CN104278082A - Specific fragment composition of mycobacterium tuberculosis drug-resistant gene of four second-line drugs and application thereof - Google Patents

Specific fragment composition of mycobacterium tuberculosis drug-resistant gene of four second-line drugs and application thereof Download PDF

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CN104278082A
CN104278082A CN201410205541.8A CN201410205541A CN104278082A CN 104278082 A CN104278082 A CN 104278082A CN 201410205541 A CN201410205541 A CN 201410205541A CN 104278082 A CN104278082 A CN 104278082A
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complementary sequence
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万康林
李桂莲
赵丽丽
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National Institute for Communicable Disease Control and Prevention of Chinese Center For Disease Control and Prevention
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Abstract

The invention provides a nucleotide acid fragment composition related to mycobacterium tuberculosis drug resistance detection of four second-line drugs, namely a fluoroquinolone drug, aminoglycoside drug kanamycin and amikacin (amikacin) and cyclopeptide drug capreomycin which are used for treating tuberculosis, and application thereof, and particularly provides a promotor mutation nucleotide acid fragment composition of a drug-resistant related gene gyrA of the fluoroquinolone drug and drug-resistant related genes rrs and eis of the aminoglycoside drug kanamycin and amikacin (amikacin) and the cyclopeptide drug capreomycin, a specific mutation detection oligonucleotide probe, a kit prepared through the specific mutation detection oligonucleotide probe and a related drug resistance detection method.

Description

The four kinds of Second line Drug drug resistant gene specific fragment combinations of a kind of mycobacterium tuberculosis and application thereof
Invention field
The application relates generally to pathogenic bacterium Resistance detection technical field.Specifically, the application relates to tuberculosis four kinds of Second line Drug fluoroquinolones, the Drug Resistance of Mycobacterium Tuberculosis of aminoglycoside medicaments kantlex and amikacin (Amikacin Sulphate) and cyclic peptide medicine capromycin detects.
Background of invention
At present, one of tuberculosis major public health problem being still most developing countries, and multi-drug resistance tuberculosis (multi-drug resistant tuberculosis, MDR-TB) and extensively the appearance of resistant tuberculosis (extensive drug resistant tuberculosis, XDR-TB) makes epidemic status lungy more severe.China is one of 22 tuberculosis high burden countries in the whole world, and annual new tuberculosis patient number of sending out occupies global second, and especially resistant tuberculosis is popular is on the rise.
Fluoroquinolones, kantlex, amikacin and capromycin are four kinds in the tuberculosis Second line Drug of world health organisation recommendations, have very important effect to treatment lungy.Can instruct clinical application to the Resistance detection that these four kinds of medicines carry out mycobacterium tuberculosis in time, avoid delay treatment, to effectively controlling the generation of resistant tuberculosis and propagating significant.At present, the Drug Resistance Detection method adopted clinically has Phenotypic examination and gene test two class, and based on traditional detection.Phenotypic examination method such as Russell medium scaling method, Roche cultivate Absolute concentration method, but these two kinds of methods have obvious limitation, and it slowly affects by M. tuberculosis growth, and even the longer time just can obtain result to need 6-8 week.
Part illustrates the molecular mechanism of drug resistance of Mycobacterium tuberculosis at present, think that the resistance to fluoroquinolones of mycobacterium tuberculosis is caused by encoding gene (DNA gyrase subunit A, the gyrA) sudden change due to its effect target molecule DNA helicase A subunit; Resistance to kantlex, amikacin and capromycin and 16S rRNA encoding gene (ribosomal RNA16S, rrs) and eis gene (enhanced invracellular survival) promoter mutation relevant.Kantlex, between amikacin and capromycin, there is cross resistance.
Current existing genotype detection method has DNA sequencing method (1.Wang H, Zhao C, He W, Zhang F, Zhang L, Cao B, et al.High prevalence of fluoroquinolone-resistant group B streptococci among clinical isolates in China and predominance of sequence type19with serotype III.Antimicrob Agents Chemother2013, 57:1538-41.), linear hybridization method (2.Ando H, Mitarai S, Kondo Y, Suetake T, Kato S, Mori T, et al.Evaluation of a line probe assay for the rapid detection of gyrA mutations associated with fluoroquinolone resistance in multidrug-resistant Mycobacterium tuberculosis.J Med Microbiol2011, 60:184-8.), real-time PCR method (3.van Doorn HR, An DD, de Jong MD, Lan NT, Hoa DV, Quy HT, et al.Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe real-time PCR.Int J Tuberc Lung Dis2008, 12:736-42.), Multiplex PCR method (4.Zhao LL, Xia Q, Lin N, Liu ZG, Zhao XQ, Wan KL.Multiplex allele-specific PCR combined with PCR-RFLP analysis for rapid detection of gyrA gene fluoroquinolone resistance mutations in Mycobacterium tuberculosis.J Microbiol Methods.2012, 88 (1): 175-8.) etc.HAIN company of Germany has the GenoType MTBDRs1 test kit that the detected testing afloqualone medicament drug resistance related gene gyrA based on linear hybridization technology suddenly change, but this test kit is only limitted to the resistance of testing afloqualone medicament in detection Second line Drug.
At present, this area still need can high-throughput, Rapid Detection of Mycobacterium Tuberculosis to the testing tool of the resistance of multi-medicament, particularly can for the testing tool of the region resistance difference of mycobacterium tuberculosis.
Summary of the invention
First aspect, the nucleic acid fragment that this application provides for mycobacterium tuberculosis (Mycobacterium tuberculosis) Resistance detection combines, and comprises following (1) to two in (3) or all:
(1) mutant of the nucleotide sequence of 208 to 390 of SEQ ID NO:1, wherein sudden change is selected from:
(1.1): the C of the 269th of SEQ ID NO:1 sports T, it causes the 90th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Val by Ala;
(1.2): the T of the 271st of SEQ ID NO:1 sports C, it causes the 91st of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Pro by Ser;
(1.3): the A of the 281st of SEQ ID NO:1 sports G, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Gly by Asp;
(1.4): the A of the 281st of SEQ ID NO:1 sports C, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Ala by Asp;
(1.5): the G of the 280th of SEQ ID NO:1 sports T, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Tyr by Asp;
(1.6): the G of the 280th of SEQ ID NO:1 sports A, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Asn by Asp;
(1.7): the G of the 280th of SEQ ID NO:1 sports C, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport His by Asp; And
(1.8): the arbitrary combination of sudden change described in (1.1) to (1.7);
(2) mutant of the nucleotide sequence of the 1390-1490 position of SEQ ID NO:3, wherein sudden change is selected from:
(2.1): the A of the 1401st of SEQ ID NO:3 sports G;
(2.2): the C of the 1402nd of SEQ ID NO:3 sports T;
(2.3): the G of the 1484th of SEQ ID NO:3 sports T; And
(2.4): the arbitrary combination of sudden change described in (2.1) to (2.3);
(3) mutant of-50 to-1 of SEQ ID NO:4, wherein sudden change is selected from:
(3.1): the C of the-14 of SEQ ID NO:4 sports T;
(3.2): the C of the-12 of SEQ ID NO:4 sports T;
(3.3): the G of the-10 of SEQ ID NO:4 sports A; And
(3.4): the arbitrary combination of sudden change described in (3.1) to (3.3).
Second aspect, this application provides can specifically in conjunction with the oligonucleotide probe of the nucleic acid fragment combination described in first aspect.
In some embodiments, oligonucleotide probe be selected from following at least two or all:
SEQ ID NO:9 or its complementary sequence, SEQ ID NO:10 or its complementary sequence, SEQ ID NO:11 or its complementary sequence, SEQ ID NO:12 or its complementary sequence, SEQ ID NO:13 or its complementary sequence, SEQ ID NO:14 or its complementary sequence, SEQ ID NO:15 or its complementary sequence, SEQ ID NO:17 or its complementary sequence, SEQ ID NO:18 or its complementary sequence, SEQ ID NO:20 or its complementary sequence, SEQ ID NO:22 or its complementary sequence, SEQ ID NO:23 or its complementary sequence, SEQ ID NO:24 or its complementary sequence.
The third aspect, this application provides the purposes of oligonucleotide probe in the testing tool preparing Drug Resistance of Mycobacterium Tuberculosis described in second aspect.
Fourth aspect, this application provides Drug Resistance of Mycobacterium Tuberculosis detection kit, it comprises solid phase carrier, described solid phase carrier secures can specific detection following in the oligonucleotide probe of at least two or more or all sudden changes:
(a): the C of the 269th of SEQ ID NO:1 sports T, it causes the 90th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Val by Ala;
(b): the T of the 271st of SEQ ID NO:1 sports C, it causes the 91st of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Pro by Ser;
(c): the A of the 281st of SEQ ID NO:1 sports G, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Gly by Asp;
(d): the A of the 281st of SEQ ID NO:1 sports C, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Ala by Asp;
(e): the G of the 280th of SEQ ID NO:1 sports T, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Tyr by Asp;
(f): the G of the 280th of SEQ ID NO:1 sports A, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Asn by Asp;
(g): the G of the 280th of SEQ ID NO:1 sports C, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport His by Asp;
(h): the A of the 1401st of SEQ ID NO:3 sports G;
(i): the C of the 1402nd of SEQ ID NO:3 sports T;
(j): the G of the 1484th of SEQ ID NO:3 sports T;
(k): the C of the-14 of SEQ ID NO:4 sports T;
(l): the C of the-12 of SEQ ID NO:4 sports T; And
(m): the G of the-10 of SEQ ID NO:4 sports A.
In some embodiments, described oligonucleotide probe be selected from following in one or more or whole:
SEQ ID NO:9 or its complementary sequence, SEQ ID NO:10 or its complementary sequence, SEQ ID NO:11 or its complementary sequence, SEQ ID NO:12 or its complementary sequence, SEQ ID NO:13 or its complementary sequence, SEQ ID NO:14 or its complementary sequence, SEQ ID NO:15 or its complementary sequence, SEQ ID NO:17 or its complementary sequence, SEQ ID NO:18 or its complementary sequence, SEQ ID NO:20 or its complementary sequence, SEQ ID NO:22 or its complementary sequence, SEQ ID NO:23 or its complementary sequence, SEQ ID NO:24 or its complementary sequence.
In some embodiments, solid phase carrier also secures the wild-type probe that can correspond to Mutational part sequence in the specific detection wild-type sequence of not undergoing mutation, such as, wild-type probe be selected from following in one or more: SEQ ID NOs:8,16,19,21.
In some embodiments, test kit also comprise following in one or more components:
(i) for the primer pair of the gene fragment at place, described mutational site of increasing, such as, primer pair be selected from following in one or more pairs of: SEQ ID NOs:25 and 26,27 and 28,29 and 30,31 and 32;
(ii) for the identification of the probe of mycobacterium tuberculosis complex, such as, described probe sequence is SEQ ID NOs:6 and 7 or their complementary sequence;
(iii) for monitoring the DNA probe being marked with vitamin H of hybridization quality, such as, the sequence of described probe is SEQ ID NO:5 or its complementary sequence.
In some embodiments, detect sample and be selected from the culture, phlegm, ascites pleural fluid, Bronchial washing, cerebrospinal fluid, peripheral blood and the pathological tissue specimen that comprise mycobacterium tuberculosis.
In some embodiments, solid phase carrier is nylon membrane.
5th aspect, this application provides the detection method of Drug Resistance of Mycobacterium Tuberculosis, it comprises makes sample contact with the solid phase carrier of the oligonucleotide probe described in second aspect or the test kit described in fourth aspect, and the hybridization of determination and analysis sample and oligonucleotide probe or solid phase carrier.
Brief Description Of Drawings
Fig. 1 utilizes the exemplary detection film bar of the application to mycobacterium tuberculosis fluoroquinolones drug resistance related gene gyrA, kantlex, amikacin and capromycin drug resistance related gene rrs, the abrupt climatic change result of eis promotor.
Fig. 2 is the schematic diagram of the exemplary solid phase carrier of the application, and it incorporates the probe of the sudden change for detecting gyrA gene, rrs gene and eis gene promoter.
Sequence brief description
The cDNA/ encoding sequence of SEQ ID NO:1: wild-type gyrA;
The aminoacid sequence of SEQ ID NO:2: wild-type gyrA;
SEQ ID NO:3: wild-type rrs sequence;
SEQ ID NO:4: wild-type eis promotor (-139 to-1, wherein transcription initiation site is the 1st);
Wherein SEQ ID NO:1-4 all derives from mycobacterium tuberculosis, and SEQ ID NO:8-30 is based on SEQ ID NO:1-4;
SEQ ID NO:5:Bio sequence, whether it is the DNA probe being marked with vitamin H, successful for monitoring hybrid experiment;
SEQ ID NO:6 and 7:16S rRNA-1 and 16S rRNA-2 probe, for the identification of mycobacterium tuberculosis complex;
SEQ ID NO:8:gyrA90-94WT probe, the wild-type probe that the codon for the 90-94 amino acids of specific detection gyrA is not undergone mutation;
SEQ ID NO:9:gyrA90GCG-GTG probe, there is the abrupt climatic change probe of GCG-GTG sudden change in the codon for the 90th amino acids of specific detection gyrA;
SEQ ID NO:10:gyrA91TCG-CCG probe, there is the abrupt climatic change probe of TCG-CCG sudden change in the codon for the 91st amino acids of specific detection gyrA;
SEQ ID NO:11:gyrA94GAC-GGC probe, there is the abrupt climatic change probe of GAC-GGC sudden change in the codon for the 94th amino acids of specific detection gyrA;
SEQ ID NO:12:gyrA94GAC-GCC probe, there is the abrupt climatic change probe of GAC-GCC sudden change in the codon for the 94th amino acids of specific detection gyrA;
SEQ ID NO:13:gyrA94GAC-TAC probe, there is the abrupt climatic change probe of GAC-TAC sudden change in the codon for the 94th amino acids of specific detection gyrA;
SEQ ID NO:14:gyrA94GAC-AAC probe, there is the abrupt climatic change probe of GAC-AAC sudden change in the codon for the 94th amino acids of specific detection gyrA;
SEQ ID NO:15:gyrA94GAC-CAC probe, there is the abrupt climatic change probe of GAC-CAC sudden change in the codon for the 94th amino acids of specific detection gyrA;
SEQ ID NO:16:rrs1401/1402WT probe, for the 1401st and 1402 wild-type probe of not undergoing mutation of specific detection rrs;
SEQ ID NO:17:rrs1401A-G probe, for the 1401st abrupt climatic change probe A-G occurring and suddenlys change of specific detection rrs;
SEQ ID NO:18:rrs1402C-T probe, for the 1402nd abrupt climatic change probe C-T occurring and suddenlys change of specific detection rrs;
SEQ ID NO:19:rrs1484WT probe, for the 1484th wild-type probe of not undergoing mutation of specific detection rrs;
SEQ ID NO:20:rrs1484G-T probe, for the 1484th abrupt climatic change probe G-T occurring and suddenlys change of specific detection rrs;
SEQ ID NO:21:eis-17 ~+1WT probe, for the-17 to+1 wild-type probe of not undergoing mutation of specific detection eis promotor;
SEQ ID NO:22:eis-14C-T probe, for the-14 abrupt climatic change probe C-T occurring and suddenlys change of specific detection eis promotor;
SEQ ID NO:23:eis-12C-T probe, for the-12 abrupt climatic change probe C-T occurring and suddenlys change of specific detection eis promotor;
SEQ ID NO:24:eis-10G-A probe, for the-10 abrupt climatic change probe G-A occurring and suddenlys change of specific detection eis promotor;
The forward of SEQ ID NO:25 and 26:gyrA surveyed area and reverse amplimer, the fragment of the 88 to 396 of amplification SEQ ID NO:1;
The forward of SEQ ID NO:27 and 28:rrs surveyed area and reverse amplimer, the fragment of the 1223 to 1512 of amplification SEQ ID NO:3;
The forward of the surveyed area of SEQ ID NO:29 and 30:eis promotor and reverse amplimer, the fragment of-123 to+205 of amplification eis promoter region, wherein identical with SEQ ID NO:4, transcription initiation site is the 1st;
The forward of SEQ ID NO:31 and 32:16S rRNA and reverse amplimer, for 94 to 373 of the 16S rRNA that increases.
The detailed description of invention
The application is the analysis in the drug-tolerant gene mutation site built on Chinese mycobacterium tuberculosis clinical separation strain at least partly.Specifically, present inventor checks order to the report drug resistant gene relevant with fluoroquinolones, kantlex, amikacin and capromycin in 252 strain China mycobacterium tuberculosis clinical separation strains, drug resistant gene comprises gyrA, rrs, eis promotor, and by analyzing sequencing result at the document of CNKI database and the domestic and international drug resistance of Mycobacterium tuberculosis associated gene mutation of PUBMED database lookup.Analytical results shows: the Fluoroquinolones Resistant Strains of about 75%-94% is undergone mutation at the 74-113 amino acids codon of gyrA, the highest with the 94th, 90 and 91 bit codon mutation rates, mutation rate is respectively 61.5%, 20% and 7%, the site that other mutation rate is lower comprises the 74th, 80,83,88,95,102 and 109 amino acids codon mutations etc., in testing afloqualone persister, gyrB gene mutation rate is lower, only account for about 10%, and the relation of gyrB transgenation and testing afloqualone medicament resistance is still not clear; There is rrs sudden change in the kantlex/capromycin/amikacin persister of about 67%, 53.8% is up to the 1401st bit base mutation rate, next is the 1402nd and 1484 bit base sudden changes, mutation rate is respectively 2%, 1.4%, the site that other mutation rate is lower comprises the 1322nd, 1408,1487 and 1483 bit base sudden changes etc.; There is the sudden change of eis promoter region in the kantlex/capromycin/amikacin persister of about 46%, is up to 23.7% with the-10 bit base mutation rate, and be secondly the-12 ,-14 sudden change, mutation rate is 8%.
According to above analytical results, present inventor has identified the drug resistance related gene and mutational site that meet Chinese mycobacterium tuberculosis persister character targetedly from numerous Drug Resistance of Mycobacterium Tuberculosis gene and drug-fast mutant sites, and provides corresponding detection method and instrument.Therefore, involved by the application each invention advantage comprise following in one or more:
(1) with strong points, be suitable for the clinical practice of CHINESE REGION;
(2) realize high-throughput and rapid detection, can disposable detection mycobacterium tuberculosis to one or more resistance in fluoroquinolones, kantlex, amikacin and capromycin;
(3) reducing testing cost by rejecting the detection in uncommon mutational site in Chinese mycobacterium tuberculosis persister, can higher susceptibility be kept simultaneously.
First aspect, the nucleic acid fragment that this application provides for mycobacterium tuberculosis (Mycobacterium tuberculosis) Resistance detection combines, and comprises following (1) to two in (3) or all:
(1) mutant of the nucleotide sequence of 208 to 390 of SEQ ID NO:1, wherein sudden change is selected from:
(1.1): the C of the 269th of SEQ ID NO:1 sports T, it causes the 90th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Val by Ala;
(1.2): the T of the 271st of SEQ ID NO:1 sports C, it causes the 91st of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Pro by Ser;
(1.3): the A of the 281st of SEQ ID NO:1 sports G, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Gly by Asp;
(1.4): the A of the 281st of SEQ ID NO:1 sports C, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Ala by Asp;
(1.5): the G of the 280th of SEQ ID NO:1 sports T, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Tyr by Asp;
(1.6): the G of the 280th of SEQ ID NO:1 sports A, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Asn by Asp;
(1.7): the G of the 280th of SEQ ID NO:1 sports C, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport His by Asp; And
(1.8): the arbitrary combination of sudden change described in (1.1) to (1.7);
(2) mutant of the nucleotide sequence of the 1390-1490 position of SEQ ID NO:3, wherein sudden change is selected from:
(2.1): the A of the 1401st of SEQ ID NO:3 sports G;
(2.2): the C of the 1402nd of SEQ ID NO:3 sports T;
(2.3): the G of the 1484th of SEQ ID NO:3 sports T; And
(2.4): the arbitrary combination of sudden change described in (2.1) to (2.3);
(3) mutant of-50 to-1 of SEQ ID NO:4, wherein sudden change is selected from:
(3.1): the C of the-14 of SEQ ID NO:4 sports T;
(3.2): the C of the-12 of SEQ ID NO:4 sports T;
(3.3): the G of the-10 of SEQ ID NO:4 sports A; And
(3.4): the arbitrary combination of sudden change described in (3.1) to (3.3).
(1) is above the mutant nucleic acid fragments for fluoroquinolones drug resistant gene gyrA, and (2)-(3) are the mutant nucleic acid fragments for kantlex, amikacin and capromycin drug resistant gene rrs, eis promotor.Fluoroquinolones is the conventional medicine used in tuberculotherapy, includes but not limited to, Ofloxacine USP 23, levofloxacin, Ciprofloxacin and Moxifloxacin.According to report before, kantlex, have cross resistance between amikacin and capromycin, therefore detecting its resistance can for common drug resistant gene.
Should be appreciated that any one nucleotide sequence mutant above-mentioned also comprises its complementary sequence.
In some embodiments, nucleic acid fragment combination can comprise the whole of (1)-(3), thus realizes the whole detections to the resistance of four kinds of medicines.In a particular embodiment, nucleic acid fragment combination can comprise (1.1)-(1.7), (2.1)-(2.3), (3.1)-(3.3) whole.
In other embodiments, nucleic acid fragment combination can comprise in (1)-(3) one or more, thus realize the detection to the resistance of one or more drug targets.
Be to be understood that, (1) mutant of the nucleotide sequence described in-(3) is the part that have chosen complete encoding sequence or promoter sequence, and such section belongs to " occurred frequently " section of resistance related mutation or is called " hypervariable region " or " resistance determining area ".Therefore, present invention also provides the variant of above-mentioned arbitrary nucleotide sequence mutant, described variant can be above-mentioned nucleotide sequence mutant through the replacement of one or more nucleosides section, disappearance (such as at one end or two ends brachymemma) or the variant that adds (such as at one end or two ends extend) and obtain, as long as variant can retain described mutational site.In some embodiments, variant can also be the fragment of the nucleotide sequence mutant remaining described mutational site.
Second aspect, this application provides can specifically in conjunction with the oligonucleotide probe of the nucleic acid fragment combination described in first aspect.
In some embodiments, oligonucleotide probe be selected from following at least two or all:
SEQ ID NO:9 or its complementary sequence, SEQ ID NO:10 or its complementary sequence, SEQ ID NO:11 or its complementary sequence, SEQ ID NO:12 or its complementary sequence, SEQ ID NO:13 or its complementary sequence, SEQ ID NO:14 or its complementary sequence, SEQ ID NO:15 or its complementary sequence, SEQ ID NO:17 or its complementary sequence, SEQ ID NO:18 or its complementary sequence, SEQ ID NO:20 or its complementary sequence, SEQ ID NO:22 or its complementary sequence, SEQ ID NO:23 or its complementary sequence, SEQ ID NO:24 or its complementary sequence.
In a particular embodiment, the oligonucleotide probe detecting (1.1) described sudden change in first aspect is as shown in SEQ ID NO:9 or be its complementary sequence; The oligonucleotide probe detecting (1.2) described sudden change in first aspect is as shown in SEQ ID NO:10 or be its complementary sequence; The oligonucleotide probe detecting (1.3) described sudden change in first aspect is as shown in SEQ ID NO:11 or be its complementary sequence; The oligonucleotide probe detecting (1.4) described sudden change in first aspect is as shown in SEQ ID NO:12 or be its complementary sequence; The oligonucleotide probe detecting (1.5) described sudden change in first aspect is as shown in SEQ ID NO:13 or be its complementary sequence; The oligonucleotide probe detecting (1.6) described sudden change in first aspect is as shown in SEQ ID NO:14 or be its complementary sequence; The oligonucleotide probe detecting (1.7) described sudden change in first aspect is as shown in SEQ ID NO:15 or be its complementary sequence; The oligonucleotide probe detecting (2.1) described sudden change in first aspect is as shown in SEQ ID NO:17 or be its complementary sequence; The oligonucleotide probe detecting (2.2) described sudden change in first aspect is as shown in SEQ ID NO:18 or be its complementary sequence; The oligonucleotide probe detecting (2.3) described sudden change in first aspect is as shown in SEQ ID NO:20 or be its complementary sequence; The oligonucleotide probe detecting (3.1) described sudden change in first aspect is as shown in SEQ ID NO:22 or be its complementary sequence; The oligonucleotide probe detecting (3.2) described sudden change in first aspect is as shown in SEQ ID NO:23 or be its complementary sequence; The oligonucleotide probe detecting (3.3) described sudden change in first aspect is as shown in SEQ ID NO:24 or be its complementary sequence.
When the sequence of known mutations site and place, mutational site section, those skilled in the art can design according to the routine techniques of biology field and prepare can the oligonucleotide probe in mutational site described in specific detection.Such as, as known in the art, following one or more principle can be considered during designing probe: G/C content is between 40%-70%; Probe sequence is between 13-25 base; Avoid same base to repeat too much, particularly G may not exceed 4 or more; Use C more than the probe of G as far as possible; Mutational site is positioned at probe center as far as possible; The Tm value using clone manager to design should as far as possible between 60-65 DEG C.
In this article, " wild type strain ", " wild-type sequence ", " wild-type probe " etc. are for " saltant ", " mutant nucleotide sequence ", " abrupt climatic change probe " etc.Such as, " wild type strain " can refer to medicine (such as fluoroquinolones, kantlex, amikacin and/or capromycin) sensitive strains, and " saltant " can refer to Resistant strain; " wild-type sequence " can refer to the sequence that resistance related mutation does not occur, and " mutant nucleotide sequence " or " nucleotide sequence mutant " can refer to the sequence that resistance related mutation as herein described occurs; " wild-type probe " can refer to the probe of the sequence that resistance related mutation as herein described does not occur for specific detection, and " abrupt climatic change probe " can refer to the probe of the sequence that there occurs resistance related mutation as herein described for specific detection.
In this article, whether " specifically detect ", " combining specifically " or term similar refer to and can detect target sequence and exist, can not detect simultaneously or in conjunction with the character of non-targeted sequence.Specifically, can to detect specifically or refer in conjunction with the probe of wild-type sequence can with wild-type sequence or its section be combined, simultaneously not in conjunction with the probe in other sites beyond wild-type sequence.Correspondingly, can to detect specifically or refer in conjunction with the probe of mutant sequences can with mutant sequences or its section be combined, simultaneously not in conjunction with the probe in the site of other sequences beyond wild-type sequence or wild-type sequence and mutant sequences.Under normal circumstances, can to detect specifically or probe in conjunction with wild-type sequence or mutant sequences designs for the sequence of region, mutational site.It is also understood that and other technologies means can be used to help realize detecting specifically or combining specifically.Such as, the gene fragment of target sequence can be comprised by increasing from the genome sequence of sample in advance, getting rid of the existence of " other sequences ", then making the probe of design contact with amplified production, thus realizing detecting specifically or combining specifically.
The third aspect, this application provides the purposes of oligonucleotide probe in the testing tool preparing Drug Resistance of Mycobacterium Tuberculosis described in second aspect.
The application of oligonucleotide probe in molecular Biological Detection field, such as but not limited to, reverse dot blot hybridization Diaphragm Method, reverse dot blot hybridization film bar method, linear inverse spot hybridization, biochip method, liquid chip method, fluid chip method etc.Therefore, those skilled in the art also can utilize oligonucleotide probe to prepare different testing tools according to the concrete detection method that will apply.In some embodiments, testing tool is the diaphragm or the film bar that utilize reverse dot blot hybridization method.
Fourth aspect, this application provides Drug Resistance of Mycobacterium Tuberculosis detection kit, it comprises solid phase carrier, described solid phase carrier secures can specific detection following in the oligonucleotide probe of at least two or more or all sudden changes:
(a): the C of the 269th of SEQ ID NO:1 sports T, it causes the 90th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Val by Ala;
(b): the T of the 271st of SEQ ID NO:1 sports C, it causes the 91st of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Pro by Ser;
(c): the A of the 281st of SEQ ID NO:1 sports G, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Gly by Asp;
(d): the A of the 281st of SEQ ID NO:1 sports C, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Ala by Asp;
(e): the G of the 280th of SEQ ID NO:1 sports T, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Tyr by Asp;
(f): the G of the 280th of SEQ ID NO:1 sports A, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Asn by Asp;
(g): the G of the 280th of SEQ ID NO:1 sports C, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport His by Asp;
(h): the A of the 1401st of SEQ ID NO:3 sports G;
(i): the C of the 1402nd of SEQ ID NO:3 sports T;
(j): the G of the 1484th of SEQ ID NO:3 sports T;
(k): the C of the-14 of SEQ ID NO:4 sports T;
(l): the C of the-12 of SEQ ID NO:4 sports T; And
(m): the G of the-10 of SEQ ID NO:4 sports A.
As described above, when the sequence of known mutations site and place, mutational site section, those skilled in the art can design according to the routine techniques of biology field and prepare can the oligonucleotide probe in mutational site described in specific detection.
In some embodiments, described oligonucleotide probe be selected from following in one or more or whole:
SEQ ID NO:9 or its complementary sequence, SEQ ID NO:10 or its complementary sequence, SEQ ID NO:11 or its complementary sequence, SEQ ID NO:12 or its complementary sequence, SEQ ID NO:13 or its complementary sequence, SEQ ID NO:14 or its complementary sequence, SEQ ID NO:15 or its complementary sequence, SEQ ID NO:17 or its complementary sequence, SEQ ID NO:18 or its complementary sequence, SEQ ID NO:20 or its complementary sequence, SEQ ID NO:22 or its complementary sequence, SEQ ID NO:23 or its complementary sequence, SEQ ID NO:24 or its complementary sequence.
In a particular embodiment, the oligonucleotide probe detecting (a) described sudden change in fourth aspect is as shown in SEQ ID NO:9 or be its complementary sequence; The oligonucleotide probe detecting (b) described sudden change in fourth aspect is as shown in SEQ ID NO:10 or be its complementary sequence; The oligonucleotide probe detecting (c) described sudden change in fourth aspect is as shown in SEQ ID NO:11 or be its complementary sequence; The oligonucleotide probe detecting (d) described sudden change in fourth aspect is as shown in SEQ ID NO:12 or be its complementary sequence; The oligonucleotide probe detecting (e) described sudden change in fourth aspect is as shown in SEQ ID NO:13 or be its complementary sequence; The oligonucleotide probe detecting (f) described sudden change in fourth aspect is as shown in SEQ ID NO:14 or be its complementary sequence; The oligonucleotide probe detecting (g) described sudden change in fourth aspect is as shown in SEQ ID NO:15 or be its complementary sequence; The oligonucleotide probe detecting (h) described sudden change in fourth aspect is as shown in SEQ ID NO:17 or be its complementary sequence; The oligonucleotide probe detecting (i) described sudden change in fourth aspect is as shown in SEQ ID NO:18 or be its complementary sequence; The oligonucleotide probe detecting (j) described sudden change in fourth aspect is as shown in SEQ ID NO:20 or be its complementary sequence; The oligonucleotide probe detecting (k) described sudden change in fourth aspect is as shown in SEQ ID NO:22 or be its complementary sequence; The oligonucleotide probe detecting (l) described sudden change in fourth aspect is as shown in SEQ ID NO:23 or be its complementary sequence; The oligonucleotide probe detecting (m) described sudden change in fourth aspect is as shown in SEQ ID NO:24 or be its complementary sequence.
In some embodiments, solid phase carrier also secures the wild-type probe that can correspond to Mutational part sequence in the specific detection wild-type sequence of not undergoing mutation, such as, wild-type probe can be selected from following in one or more: SEQ ID NOs:8,16,19,21.The structure and function of above-mentioned concrete sequence can see " sequence brief description " part herein.The results of hybridization obtained from wild-type probe and abrupt climatic change probe can be confirmed mutually, thus determines mutation type further.
In some embodiments, the 5 ' end or 3 ' of probe is held and is modified through amination.
In some embodiments, the 5 ' end or 3 ' of probe is held and is marked with vitamin H.
In some embodiments, detect sample and be selected from the culture, phlegm, ascites pleural fluid, Bronchial washing, cerebrospinal fluid, peripheral blood and the pathological tissue specimen that comprise mycobacterium tuberculosis.Under normal circumstances, before hybridization check, need to process sample, such as, cultivate and be separated mycobacterium tuberculosis, the steps such as the genomic dna of separation and extraction M. txberwlosis cultures or RNA.Therefore, test kit can also comprise above-mentioned steps reagent used.
In some embodiments, test kit can be used in situ detection, such as, and the detection of genomic level.
In some embodiments, because the copy number detecting drug resistant gene in sample or M. txberwlosis cultures is lower, therefore before hybridization check, need to increase to the fragment of drug resistant gene.In some embodiments, amplification method used is PCR method.It will be appreciated by those skilled in the art that amplified production should have specificity (that is, the fragment outside the target gene that do not increase), and the position at place, mutational site should be comprised.Those skilled in the art can according to above-mentioned requirements, designs suitable primer and amplification obtains required gene fragment.Therefore, in some embodiments, test kit also comprises the primer pair of the gene fragment for place, described mutational site of increasing, such as, primer pair can be selected from following in one or more pairs of: SEQ ID NOs:25 and 26,27 and 28,29 and 30,31 and 32.Test kit can also comprise amplification step reagent used.In some embodiments, 5 ' end of primer is marked with vitamin H.
In some embodiments, test kit also comprises mycobacterium tuberculosis complex 16S rRNA sequence probes, whether for monitoring PCR system and being mycobacterium tuberculosis complex, such as, 16S rRNA sequence probes can be SEQ ID NOs:6 and 7 or their complementary sequence.If two 16S rRNA sequence probes results of hybridization is the positive, illustrate that test strain is mycobacterium tuberculosis complex bacterial strain.The design and use of 16S rRNA sequence probes are known in those skilled in the art.Those skilled in the art are to be understood that, except 16S rRNA probe method, differentiate that the method for mycobacterium tuberculosis complex also has a lot, include but not limited to house-keeping gene hsp65, rpoB, gyrA, gyrB, 16s rRNA sequencing and Multiplex PCR method etc.
Whether in some embodiments, test kit also comprises the DNA probe being marked with vitamin H, successful for monitoring hybridization, such as, can be SEQ ID NO:5 or its complementary sequence.If the results of hybridization of this probe is positive, then confirm that hybridization is successful.The design and use being marked with vitamin H DNA probe are known in those skilled in the art.For monitoring the sequence that the sequence of hybrid experiment can be any one section of 15-30 the base in the application beyond multiple pcr amplification sequence.It will be appreciated by those skilled in the art that except biotin labeling, other marking methods can also be used, include but not limited to digoxigenin labeled etc.
In some embodiments, solid phase carrier is film bar or diaphragm.In a particular embodiment, solid phase carrier is nylon membrane bar or nylon membrane.
The method preparing solid phase carrier as herein described or test kit is technology as known in the art, such as, and can see the illustrative methods in this paper embodiment.
Fig. 2 shows the schematic diagram of the solid phase carrier of the application, this solid phase carrier incorporates can the abrupt climatic change probe of all sudden changes of specific detection (a)-(m) mentioned above, and wild-type probe mentioned above, 16S rRNA probe and be marked with the DNA probe of vitamin H.
5th aspect, this application provides the detection method of Drug Resistance of Mycobacterium Tuberculosis, it comprises makes sample contact with the solid phase carrier of the oligonucleotide probe described in second aspect or the test kit described in fourth aspect, and the hybridization of determination and analysis sample and oligonucleotide probe or solid phase carrier.Under applicable circumstances, the technical characteristic of detection method can according to the technical characteristic disclosed in the oligonucleotide probe described in second aspect or the test kit described in fourth aspect.
Should be appreciated that the content of above detailed description only in order to make those skilled in the art more clearly understand the application, and be not intended in office where face is limited.Those skilled in the art can carry out various change and change to described embodiment.
Embodiment
Following examples are provided to further describe the application, and not in addition any restriction.
Key instrument or the equipment of employing are as follows:
The solution of main preparation is as follows:
1) 16%EDAC:1.6g EDAC+10ml distilled water;
2) 0.5M NaHCO 3: 4.2g NaHCO 3+ 100ml distilled water, adds NaOH and regulates PH to 8.4;
3) 20 × SSPE:17.5gNaCl, 27.6gNa 2hPO 42H 2o, 7.4g (0.02M) EDTA, above powder adding distil water, in 1000ml, adds NaOH and regulates PH to 7.4;
4) 2 × SSPE/0.1%SDS (1L): 100ml20 × SSPE+10ml10%SDS+890ml distilled water, mixing;
5) 2 × SSPE/0.5%SDS (1L): 100ml20 × SSPE+50ml10%SDS+850ml distilled water, mixing;
6) 2 × SSPE (1L): 100ml20 × SSPE+900ml distilled water, mixing;
7) 10%SDS (100ml): SDS10g+100ml distilled water;
8) 20mM EDTA (pH8.0,250ml): 0.5M EDTA (pH8.0) 10ml+240ml distilled water.
The preparation of embodiment 1. fluoroquinolones, kantlex, amikacin and capromycin sensitivity Detection film bar
1) Clone Manager (Professional Suite) is applied, for the mutational site of fluoroquinolones drug resistance related gene gyrA, kantlex, amikacin and capromycin drug resistance related gene rrs, the mutational site designing probe sequence of eis promotor, probe sequence is SEQ ID NO:8-24;
2) apply Clone Manager (Professional Suite) and design mycobacterium tuberculosis complex species specificity 16S rRNA probe sequence, probe sequence is SEQ ID NO.6-7;
3) according to prior art composition sequence each probe as shown in SEQ ID NO.6-24, and amino labeled is done at 5 ' end of probe;
4) design and synthesize vitamin H sequence probes (SEQ ID NO.5), do amino labeled at 5 ' end, do biotin labeling at 3 ' end;
5) with the probe of the new synthesis of TE damping fluid dilution, its final concentration is made to be 10pmol/ μ L;
6) nylon membrane (Biodyne C film) 15 minutes is hatched to make film activate with freshly prepared 16% (w/v) EDAC under room temperature;
7) film is placed in plastic containers and washes 2 minutes with deionized water, then film is placed on the support pad of clean miniblotter, tighten the screws, removes residual liquid;
8) with the 0.5M NaHCO of 150 μ L 3(pH8.4) probe (10 μMs) 1.9 μ L to 0.125 μM is diluted, mixing;
9) by NaHCO 3the probe (totally 150 μ L) of dilution is added in the groove of miniblotter successively, and wherein first adds with last groove the ink that 2 × SSPE dilutes and do border;
10) after application of sample, incubated at room temperature 1h;
11) according to probe solution addition sequence liquid-transfering gun, probe solution is sucked;
12) with tweezers, film is taken out from miniblotter, be placed in plastic containers, under room temperature, hatch 10 minutes with freshly prepared 0.1M NaOH;
13) film is washed 5 minutes with 2 × SSPE/0.1%SDS at 60 DEG C;
14) film is at room temperature washed 15 minutes with 20mM EDTA (pH8.0);
15) film phonograph seal is saved backup in mid-4 DEG C of plastics bag.
The fluoroquinolone of embodiment 2. mycobacterium tuberculosis, kantlex, amikacin and capromycin Resistance detection
The present embodiment carries out performance test to prepared fluoroquinolone, kantlex, amikacin and capromycin sensitivity Detection film bar.
the acquisition of Resistant strain and qualification
14 strain mycobacterium tuberculosis are obtained from the 14 routine pulmonary tuberculosis patient sputum specimens from Fujian, Tibet and tuberculosis prevention and treatment mechanism of Gansu Province, wherein 11 strains are to quinolones resistance, 3 strains are simultaneously to quinolone and kantlex resistance, and drug-resistant phenotype is determined by following test.
Traditional Russell medium scaling method is adopted to carry out medicament sensitivity test.Be taken at tubercule bacillus 1 ring Russell medium growing 4 weeks-5 weeks, mill bacterium is mixed with 1 Maxwell turbidity, then uses physiological saline furnishing 10 -2mg/ml and 10 -4mg/ml bacterium liquid, inoculation 10 -3mg and 10 -5mg bacterium liquid, to containing on the substratum of antitubercular agent, is put 37 DEG C and is cultivated 4 weeks observationss.When pastille substratum growing colony number and being more than or equal to 1% of colony number in control medium, be judged as that bacterial strain is to this Drug-resistant.In test in the susceptibility of different pharmaceutical, Russell medium drug concentration is as follows: fluoroquinolone 3 μ g/ml, kantlex 30 μ g/ml, capromycin 40 μ g/ml, amikacin 30 μ g/ml. fluoroquinolone, kantlex, amikacin and capromycin drug susceptibility detect the preparation of film bar
As described in Example 1.
bacterial strain DNA extraction
1) 500 μ L TE damping fluids are got in 1.5ml centrifuge tube;
2) scraping 1 ring M. txberwlosis cultures is placed in above-mentioned 1.5ml centrifuge tube, carries out (precaution: bacterium liquid can not be polluteed pipe outer) in Biohazard Safety Equipment;
3) centrifuge tube of splendid attire bacterium liquid puts 20min in 95 DEG C of water-baths or constant-temperature metal bath;
4) centrifuge tube is put ultrasonic 15min in ultrasonic pot;
5) at 4 DEG C, the centrifugal 5min of 10000rpm, without the need to sucking-off supernatant after centrifugal, directly places 4 DEG C of Refrigerator stores for subsequent use by bacterium liquid.
pcr amplification
1) reaction system cumulative volume is 50 μ L;
2) in 2 0.2ml plastic centrifuge tubes, following reagent is added respectively:
3) after mixing, put in the PCR amplification instrument of Eppendorf company production, first 94 DEG C of sex change 5min, then 94 DEG C of sex change 1min, 54 DEG C of annealing 1min, 72 DEG C extend 1min, circulate 35 times, and last 72 DEG C extend 7min;
4) get 5 μ l pcr amplification products electrophoresis detection in the low melting-point agarose gel of 1.5% respectively, result can see the amplified fragments of 309bp, 289bp, 328bp and 280bp respectively.
order-checking
Order-checking company is sent to check order above-mentioned amplified production.
hybridization check
1) in 150 μ L2 × SSPE/0.1%SDS, 45 μ LPCR products are added, mixing;
2) PCR primer of dilution is heated 10 minutes, immediately in cooled on ice at 100 DEG C;
3) prehybridization: hatch 5 minutes at 50 DEG C with 2 × SSPE/0.1%SDS (hybridization solution);
4) film and support pad are put into miniblotter, make groove vertical with the direction adding probe;
5) PCR primer cooled is added in groove, hybridizes 60 minutes for 60 DEG C;
6) sample is sucked from miniblotter, take out film;
7) film twice is washed with 2 × SSPE/0.5%SDS (washing lotion) at 60 DEG C, each 10 minutes;
8) 2.5 μ L antibiotin-Ap coupling proteins add 20ml2 × SSPE/0.5%SDS, film are placed in this solution and hatch 40 minutes at 42 DEG C;
9) film is washed 10 minutes at 42 DEG C, twice with 2 × SSPE/0.5%SDS;
10) with washing film under 2 × SSPE room temperature 5 minutes, twice is washed;
11) washed diaphragm is put into tank, add appropriate nitrite ion (NBT:BCIP:Tris:1:1:10), 42 DEG C of lucifuges develop the color about 15-30min, visible bluish voilet spot, take out diaphragm, after distilled water rinsing, dry preservation.
result
Bluish voilet signal power according to probe hybridization judges, hybridization check result as shown in Figure 1.1 is aqua sterilisa negative control, and 2 is H 37r vstandard sensitive strain, for checking the results of hybridization of acquisition whether correct, 3,7-16 be the single bacterial strain of resistance to quinolone, 4-6 is quinolone and the dual Resistant strain of kantlex.A-B is 16S rRNA probe (SEQ ID NO:6-7), when A-B all hybridizes the positive, illustrates that this bacterial strain is mycobacterium tuberculosis complex bacterial strain, shows that the result of C-R is reliable thus.C is gyrA90-94 wild-type probe (SEQ ID NO:8), D-J is the 90th, the 91 and 94 amino acids saltant type probes (SEQ ID NO:9-15) of gyrA, be judged as that gyrA has sudden change when disappearance appears in the wild-type probe position of C, if when simultaneously hybridization signal appears in D-J saltant type probe corresponding position and the particular type that suddenlys change of known gyrA; K is rrs1401/1402 wild-type probe (SEQ ID NO:16), L-M is rrs1401/1402 saltant type probe (SEQ ID NO:17-18), O is rrs1484 wild-type probe (SEQ ID NO:19), N is rrs1484 saltant type probe (SEQ ID NO:20), be judged as that rrs has sudden change when disappearance appears in K and O wild-type probe position, if when simultaneously hybridization signal appears in L-M and N saltant type probe corresponding position and the particular type that suddenlys change of known rrs; P is eis promotor-17 ~+1 wild-type probe (SEQ ID NO:21), Q-S is eis promoter mutation type probe (SEQ ID NO:22-24), be judged as that eis promotor has sudden change when disappearance appears in P wild-type probe position, if when simultaneously hybridization signal appears in Q-S saltant type probe corresponding position and the particular type that suddenlys change of known rrs.
3,7-11 is that the codon of the 90th amino acids of the aminoacid sequence (SEQ ID NO:2) of gyrA is undergone mutation, and mutation type is GCG (Ala)-GTG (Val); 4 be gyrA the codons of the 90th amino acids of aminoacid sequence (SEQ ID NO:2) ,-10 bit bases of eis promoter sequence (SEQ ID NO:4) are undergone mutation, and mutation type is respectively GCG (Ala)-GTG (Val) and G-A; 5 be gyrA the codons of the 91st amino acids of aminoacid sequence (SEQ ID NO:2) ,-14 bit bases of eis promoter sequence (SEQ ID NO:4) are undergone mutation, and mutation type is respectively TCG (Ser)-CCG (Pro) and C-T; 6 be gyrA the codons of the 90th amino acids of aminoacid sequence (SEQ ID NO:2), 1401 bit bases of rrs sequence (SEQ ID NO:3) are undergone mutation, and mutation type is respectively GCG (Ala)-GTG (Val) and A-G; The codon of the 91st amino acids of 12 aminoacid sequences (SEQ ID NO:2) that are gyrA is undergone mutation, and mutation type is respectively TCG (Ser)-CCG (Pro); 13-16 is that the codon of the 94th amino acids of the aminoacid sequence (SEQ ID NO:2) of gyrA is undergone mutation, and mutation type is respectively GAC (Asp)-GGC (Gly).
The fluoroquinolones of embodiment 3. mycobacterium tuberculosis, kantlex, amikacin and capromycin Resistance detection and with the comparing of other testing method
The present embodiment tests the effect of fluoroquinolones, kantlex, amikacin and the capromycin sensitivity Detection film bar prepared according to the application on a larger scale, and carries out intersecting comparing with the result that Conventional solid substratum scaling method (Phenotypic examination) and DNA sequencing method obtain.
Fluoroquinolones, kantlex, amikacin and capromycin drug susceptibility detection film bar preparation method and detection method are as shown in embodiment 1 and 2.
The fluoroquinolones utilizing the application to prepare, kantlex, amikacin and capromycin sensitivity Detection film bar carry out gyrA gene mutation analysis to 214 strain mycobacterium tuberculosis clinical separation strains, result shows 40 strains in 59 strain Fluoroquinolones Resistant Strainss and sudden change (comprising the sudden change of the 90th, 91,94 of gyrA aminoacid sequence mentioned above) detected, has 6 strains sudden change to be detected in 174 strain Rifampin sensitive strains; Cultivate scaling method for reference with Conventional solid, susceptibility and specific degree are respectively 67.8% (40/59) and 96.6% (168/174).With DNA sequencing results contrast, 46 strain order-checking mutant strains have 40 strains to be judged as persister, and 187 strain order-checkings do not find that in the bacterial strain suddenlyd change, 174 strains are judged as sensitive strain; Susceptibility and specific degree are respectively 87.0% (40/46) and 93.0% (174/187).
Utilize fluoroquinolones prepared by the application, kantlex, amikacin and capromycin sensitivity Detection film bar carry out the analysis of rrs and eis promoter mutation to 55 strain mycobacterium tuberculosis clinical separation strains, result shows 12 strains in 25 strain capromycin persisters and detects that sudden change (comprises the 1401st of rrs sequence mentioned above the, the sudden change of 1402 and 1484 and-14 of eis promotor,-12 and the sudden change of-10), 0 strain is had sudden change to be detected in 30 strain capromycin sensitive strains, susceptibility and specific degree are respectively 48.0% (12/25) and 100.0% (30/30), with DNA sequencing results contrast, 12 strain order-checking mutant strains have 12 strains to be judged as persister, 43 strain order-checkings do not find that in the bacterial strain suddenlyd change, 30 strains are judged as sensitive strain, and susceptibility and specific degree are respectively 100.0% (12/12) and 69.8% (30/43), in 20 strain kantlex persisters, 17 strains detect sudden change, and have 0 strain sudden change to be detected in 35 strain kantlex sensitive strains, susceptibility and specific degree are respectively 85.0% (17/20) and 100.0% (35/35), with DNA sequencing results contrast, 17 strain order-checking mutant strains have 17 strains to be judged as persister, 38 strain order-checkings do not find that in the bacterial strain suddenlyd change, 35 strains are judged as sensitive strain, and susceptibility and specific degree are respectively 100.0% (17/17) and 92.1% (35/38).
As can be seen from the above results, the detection method set up according to the mutational site identified each drug resistant gene in the application and instrument can realize higher detection specificity and susceptibility.
The application completes under national transmissible disease key special subjects is subsidized, and can be widely used in the association areas such as the diagnosis of drug resistant tuberculosis, epidemiology survey and monitoring based on the drug susceptibility detection method of the application and instrument.

Claims (10)

1. the nucleic acid fragment for mycobacterium tuberculosis (Mycobacterium tuberculosis) Resistance detection combines, and comprises following (1) to two in (3) or all:
(1) mutant of the nucleotide sequence of 208 to 390 of SEQ ID NO:1, wherein sudden change is selected from:
(1.1): the C of the 269th of SEQ ID NO:1 sports T, it causes the 90th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Val by Ala;
(1.2): the T of the 271st of SEQ ID NO:1 sports C, it causes the 91st of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Pro by Ser;
(1.3): the A of the 281st of SEQ ID NO:1 sports G, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Gly by Asp;
(1.4): the A of the 281st of SEQ ID NO:1 sports C, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Ala by Asp;
(1.5): the G of the 280th of SEQ ID NO:1 sports T, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Tyr by Asp;
(1.6): the G of the 280th of SEQ ID NO:1 sports A, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Asn by Asp;
(1.7): the G of the 280th of SEQ ID NO:1 sports C, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport His by Asp; And
(1.8): the arbitrary combination of sudden change described in (1.1) to (1.7);
(2) mutant of the nucleotide sequence of the 1390-1490 position of SEQ ID NO:3, wherein sudden change is selected from:
(2.1): the A of the 1401st of SEQ ID NO:3 sports G;
(2.2): the C of the 1402nd of SEQ ID NO:3 sports T;
(2.3): the G of the 1484th of SEQ ID NO:3 sports T; And
(2.4): the arbitrary combination of sudden change described in (2.1) to (2.3);
(3) mutant of-50 to-1 of SEQ ID NO:4, wherein sudden change is selected from:
(3.1): the C of the-14 of SEQ ID NO:4 sports T;
(3.2): the C of the-12 of SEQ ID NO:4 sports T;
(3.3): the G of the-10 of SEQ ID NO:4 sports A; And
(3.4): the arbitrary combination of sudden change described in (3.1) to (3.3).
2. can specifically in conjunction with the oligonucleotide probe of nucleic acid fragment combination according to claim 1.
3. oligonucleotide probe as claimed in claim 2, is selected from following at least two or whole:
SEQ ID NO:9 or its complementary sequence, SEQ ID NO:10 or its complementary sequence, SEQ ID NO:11 or its complementary sequence, SEQ ID NO:12 or its complementary sequence, SEQ ID NO:13 or its complementary sequence, SEQ ID NO:14 or its complementary sequence, SEQ ID NO:15 or its complementary sequence, SEQ ID NO:17 or its complementary sequence, SEQ ID NO:18 or its complementary sequence, SEQ ID NO:20 or its complementary sequence, SEQ ID NO:22 or its complementary sequence, SEQ ID NO:23 or its complementary sequence, SEQ ID NO:24 or its complementary sequence.
4. the purposes of the oligonucleotide probe described in Claims 2 or 3 in the testing tool preparing mycobacterium tuberculosis (Mycobacterium tuberculosis) resistance.
5. mycobacterium tuberculosis (Mycobacterium tuberculosis) Resistance detection test kit, it comprises solid phase carrier, described solid phase carrier secures can specific detection following in the oligonucleotide probe of at least two or more or all sudden changes:
(a): the C of the 269th of SEQ ID NO:1 sports T, it causes the 90th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Val by Ala;
(b): the T of the 271st of SEQ ID NO:1 sports C, it causes the 91st of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Pro by Ser;
(c): the A of the 281st of SEQ ID NO:1 sports G, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Gly by Asp;
(d): the A of the 281st of SEQ ID NO:1 sports C, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Ala by Asp;
(e): the G of the 280th of SEQ ID NO:1 sports T, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Tyr by Asp;
(f): the G of the 280th of SEQ ID NO:1 sports A, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport Asn by Asp;
(g): the G of the 280th of SEQ ID NO:1 sports C, it causes the 94th of the aminoacid sequence coded by SEQ ID NO:1 (SEQ ID NO:2) to sport His by Asp;
(h): the A of the 1401st of SEQ ID NO:3 sports G;
(i): the C of the 1402nd of SEQ ID NO:3 sports T;
(j): the G of the 1484th of SEQ ID NO:3 sports T;
(k): the C of the-14 of SEQ ID NO:4 sports T;
(l): the C of the-12 of SEQ ID NO:4 sports T; And
(m): the G of the-10 of SEQ ID NO:4 sports A.
6. test kit as claimed in claim 5, wherein said oligonucleotide probe be selected from following in one or more or whole:
SEQ ID NO:9 or its complementary sequence, SEQ ID NO:10 or its complementary sequence, SEQ ID NO:11 or its complementary sequence, SEQ ID NO:12 or its complementary sequence, SEQ ID NO:13 or its complementary sequence, SEQ ID NO:14 or its complementary sequence, SEQ ID NO:15 or its complementary sequence, SEQ ID NO:17 or its complementary sequence, SEQ ID NO:18 or its complementary sequence, SEQ ID NO:20 or its complementary sequence, SEQ ID NO:22 or its complementary sequence, SEQ ID NO:23 or its complementary sequence, SEQ ID NO:24 or its complementary sequence.
7. the test kit as described in claim 5 or 6, wherein said solid phase carrier also secures the wild-type probe that can correspond to Mutational part sequence in the specific detection wild-type sequence of not undergoing mutation, such as, wild-type probe be selected from following in one or more: SEQ ID NOs:8,16,19,21.
8. the test kit according to any one of claim 5-7, described test kit also comprise following in one or more components:
(i) for the primer pair of the gene fragment at place, described mutational site of increasing, such as, primer pair be selected from following in one or more pairs of: SEQ ID NOs:25 and 26,27 and 28,29 and 30,31 and 32;
(ii) for the identification of the probe of mycobacterium tuberculosis complex, such as, described probe sequence is SEQ ID NOs:6 and 7 or their complementary sequence;
(iii) for monitoring the DNA probe being marked with vitamin H of hybridization quality, such as, the sequence of described probe is SEQ ID NO:5 or its complementary sequence.
9. the test kit according to any one of claim 5-8, wherein detects sample and is selected from the culture, phlegm, ascites pleural fluid, Bronchial washing, cerebrospinal fluid, peripheral blood and the pathological tissue specimen that comprise mycobacterium tuberculosis.
10. the test kit according to any one of claim 5-9, wherein said solid phase carrier is nylon membrane.
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CN117070645A (en) * 2023-07-25 2023-11-17 鲲鹏基因（北京）科技有限责任公司 Composition, kit and method for detecting aminoglycoside drug resistance of mycobacterium tuberculosis

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