TWI303664B - - Google Patents

Description

1303664 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a primer for rapidly identifying the genus Gordenia. In particular, the present invention relates to a method for rapidly identifying the genus Gordenia by a polymerase chain reaction (PCR). [Prior Art] Gord's bacteria spp·) is a kind of radiobacteria, which belongs to the Gordcmz'aceae family and suborders. It currently contains 21 species. Its cell wall contains mycolic acid as one of the main characteristics, which can be Separation and screening in activated sludge, sewage treatment tanks, biological filter beds, industrial wastewater, steam locomotive tires or contaminated soil. Many studies have demonstrated that Gordonia has the ability to degrade aromatics (the main substance of oil stains) and can be used to develop bioremediation techniques for treating environmental oils. It is known that the degradation and metabolism characteristics of Gordonella include the removal of sulfur in the sulfur-containing compounds benzothiophene and dibenzothiophene, 3-ethylpyridine, 3- Biodegradation of 3-methylpyridine, metabolism of butyl benzyl phthalate, ether compounds such as ethyl tertiary-butyl ether (ETBE), Degradation of methyl tert-butyl ether (MTBE), tertiary-amyl methyl ether (TAME), rubber degradation and reduction of nitric acid, etc. As a carbon source for the Gordon's strain. The identification and classification of traditional bacteria mainly depend on the physiological characteristics of the strains and the difference in phenotypic characteristics such as the biochemical generation 100247.doc 1303664. The bacteria are classified into different types, including colony type, color traits, enzyme activity, carbon source utilization and serum. Immune methods such as reaction 'however, it often takes a lot of time and cost in traditional classification methods'. In the study of environmental microorganisms such as Gordonia, the classification of strains is often not known accurately due to the diversity of strains. It can be mixed by a variety of analytical methods. For example, the use of physiological characteristics (such as the characteristics of mycotic acid shared by actinomycetes) can be applied to the identification of Gordonia, and the cell wall composition of Gordonella can be distinguished by gas chromatography to distinguish it from other actinomycetes, but microorganisms. The composition of the cells is easily mutated due to changes in the culture environment, so there are still many limitations in the measurement. The development of molecular identification technology has provided a more efficient and accurate bacterial detection technology, which has become an important auxiliary tool in clinical bacterial diagnosis. Actinomycetes are also classified in the bacterial world, so the classification method is similar to that of bacteria. Many scholars have tried to use genotyping methods to identify actinomycetes' such as restriction fragment length polymorphism analysis (Restricti〇n Bu called coffee to Length) Polymorphism (RFLP), Random Amplified P〇lymorphism DNA (RApD) and BOX-PCR, REP-PCR and other analytical techniques. It is currently known as a molecular marker for bacterial identification such as the 16S rRNA gene, the 23S rRNA gene, and the DNA region between the 16S and 23 S rRNA genes (Integenic spacer, ITS). The sensitivity and specificity of the genotype analysis method are better than the traditional physiological and biochemical characteristics, which play an important role in the rapid identification of bacteria. However, there is still no suitable method for the identification and detection of Gordonia. In view of the increasing demand for environmental rehabilitation in recent years, there is a need to develop a rapid and correct method for the identification of the organism, in order to screen for biological re-cultivation of microorganisms. SUMMARY OF THE INVENTION [0001] It is an object of the present invention to provide a primer pair for identifying the genus Gordenia which is selected from the following primer pairs and their variants: (〇 forward primer G268F, a sequence having a sequence identification number 丨, and a reverse primer G1096R having a sequence as shown in sequence identification number 2; and (u) a forward primer G699F having a sequence as shown in sequence identification number 3. J and a reverse primer G1134R having a sequence as shown in SEQ ID NO: 4. Another object of the present invention is to provide a kit for identifying the genus Gordonia which comprises the above-described primer pair. The object is to provide a method for identifying the genus Gordenia comprising the steps of: (a) providing chromosomal DNA of the cell to be tested; (b) performing a polymerization enzyme chain reaction using a primer pair selected from the following a pair of primers and their variants (i) a forward primer G268F having a sequence as shown in sequence identification number ,, and a reverse primer G1096R having a sequence as shown in sequence identification number 2; Ii) positive The primer G699F has a sequence as shown in sequence identification number 3, and a reverse primer G1134R having a sequence as shown in sequence number 100247.doc 1303664; and (c) analyzing the length of the amplified amplified fragment. DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the identification of the genus Gordenella, the design of a specific nucleotide primer pair, which can be used to identify the 16S rRNA gene fragment of Gordonella in a polymerase chain reaction to identify Gordonia. And effectively screening the target microorganisms from the environment to develop microbial resources in the environment. The present invention relates to a primer pair for identifying the genus Gordenia, which is selected from the following primer pairs and their variants: (i a forward primer G268F having a sequence ' and a reverse primer G1096R' as shown in sequence identification number i having a sequence as shown in sequence identification number 2; and (11) a forward primer G699F having sequence identification The sequence shown in Figure 3 and the reverse primer G1134R have the sequence as shown in Sequence Identification Number 4. The polymerase chain reaction usually involves three steps: (1) denaturation of a template. (2) bonding the two primers to the two strands of step (1); (3) extending the primers with DNA polymerase to obtain the DNA of the two double strands. Repeating the above steps, A specific DNA fragment can be amplified. Generally, in order to fully carry out the reaction, a first-stage denaturation step is usually performed in the cyclic reaction. The term "variant" is used herein. Refers to a primer that can replace a single primer in the primer pair of the present invention and can still amplify the same specific fragment sequence together with another primer in the primer pair. Due to the nature of the polymerase chain reaction itself, the primer 100247.doc 1303664 and the template to be enlarged Even if there is variability in the sequence, it can be synthesized by adjusting the reaction temperature of the bonding step in the polymerase chain reaction (10) into the tablet and δ, which means that the similarity with the primer sequence according to the present invention is higher than 75%. . For example, if the sequence variability between the primer and the template to be enlarged is larger, the reaction temperature of the bonding step is lower; in other words, if the sequence variability between the primer and the template to be enlarged is smaller, the adhesion can be improved. The reaction temperature of the step. Therefore, according to the disclosure of the present invention, a person having general knowledge in the specific field can design different primers according to the DNA fragment to be amplified, and any subject replacement, addition or reduction for the hair (4) can still be reduced. The introduction of the primers of the present invention is the scope of the invention to be protected. The present invention further provides a method for identifying a genus Gordenella, comprising the steps of: (a) providing a chromosome dna of the cell to be tested; (b) performing a polymerization enzyme chain reaction using a primer pair selected from the group consisting of The following primer pairs and their variants: (I) a forward primer G268F having a sequence as shown in SEQ ID NO: 1, and a reverse primer G1096R having a sequence as shown in SEQ ID NO: 2; II) Forward primer 〇69917 having a sequence as shown in SEQ ID NO: 3, and a reverse primer G1134R having a sequence as shown in SEQ ID NO: 4; and (c) analyzing the length of the amplified amplified fragment. The extraction of the chromosomal DNA of the cells to be tested, the polymerase chain reaction and the polymerization 100247.doc 10 1303664 The inter-segment of each chain reaction product is achieved in Yishan and the analysing system, which is well known to those skilled in the art. The genus to be tested _chromosome DNA extraction method, polymerase chain reaction and product analysis method are illustrated in the following examples. In a preferred embodiment of the present invention, the polymerase chain reaction in step (b) is from the first stage of the denaturation temperature before the cycle; the reaction time is from 1 minute to i 〇 minutes.

. Wang...The knives in the knives, the bonding temperature in the 裱 为 is from 6 (TC to 72 C ' is better 72 〇 C, in this >, the sore τ is the degree, the adhesion reaction and extension of the polymerase chain reaction The reaction can be carried into the rod, and the juice is removed. The bonding time is more than 30 seconds; preferably from 30 seconds to 3 minutes; the extension temperature in the selection φ + and the clothes is from 70. (: to 75t; Preferably, it is 72 ° C; the number of cycles is from 5 to 5 〇. In one of the inventions, the condition of the polymerase chain reaction in step (b) is included in the case of the invention. : The first stage of initial denaturation temperature 94t lasted 5 minutes; 3 The second stage of deuteration cycle number denaturation / dish degree 94 C for 1 minute and bonding temperature 72t for 3 minutes, and 4 fox extension temperature for 7 minutes Preferably, step (c) comprises analyzing the length of the amplified fragment by electrophoresis on a guar-pyrene gel; wherein the length of the fragment analyzed in a magnification in step (b) is 829 b (), The test letter was identified as Gordonella, on the other hand, as in step (", the (11) primer pair was used, and the step (the fragment length of the analysis was 430). Bp, 佐, ΒΪ „ J Amm n P ^ phase bacteria was identified as the genus Gordonia. In the examples described in this narration, the 16 strains of the 16 strains of the genus Gordenella (a total of nine Species) and the genus (4) are similar to other fungi belonging to 11 species). After polymerase chain reaction, you can prepare a strain of sputum. After co-culture, agarose gel electrophoresis analysis 100247.doc 1303664 DNA fragment size It can be seen that the Gordonella can be accurately identified by the method of the specific primer according to the present invention, and all tested non-Gardenia are negative, showing high sensitivity and specificity of identification according to the method of the present invention. It can quickly identify such genus, which is superior to the uncertainty of traditional physiological and biochemical tests. Therefore, the primer according to the present invention can be used to detect the environment of Gordonella in the environment and contribute to the environment of Gordonella. The screening and development of the microbial resource sieve k of the Gordon strain can also be applied to the biological re-conservation of pollutants, and can effectively monitor the ethnic variation of the strain in the environment. The invention further provides a method for identifying the genus Gordenella. a set containing the above-mentioned pair of primers In a preferred embodiment of the present invention, the kit of the present invention further comprises reagents required for extracting the chromosomal DNA of the cells to be tested. In a preferred embodiment of the present invention, the kit of the present invention further comprises Reagents required for electrophoretic analysis include, but are not limited to, reagents required for discontinuous agarose gel electrophoresis. In a preferred embodiment of the invention, the kit of the invention further comprises reagents required for performing a polymerase chain reaction. The present invention will be described in detail by the following examples, which are not intended to be construed as being limited to the disclosure of the examples. [Embodiment] Example 1 Design of Gordonella specific detection primer

The region with higher variability in the 16SrDNA sequence was used as the primer design position. Firstly, search for the 16S rDNA 100247.doc 1303664 sequence of Gordonella and its similar actinomycete from the National Bioinformatics Center gene database (ncbi GenBank), and use the CLUSTAL X program for 16S rDNA multiple sequence alignment analysis. In the species with higher retention and similar sequence but different from other actinomycetes, the primers with a design size of about 2 nucleotides are found at nucleotide positions 291-3 09, 1100-1119, The four regions 722-741 and 1140-1157 are highly retained sequence regions of the genus Gordenia. Figure 1 is a schematic diagram of the design position of Gordon's specificity primer. The design of the primer includes considering the fusion temperature, secondary structure, length, G+c percentage, whether it will self-adhesive, and then predicting the primer sequence with Primer®3 software. The resulting primer sequence is the complementary sequence of the corresponding reserved region sequence. :G268F (SEQ ID NO: 1) is a 19-nucleotide forward primer (5·CGA CCT GAG AGG GTG ATC G 31), and G1096R (SEQ ID NO: 2) is a 20-nucleotide reverse primer (5) 'ATA ACC CGC TGG CAA TAC AG 31), the combination of this primer can produce a product of 829 base pairs by polymerase chain reaction; G699F (SEQ ID NO: 3) is a forward primer of 20 nucleotides ( 5, AGG CGG GTC TCT GGG TAG TA 3'), G1134R (SEQ ID NO: 4) is a 18-nucleotide reverse primer (5, CGG CAG TCT CCT GCA AGT 3f), the polymerase chain reaction product of this combination The size is approximately 436. Example 2: Gordonella specific detection conditions optimization The reference strain reference strain used in this example was mainly purchased from the German Strain Center (DSMZ), the Taiwan Bioresource Conservation and Research Center (BCRC), and some from the successful university. Professor Zhang Changquan, Department of Medical Laboratory Biotechnology, has tested 10 standard strains of Gordonia, 6 non-standard strains of Gordonia, and 9 strains of other actinomycetes, 100247.doc -13 - 1303664 13 strains of standard strain (see Table 1 below), Gordon's strains were cultured in tryptic soy broth in a growth chamber at 28 to 30 °C. Chromosomal DNA extraction The strain was inoculated into tryptic soy medium and cultured at 28 to 30 ° C. After 24 hours, the bacterial chromosomal DNA isolation kit provided by MO BI0® was used (UltraClean Microbial Genomic DNA Isolation Kits, MO BIO, USA) )extraction. Sensitivity test of two sets of specific primers In this study, 16 strains of Gordon's strains were tested, including 10 standard strains, to adjust the adhesion temperature of specific primers to control the proliferation reaction, and the chromosomal DNA of Gordonella was used as a template. The polymerase chain reaction was carried out, and the results are shown in Table 1 below. It was found that the primer G268F/G1096R can accurately proliferate a gene fragment of 829 base pairs at a binding temperature of 66 ° C, and G699F/G1134R also successfully proliferated. The 6 base pair fragment, with the increase of the bonding temperature to increase the reaction conditions, the standard strain of Gordonia still produces a specific Gordonella fragment at 72 ° C bonding temperature, however, the two strains of DSM 777 and ATCC 21930 could not produce this fragment (see Figure 2 and Figure 3). To determine whether the two strains are Gordenella, 16S rRNA was used as the target gene for sequencing reaction to identify the strains. Later, it was found that 〇8]^ 777 is closer to Rhodococcus erythropolis (7?/26^6^<9[(:1«called 1//) (similarity is 100%), and ATCC 21930 is closer to pyridine red Cocci (similarity 99.9%) and Rhodococcus rhodochrous (10) ( The degree of similarity is 99.7%. From the results of the study, it can be seen that except for the above two Gordon strains (non-standard strains) with undetermined taxonomic status, 100247.doc -14·1303664, the other tested Gordonella can produce stable and obvious. The specific fragment shows that the two sets of primers have excellent sensitivity and can be used to detect the species in the genus Gordonia. The specificity test of the two groups of specific primers was carried out by PCR after the G268F/G1096R primer combination. When the binding temperature was 66 °C, all Gordon's strains produced a fragment of 829 bp, but some of the other closely related actinomycetes also produced fragments of the same size (see Table 1 below). Such as Corynebacterium, Mycokcier/wm, Rhodococcus and Skemannella. The polymerase chain reaction product obtained by the combination of G699F/G1134R primer is also present in Gordonia and other actinomycetes. The 16S rDNA sequence in this actinomycete is retentive. However, if the adhesion temperature is raised to 72 °C, 22 non-Gardenella strains tested by G268F/G1096R or G699F/G1134R primer combination cannot. The specific fragment of the genus Gordon's strain (see Figure 2 and Figure 3) proves that the nucleotide primers designed in this study can be used to identify the strains of the genus Gordenia. The use of polymerase chain reaction technology combined with electrophoretic analysis has achieved excellent reproducibility through repeated iterations, providing a method for rapid determination of the genus Gordenia. 100247.doc 15 1303664

Table 1: Reference strain tested by this example and polymerase chain reaction proliferation results. Genus and strain name G268F/G1096R G699F/G1134R 66 °C 72 °C 66 °C 72 °C Gordonella burned by Gordonella (G a/A^m_wnms*) DSM 44369τ 1 Burned-out Gordonella (G a/A: am'wnmy) DSM 44187 1 Anaerobic Gordon (G amar sink) DSM43392T 1 and Good Gold's ( G 44461τ 1 Phytophthora bronchii (G 6rcwc/^/zX) DSM43247T 1 Desulforative Gordonella (G-like imitation #7i(my)DSM 44462τ 1 G. glabrata (G mYzWa) DSM 44499τ 1 Bright Gordonella (G m%fo) DSM777 (1) G. glabrata (G mY 〇 CC-JG39 1 Red-type Gordonia (G rw6n) 7eriz>? cft^) DSM 43197τ 1 Red-like Dengue (G force ^) DSM 43248 1 Red-like Gordonia (G such as c/zOJCM 3199 1 Red-type Gordonia (G rw &nj^r/z>?e/to) ATCC 21930 0 Jet Gordonia (G哕 / /) DSM 43896τ 1 Desktop Gordonella (G rem^) DSM 43249τ 1 Wesfarid Gordon (G wrf / Wca) DSM44215T 1 Other bacteria Corynebacterium bovis 6(mX)DSM 20582τ (1) Corynebacterium glutamicum (C g/wtomhm) DSM 20300τ (1) Corynebacterium glutamicum (C. g/z^am/cww) ATCC 21179 (1) Mycobacterium tuberculosis (both ^^erew/os^ATCC 25177 (1) Carrion sinensis car«ea) DSM 43397τ (1) Rhodococcus equine (i?· ew〇DSM 20307τ (1) Red City Red Cocci (i?. er sound/2rajw/zX) DSM 43188τ (1) Rhodococcus erythropolis (7?. ay/7wO/^to) CC-BC01 (1) Rhodococcus rhodochrous (i?. er>T72rajwto) CC-BC04 (1) Rhodococcus erythropolis (i?. CC-BC08 0 Rhodococcus erythropolis (i?. e/^/Tzrc^/^CC-BCOQ 0 Red City Rhodococcus (brother recognized rajw/CC) -BC11 0 Rhodococcus erythropolis (7?. CC-BC17 0 Rhodococcus erythropolis (i?. CC-BC20 0 Rhodococcus erythropolis (i?. erj^/z/Opo/^CC-BC] 0 Rhodococcus rhodochrous (R, erythropoIis) CC-BC25 0 Rhodococcus rhodochrous (7?· 43241τ (1) Rhodococcus (i?. rw6〇CC-N4 (1) Rhodococcus (brother n/&r) CC-7-2 (1) Pineapple-like Skemannella (iSfermamb reed 'mybrm^DSM (1) 43998τ called 卓 卓 密 t tah/a) DSM 0 20162τ 幕瑞威廉菌(奶//ζ· α·ζ·α mwra/e)DSM Φ4343τ 0 11 il 11 11 11 1X 11 PMU 11 11 11 il 11 11 11 11 11 11 11 il 11 11 11 11 11 11 ΊΧ 11 11 /i\ 11 11 11 Ίχ /( \ 11 11 11 11 11 11 11 1x 11 oo 11 11 11 11 A·^ 11 oo 100247.doc -16- 1303664 Example : Identification of Gordonella isolates in environmental samples to test whether the conditions for the linkage reaction between the two sets of nucleotide primers designed by the present invention and the polymerase can identify the environment of Gordonella, which is used in the polymerase chain reaction. The DNA template is the chromosomal DNA of each strain. Mix 0.2 mM dNTP, 20 pmol forward and reverse primer, 2U DNA polymerase, polymerase buffer and 1 μ chromosome DNA in 25 μL solution under the initial denaturation of 94 ° C for five minutes, 94 °C one minute, 72 ° C three minutes, a total of 30 cycles and extended reaction 72t: seven minutes. The size of the product after propagation by the polymerization-transition reaction was analyzed by 1% agarose gel electrophoresis. The DNA fragment proliferated by G268F/G1096R was 829 base pairs using a specific primer, and the DNA fragment proliferated by G699F/G1134 was 436 base pairs. Twenty-three strains of strains with orange, orange-red, rough surface, smooth, flat or irregular edges were screened from different locations in the central mountainous area of central Taiwan. Ten of them were tested by G268F/G1096R and G699F/G1134R primers. Three isolates (CC-S2a, CC-S2b, CC-S2c, CC-S2d, CC-S5a, CC-S5b, CC-S5d, CC-S5-Bu CC-S5-2, CC-S5-3, CC-S5-7, CC-S5-8, CC-JiJMO) can produce Gordon's specific DNA fragments in both sets of primer tests (see Table 2 below). After BOX-PCR DNA fingerprinting analysis, thirteen strains can be divided into nine categories according to genotype (please refer to Figure 4), wherein CC-S2b, CC-S2c, CC-S2d are judged to be the same strain, CC-S5b, CC-S5d and CC-S5-3 are also the same strain. Analysis of bacterial species relationship by 16S rDNA sequence

Use bacterial universal primer 1F (SEQ ID NO: 5) (5' GAG TTT GAT CAT GGC TCA G 3') and 9R (SEQ ID NO: 6) (5' AAG 100247.doc 1303664 GAG GTG ATC CAA CCG CA 3' Proliferating bacterial 16S rDNA fragment, mixing 0.2 mM dNTP, 20 pmol forward and reverse primer, 2U DNA polymerase, polymerase buffer and 1 μΐ chromosomal DNA in 25 pL solution, the reaction conditions are starting Denaturation at 94 ° C for five minutes, 94 ° C for one minute, 45 ° C - for thirty seconds, 72 ° C for two minutes, a total of 35 cycles and an extension reaction of 72 ° C for seven minutes. The sequence of 16S rDNA was sequenced using a fluorescent termination cycle reaction, and the sequencing results were interpreted using an automated DNA sequencer (ABI® PRISM 310, PE Applied Biosystems, CA, USA). The MEGA2 molecular evolution genetic analysis program was used to analyze the phylogenetic relationship and to map the phylogenetic tree. The results are shown in Fig. 5.

According to the 16S rDNA sequence analysis, the above nine strains were all named Gordonella (please refer to Figure 5). According to the similarity of 16S rDNA, the strains can be classified as G. and Namibia Gordon. G (CC-S5a) and G. ierrw (CC-S2a, CC_S2d, CC-S5b, CC-S5-1, CC-S5-2, CC-S5-8, CC-JiJMO). The Gordant strains screened and isolated from the environment can also be verified under the conditions of this analysis, indicating that this combination of oligonucleotide primers and polymerase chain reaction conditions for the identification of Gordonella is not only available. It can be detected by the standard strain of Gordonia, and can also be applied to the environment of Gordonia isolate. 100247.doc -18- 1303664

^ 2 * f research nine! Hypoplastic strain and polymerase chain reaction proliferation result strain ^68F/gT〇96R G699F/G1134R _72 °C 72 °C CC-S2a CC-S2b CC-S2c CC-S2d CC-S2-2 CC-S2-5 CC- S5a CC-S5b CC-S5d CC-S5e CC-S5h CC-S5-1 CC-S5-2 CC-S5-3 CC-S5-6 CC-S5-7 CC-S5-8 CC-JiJi-7 CC- JiJi-8 CC-JiJi-10 CC-JiJi-11 S32a S32-1 11 11 1χ 11 ^ow Ίχ 11 11 1χ Ίχ 11 IX 1χ Λν Λν 11 11 11 11 Λυ 11 Ί1 11 11 11 11 11 IX 11 The examples are merely illustrative of the principles of the invention and its effects, and are not intended to limit the invention. Modifications and variations of the embodiments described above will be apparent to those skilled in the art without departing from the spirit of the invention. The scope of the invention should be as set forth in the appended claims. [Simple description of the diagram] Figure 1 is a schematic diagram of the design position of the Gordonella specific primer. The number above the sequence represents the nucleotide position corresponding to the E. coli 16S rRNA gene. Figure 2 shows the electrophoresis pattern of the Gordonella species specific polyglycoside using the G268F/G1096R specific primer. Μ is 1 〇〇 bp DNA ladder, number 1-38 戈 囷 different species · 1 · Burning Gordonella (G. α / Α: (4) 44369, 2: Alternate Gordonella (G α /Α^ζ·read)DSM 44187 ; 3 : I00247.doc •19- 1303664 Anaerobic Gordonella (G·43392τ ; 4 : and G. genus (G·

44461τ; 5: bronchial Gordon's bacteria (G 43247τ; 6: desulforidation of Gordonia (G j (10) / / said · (: Xie) DSM 44462τ; 7 · · G. glabrata (G · m7Wa) DSM 44499τ 8 : G. glabrata (G· m7z·heart) DSM 777 ; 9 : G. glabrata (G. m·如如) CC-JG39 ; 10 : Red-like Gordonia (G·like) DSM 43 197T ; 11 : Red-like Gordonia (G·43248; 12: Red-like Gordonia)

(G. rubripertinctus) JCM 3199; 13: G. rubripertinctus ATCC 21930; 14: G. sputi OSM 43896τ; 15: Desktop Gordonella (G· Krrw)DSM 43 249τ; 16 : # ^ ^ ^ ^ ^ g (G. westfalica) OSM 44215τ ; 17 Corynebacterium bacillus, ζ 'ι^δ(9νζ··$·)Β8Μ 20582τ; 18: Valley Corynebacterium aureus (C·g/wiamz_ewm) DSM 20300τ; 19: C. glutamicum ATCC 21179; 20: Mycobacterium tuberculosis iwZ^rew/c^z^ATCC 25177; 21: meat slave Cargill 43 397 Γ; 22: Rhodococcus equine (i?. called w/) DSM 20307τ; 23: Rhodococcus erythropolis (7?· (10) him) DSM 43 1 88Τ; 24: Rhodococcus rhodochrous (brother eryi/ Zrapo/WCC-BCOl; 25: Rhodococcus erythropolis (brother erythropolis) CC-BC04; 26: Rhodococcus erythropolis (brother erythropolis) CC-BC08; 27: Rhodococcus erythropolis (brother ^yArapo/z^s^CC -BCOQ ; 28 : Rhodococcus rhodochrous (where erj^i/zrcT^/z'OCC-BC 11 ; 29 : Rhodococcus rhodochrous (i?· CC-BC17; 30: Rhodococcus rhodochrous (i?· erythropolis) ) CC-BC20 ; 31 : Rhodococcus erythropolis (brother er less, /7Mpo/b) CC-BC22 ; 32 : Rhodococcus erythropolis i?· ; 33 : Rhodococcus erythropolis (7?· 100247.doc -20- 1303664 r/zo as Mrow) DSM 43241τ; 34: Rhodococcus (i?·rMer) CC-N4; 35: Rhodococcus (i ?·rwZ?w)CC-7-2; 36: pineal-like Skekeman's pz_mybrmb) DSM 43998τ; 37: Tsukamurella paurometabola^DSM 20ί62τ I 3S · · 瑞瑞蒹菌 mwra /e)DSM 44343τ 〇

Figure 3 is an electropherogram showing the specificity of the Gordonella species specific strains using the G699F/G1134R specific primer. Μ is a 100 bp DNA ladder, and the number 1-38 represents different strains: 1: burned Gordonella (Gl 44369τ; 2: burned Gordonella (G·like) DSM44187; 3 : anaerobic G. amww DSM 43392τ ; 4 : G. amz'ca / z' ODSM 44461τ ; 5: Bronchial Gordon (G.

DSM 43247τ ; 6 : Desulforative Gordon (G·like) DSM 44462τ ; 7 : G. glabrata (G· 44499τ ; 8 : G. wzWa) DSM 777 ; 9 : Bright Dengue (G· /7ζ·ίζ·ί/β) CC-JG39; 10 ··Red-like Gordonia (G·43197τ; 11: Red-like Gordonia (G. 43248; 12: red-like) Gordonella (G. rwZ?rz>eriz-??ciz^) JCM 3 199 ; 13 : Red-like Gordonia (G·rwZ>ri/7eri/^7ciz^) ATCC 21930; 14: Jet Ge Dengue (G· mW/) DSM 43896τ ; 15 : G. ierrae DSM 43249τ; 16 : Wesfarid Gordon (G·body, /a"ca) DSM 44215τ 17 Corynebacterium bovis ((7 < 9 7 /? ^ (2 (7 chemical plant / 1772 office 6 ^)) 〇 8] ^ 20582 butyl; 18: Corynebacterium glutamicum (C. 20300τ; 19: C glutamicum ATCC 21179; 20: Mycobacterium tuberculosis iwZ^rcw/c^z^ATCC 25 177 ; 2 1 : Meat color Nocardi 100247.doc -21 - 1303664 bacteria (TV^ar Mountain 43397τ; 22: Rhodococcus equine (7?·called wj?) DSM 20307τ; 23: Rhodococcus rhodochrous (i?· eryi/z~ household<9/z,) DSM 43 188τ; 24: Red City Red Cocci (brother er;; i/^q ^/&)CC-BC01; 25: Rhodococcus erythropolis (7?. er less i/zrc^0/h) CC_BCO4; 26: Rhodococcus rhodochrous (7?. e^yi/zrc^c^/ OCC-BCOS ; 27 : Rhodococcus erythropolis (several erythropolis) CC-BC09 ; 28 : Rhodococcus erythropolis (i? · to) CC-BC11; 29: Rhodococcus erythropolis (i?· ; 30: Red City Red Cocci (i?·

Eai/zropo/iOCC-BCSO; 31: Rhodococcus erythropolis (several erythropolis) CC-BC22 ·, 32: Rhodococcus erythropolis (i?.; 33: Rhodococcus erythropolis (i?·r/zc^oc/ zrowdDSM 43241T ; 34 : Rhodococcus erythropolis (and · rwZ > er) CC-N4 ; 35 : Rhodococcus erythropolis (/?· rwZ?er) CC-7-2; 36: Pineal-shaped Skemannella (Skr 0(1)a p /"zybrmb"DSM 43998τ ; 37 : slightly changed ^ ^ @1 {Tsukamurella paurometabola) OSM 20162τ ; 38 · ^ S. wilfordii mwra / e) DSM 44343τ ο

Figure 4 is an electropherogram of the Gordonella isolate after proliferation by BOX-PCR. Μ is a 100 bp DNA ladder, and numbers 1 to 13 represent different strains: 1: CC-S2a; 2: CC-S2b; 3: CC-S2c; 4: CC-S2d; 5: CC-S5a; 6: CC_S5b 7 : CC-S5d ; 8 : CC-S5-3 ; 9 : CC-S5-1 ; 10 : CC-S5-2 ; 11 : CC-S5-7 ; 12 : CC-S5-8 ; 13 : CC -JiJi-10. Figure 5 is a tree diagram of the germline generation of Gordon's bacteria, which is based on the 16S rDNA sequence difference. 100247.doc -22-

Claims (1)

•-1303秘116224_ On the Chinese patent application scope to replace this year 5, '#修(more) original | ', the scope of the patent application: — ^ A kind of primer (four) (- (4) the pair of primers, which is selected from The following pairs of primers: W forward primer (10), which has the sequence "redirect primer G1096R" as shown in sequence identification number!, which has a sequence as shown in sequence identification number 2; and (11) forward primer 06991? It has a sequence J and a reverse primer G1134R as shown in SEQ ID NO: 3, which has a sequence as shown in SEQ ID NO: 4. A method for identifying the genus Gordonella (Gw and mh), which includes the following Step (a) provides the chromosomal DNA of the cell to be tested; (b) performs a polymerase chain reaction using a primer pair selected from the following primer pairs: (1) a forward primer G268F having a sequence identification number a sequence 'and a reverse primer G1096R having a sequence as shown in sequence identification number 2; and (!i) a forward primer G699F having a sequence as shown in sequence identification number 3, and a reverse primer G1134R, which has the sequence identification number 4 The sequence; wherein the polymerase chain reaction has a bonding temperature of 72. (:; and 13〇3$ round I6224 material application application Chinese patent application scope is replaced by May 1997) (c) Analysis of the fragment length of the amplification. 3. The method of claim 2, wherein the binding time of the polymerase chain reaction in step (8) is greater than 3 〇 seconds. 4. The method of claim 3, wherein the bonding time of the polymerase chain reaction in step _ is from 3 0 The method of claim 2, wherein the extension temperature of the polymerase chain reaction in step (b) is from 70 ° C to 75 ° 6. 6. The method of claim 5, wherein step (8) The extension temperature of the polymerase chain reaction is 72 ° C. 7. The method of claim 2 wherein (4) (b) the number of cycles of the polymerase chain reaction is from 5 to 50. 8. The method of claim 2, The condition of the towel step (8) towel polymerase chain reaction includes: the first stage initial denaturation temperature 94 < t duration of 5 minutes; 3 〇 cycle number of the first section of the denaturation temperature 9 generations of the chronograph clock and bonding temperature 72 ° C duration 3 Minutes; and X extension temperature lasts 7 minutes. 9. As requested in item 2 Method [4] wherein step (4) comprises analyzing the length of the fragment amplified by the agarose gel electrophoresis. The method of claim 2, wherein the method is as follows: (1) the pair of primers, and the length of the fragment of the bovine sputum analysis _ 9bp, (4) to be tested _ is determined to be Ge 2 13 03 6224 patent application Chinese application patent scope replacement this \97 May) genus. U. If the method of claim 2 'which is used in step (8) ( U gift pair, and the length of the fragment analyzed in step (4) is 436 bp, then the test is _ genus. X A kit for the identification of the genus Gordonella ("(4), which consists of a primer pair and its required reagents. Cell staining 13. The kit of claim 12] wherein the reagent contains an extract to be tested Reagents required for DNA. Synthase chain reaction 15. The kit of claim 12, wherein the test reagent is required for the agarose gel electrophoresis. ^ 电永 14. As requested in claim 12 Kit 'where the reagent contains the reagents needed to carry out the polymerization. A