, where The sequence of SEQ ID NO: 1 is as follows: GTCGT AACAA GGTAG CCGTA. 3. The method according to item 1 of the scope of patent application, wherein the aforementioned primer system includes the nucleotide sequence shown in SEQ ID NO: 2, its complementary strand, derivative or degenerate sequence, wherein the SEQ ID NO: 2 The sequence is as follows: GGGTT YCCCC RTTCR GAAAT, the aforementioned Y is cytosine or thymine, and R is adenine or guanine. 4. The method according to item 2 or 3 of the scope of patent application, wherein the aforementioned derivative refers to the 3 'end or 5' end of SEQIDNO: 1, SEQIDN02 or its complementary strand to modify other nucleotide sequences, It still has a nucleotide sequence that is 70% or more similar to the original sequence. 5. The method according to item 2 or 3 of the scope of patent application, wherein the aforementioned degenerate sequence means that 30% or less of the nucleotides in SEQ ID NO: 1, SEQ ID NO: 2, or the complementary strand sequence thereof are Replaced by other nucleotides. 6. The method according to item 1 of the scope of the patent application, wherein the aforementioned step (b) can be amplified using a polymerase chain reaction. 7. The method according to item 1 of the scope of patent application, wherein the sequence of step (d) 31 200533757 is made by collecting the internal transcription region (ITS) sequence of the experimental strain 16S-23S ribosomal DNA. • The method according to item 7 of the stated patent scope, wherein the aforementioned experimental strains include Acinetobacter model strains and reference strains. 9. The method according to item 丨 in the scope of the patent application, wherein the aforementioned Acinetobacter strains include Acinetobacter complex populations. 10. A method for identifying Acinetobacter strains, comprising the following steps: (a) obtaining and transferring a plurality of DNA probes containing ITS sequences of Acinetobacter strains, wherein the aforementioned DNA probes are single-stranded DNA sequences and can be used in Hybridize with its complementary strand under appropriate circumstances; (b) extract DNA from the test strain; (0) use the appropriate primers to amplify the 16S-23S ribosomal DNA internal transcription region (ITS) sequence of the DNA extracted in step (b) (D) hybridizing the DNA of step (c) with the dNA probe of step (a) under appropriate circumstances for a period of time; and (e) detecting the results of DNA hybridization to determine the strains to be tested Gene species. U. The method as described in item 10 of the scope of patent application, wherein the DNA probe of step (a) is prepared by collecting the internal transcription region sequence of 16S_23S ribosomal DNA. The method according to item 10, wherein the aforementioned Acinetobacter strains include Acinetobacter dci) complex group strains. U. The method according to item 10 of the scope of patent application, wherein the DNA probe of step (a) can be further fixed on the wafer. 14. The method according to item 10 of the scope of patent application, wherein the foregoing step (0 is amplifying using a polymerase chain reaction. 15. The method according to item 10 of the scope of patent application, wherein the above step (c) The primer includes the nucleotide sequence of SEQ ID NO], its complementary strand, derivative 32 200533757 or degenerate sequence 'its + SEQ ID NO: i The sequence is: gtcgt aacaa GGTAG CCGTA. 1 6. According to the scope of patent application In the method described in item 10, the primer includes a nucleoside lysate or a degenerate sequence of SEQ ID NO: 2, wherein the sequence of SEQ ID NO: 2 is the sequence of the aforementioned step (c), which is complementary Stock, derivative: GGGTT YCCCC RTTCR GAAAT, the aforementioned Y is cytodine or thymic sprain, & is glandular chant or guanopurine. 17. The method according to item 15 or 16 of the scope of patent application, wherein Derivatives refer to nucleotides that modify other nucleotide sequences at 3, 5 or 5 ends of SEQIDNO: 1, SEQIDNO: 2, or their complementary strands, so that they are still 70% or more similar to the original sequence. 18. The method described in item 15 or 16 of the scope of patent application, The aforementioned degenerate sequence means that 30% or less of the nucleotides in SEQ ID NO: 1, SEQ ID NO: 2 or the complementary strand sequence thereof are replaced by other nucleotides.

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