CN112725486A - Mycobacterium tuberculosis complex rapid detection and diagnosis kit and preparation method thereof - Google Patents
Mycobacterium tuberculosis complex rapid detection and diagnosis kit and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a rapid detection and diagnosis kit for mycobacterium tuberculosis complex, which comprises a primer pair and a probe, wherein the primer pair comprises: upstream primer IS1081-F1And the downstream primer IS1081-R1The sequences of the two are shown in SEQ ID NO. 1-2; the probe IS a probe IS1081-P1The sequence is shown as SEQ ID NO. 3; the invention also provides a preparation method of the kit for rapidly detecting and diagnosing the mycobacterium tuberculosis complex, which comprises the following steps: s1 designing a primer pair; s2 forming a mixed system; amplifying an S3 standard sample; s4 judging the sensitivity and specificity; s5 judging the designed probe site; s6 screening to obtain the primer pair and the probe; the invention is provided withThe beneficial effects are that; the specific primer pair and the arranged probe can be used for realizing the large-scale amplification of the standard gene and the sample gene, and meanwhile, the specific gene locus IS1081 IS adopted to realize high accuracy.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a kit for rapidly detecting and diagnosing mycobacterium tuberculosis complex and a preparation method thereof.
Background
Tuberculosis is one of major infectious diseases in the world, and is a chronic infectious disease caused by Mycobacterium tuberculosis (m. tuberculosis), Mycobacterium microti (m. microti), Mycobacterium bovis (m. bovis), etc., which can spread throughout the whole body organs and mainly controls the lung.
As early as 17000 years ago, some scholars found mycobacterium tuberculosis in the remains of bison, which was the earliest source of tuberculosis found so far [ lucentin. University of Shandong, 2017 ]. Mycobacterium tuberculosis has been found in humans almost 7000 years ago [ 1. Herrera L, et al, saez-Nieto: Molecular analysis of rifaximin-resistant Mycobacterium tuberculosis isolated in Spain (1996-2001) -Description of new microorganisms in the rpoB gene and review of the performance [ J ]. Int J antimicrobial Agents,2003,21:403-8 ]. However, whether tuberculosis in humans originates in cattle has not been reported clearly [2 Huang H, et al, mutation of rpoB mutations in rifampin-resistant Mycobacterium tuberculosis isolates in China [ J ] Tuberculosis (Edinb) 2003.21:403-8 ]. Until 1882, the tuberculosis pathogen Mycobacterium tuberculosis was isolated by Robert Koch and established the foundation for the development of antibiotics and vaccines later [ 1. Herrera L, et al, saez-Nieto: Molecular analysis of rifapeuticin-resistant Mycobacterium tuberculosis isolatedin Spain (1996) 2001. Description of new microorganisms in the rpb gene and review of the last microorganisms [ J ]. Int J antibiotic Agents,2003,21:403-8 ] [3 Zhang M, Yue J, Yang Y, et al, Detection of mutation with tuberculosis microorganisms in Mycobacterium tuberculosis isolates [ 43. Cl547.7: 82 ]. In 1906, Albert Calmette and Camile Guerin successfully developed the first anti-tuberculosis vaccine, BCG, and first inoculated into humans in France in 1921 [ Ramasumamy S, Musser JM. Molecular genetic basis of anti-microbial agent resistance in Mycobacterium tuberculosis, 1998, update [ J ]. Tuber Lung Dis. 1998,79:3-29 ]. The advent of streptomycin 1945 has controlled tuberculosis. Subsequently, drugs such as rifampicin, ethambutol, etc. were beginning to be used to treat tuberculosis, reducing the number of deaths worldwide in patients with pulmonary tuberculosis. Particularly, isoniazid appears in 1952, has the advantages of strong sterilization capability, small side effect, economy, convenience for taking and the like, and reduces the morbidity of tuberculosis patients by 50-60 percent within 10 years of preventive treatment of isoniazid. Therefore, in the 20 th century and the 80 s, it was considered that tuberculosis could be completely destroyed at the end of the 20 th century. However, tuberculosis has increased again worldwide due to drug abuse, HIV epidemic and emergence of drug-resistant Mycobacterium tuberculosis. Currently, about 20 hundred million people worldwide infect mycobacterium tuberculosis, and 2015 reports of global tuberculosis indicate that about 960 ten thousand of new cases worldwide in 2014 and about 150 thousand of death cases, and the occurrence and prevalence of aids and drug-resistant mycobacterium tuberculosis cause the incidence of global tuberculosis to rise continuously, which makes the prevention, control, diagnosis and treatment of tuberculosis face very serious challenges. Tuberculosis has become one of global important public health problems, data published in WHO 2015 show that China is one of world tuberculosis high-burden countries, is in the third place of the world, and about 98 thousands of 2015 new tuberculosis cases.
Once a person is infected, the problems of great toxic and side effects of a medicine, long treatment period, prevalence of drug-resistant strains, combined attack of various diseases and the like can be faced, the treatment difficulty of tuberculosis is greatly increased, and the health of the human is seriously threatened. Therefore, early diagnosis and accurate diagnosis are of great significance for the treatment, prevention and control of tuberculosis, but at present, because of lack of pathogenic basis, clinical diagnosis is extremely difficult, and missed diagnosis and misdiagnosis exist, so that the disease is widely spread. Therefore, the early, rapid and accurate diagnosis has extremely important significance for the treatment and the control of the spread of tuberculosis.
With the development of gene detection technology, nucleic acid detection methods are also gradually applied to the rapid diagnosis of tuberculosis, and after a kit containing a gene probe is mainly used for detecting a standard target gene, the kit data of the gene to be detected is compared, so that the primer design and the probe reagent surrounding the detection kit play a relevant role.
However, most of the current nucleic acid detection combined with mycobacteria focuses on the respective detection of various types of the mycobacterium tuberculosis to improve the precision, but most of the current detection aiming at various mycobacteria has the problems of low precision and low sensitivity, and meanwhile, the subsequent verification of the current primer design is not accurate, so that the judgment of the precision is influenced.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a kit for rapidly detecting and diagnosing mycobacterium tuberculosis complex and a preparation method thereof, so as to at least achieve the aims of high precision and high sensitivity.
The purpose of the invention is realized by the following technical scheme:
a kit for rapidly detecting and diagnosing Mycobacterium tuberculosis complex comprises a primer pair and a probe, wherein the primer pair comprises: upstream primer IS1081-F1And the downstream primer IS1081-R1The sequences of the two are shown in SEQ ID NO. 1-2; the corresponding probe IS probe IS1081-P1The sequence is shown in SEQ ID NO. 3.
Preferably, for the purpose of further achieving high accuracy, the primer pair IS designed according to the nucleotide sequence of a specific target gene IS1081 gene of the Mycobacterium tuberculosis complex known from the Pub-Med gene bank; the mycobacterium tuberculosis complex comprises one or more of standard mycobacterium tuberculosis, mycobacterium bovis, mycobacterium africanum, mycobacterium microti and mycobacterium capricolum; by defining the species in the mycobacterium tuberculosis complex, the common mycobacterium tuberculosis IS taken as the basis, the target gene IS1081 IS taken as the reference for design, the obtained primer pair and the corresponding probe can provide better accuracy, and the detection specificity can cover the common mycobacterium tuberculosis, so that certain representativeness IS achieved, and the purpose of high precision IS further achieved.
The invention also provides a preparation method of the kit for rapidly detecting and diagnosing the mycobacterium tuberculosis complex, which comprises the following steps:
s1, according to the gene sequence of the Mycobacterium tuberculosis complex provided by the Pub-Med gene library, carrying out multi-sequence comparison by using the H37Rv whole genome of the Mycobacterium tuberculosis complex, selecting a conserved region, and designing an upstream primer IS1081-F, a downstream primer IS1081-R and a probe IS1081-P shown as SEQ ID No. 1-21 aiming at a specific target gene IS1081 of the Mycobacterium tuberculosis complex;
s2, selecting a qPCR MIX reaction system by using the primer and the probe to form a mixed system;
s3, adding a standard sample into the mixed system, selecting a PCR amplification system, uniformly mixing, and placing the mixture in a fluorescent quantitative PCR instrument for PCR amplification to obtain an amplification result;
s4, respectively testing the sensitivity and the specificity of the obtained amplification result, and judging the sensitivity and the specificity of the designed probe;
s4, judging whether the designed probe site is reasonable or not by taking the standard gene as a reference according to the result of the sensitivity and specificity test, executing the next step if the designed probe site is reasonable, and returning to S1 for redesign if the designed probe site is not reasonable;
s5 screening to obtain an upstream primer IS1081-F1And the downstream primer IS1081-R1The primer set and the probe IS1081-P1Namely the preparation method.
Preferably, for the purpose of further achieving high sensitivity, the sensitivity test is to perform 10-fold gradient dilution of the Mycobacterium tuberculosis H37Rv whole genome DNA strain, 10 times each0-106Taking 2 mu L of each concentration into the mixed system, using ultrapure water as a negative control, carrying out sensitivity test, and establishing and applying the results with the existing literature (Leyahui; Lulisna; Doboping et al. Mycobacterium tuberculosis IS6110 and IS1081 microdroplet digital PCR detection system [ J]Beijing medicine 2018,40(4): 314-; by comparing the method and the primers disclosed in the literature, the sensitivity is accurately compared with the existing sensitivity, so that the sensitivity data is accurately obtained, and the reliability of the sensitivity is improved.
Preferably, for the purpose of further achieving high accuracy, the specificity test IS to take mycobacterium tuberculosis of mycobacterium tuberculosis H37Rv whole genome DNA as a positive control, take DNA of mycobacterium bovis, mycobacterium africanum, mycobacterium microti, mycobacterium caprine, escherichia coli, mycobacterium baumannii, klebsiella pneumoniae, staphylococcus aureus and pseudomonas aeruginosa as a sample for specificity detection, take ultrapure water as a negative control, respectively perform PCR amplification on a primer pair consisting of an upstream primer IS1081-F and a downstream primer IS1081-R and a probe IS1081-P, and judge the specificity of the primer pair to various floras; the mycobacterium bovis is selected from mycobacterium bovis with BAA-935D-2 standard DNA, the mycobacterium africanum is selected from mycobacterium africanum with 25420DQ standard DNA, the mycobacterium microti is selected from mycobacterium microti with 19422DQ standard DNA, and the mycobacterium caprine is selected from mycobacterium caprine with BAA-824D-2 standard DNA;
the qPCR amplification system and the program are provided by a Vazyme qPCR premix kit, and the amplification system specifically comprises the following components: 10 μ L of qPCR MIX, 0.4 μ L of 10 μmol/L primer pair, 0.2 μ L of 10 μmol/L probe IS1081-P, 2 μ L of DNA template, 7 μ L of ddH2O; the amplification procedure is specifically as follows: carrying out pollution digestion at 37 ℃ for 2 min, pre-denaturation at 95 ℃ for 5 min, denaturation at 95 ℃ for 10 s, annealing at 60 ℃ for 30s, and carrying out 45 cycles of denaturation and annealing; the specific amplification system and the specific amplification program are utilized to cooperate with different bacteria detection to determine the specificity of the whole amplification system and the mixed system, thereby realizing the purpose of high precision.
The invention has the beneficial effects that:
1. the specific primer pair IS utilized by a primer pair consisting of a specific upstream primer IS1081-F and a downstream primer IS1081-R and a set probe IS1081-P, so that a standard gene and a sample gene can be amplified in a large amount, and meanwhile, a specific reaction can be carried out on mycobacterium tuberculosis due to the adoption of a large amount of specific gene sites IS1081 determined by the mycobacterium tuberculosis, so that the aim of high precision IS fulfilled.
2. By defining the species in the mycobacterium tuberculosis complex, the common mycobacterium tuberculosis IS taken as the basis, the target gene IS1081 IS taken as the reference for design, the obtained primer pair and the corresponding probe can provide better accuracy, and the detection specificity can cover the common mycobacterium tuberculosis, so that certain representativeness IS achieved, and the purpose of high precision IS further achieved.
3. By comparing the method and the primers disclosed in the literature, the sensitivity is accurately compared with the existing sensitivity, so that the sensitivity data is accurately obtained, and the reliability of the sensitivity is improved.
4. The specific amplification system and the specific amplification program are utilized to cooperate with different bacteria detection to determine the specificity of the whole amplification system and the mixed system, thereby realizing the purpose of high precision.
Drawings
FIG. 1 IS a diagram showing the results of screening primers and probes for IS1081 gene,
wherein, 1 IS an upstream primer IS1081-F2And the downstream primer IS1081-R2And probe IS1081-P2，
2 IS an upstream primer IS1081-F2And the downstream primer IS1081-R2And probe IS1081-P2，
3 IS an upstream primer IS1081-F3And the downstream primer IS1081-R3And probe IS1081-P3，
4 IS an upstream primer IS1081-F4And the downstream primer IS1081-R4And probe IS1081-P4，
5 IS an upstream primer IS1081-F5And the downstream primer IS1081-R5And probe IS1081-P5，
6 IS an upstream primer IS1081-F6And the downstream primer IS1081-R6And probe IS1081-P6，
7 IS an upstream primer IS1081-F7And the downstream primer IS1081-R7And probe IS1081-P7，
8 is a negative control of distilled water;
FIG. 2 IS a graph showing the results of sensitivity tests of primers and probes for the IS1081 gene,
wherein 1-7 are respectively 10 times of the whole genome DNA strain of mycobacterium tuberculosis H37Rv0、101、102、103、104、105、106The secondary dilution amplification curve, 8 is a negative control;
FIG. 3 is a graph showing the results of the sensitivity test of the literature,
wherein 1-7 are respectively 10 times of the whole genome DNA strain of mycobacterium tuberculosis H37Rv0、101、102、103、104、105、106The amplification curve was diluted by a factor of 8, which is a negative control.
FIG. 4 IS a graph showing the results of fluorescent quantitative qPCR specific assay of IS1081 gene,
wherein, 1-plasmid, 2-mycobacterium tuberculosis H37Rv whole genome DNA strain, 3-mouse mycobacterium, 4-African mycobacterium, 5-bovine mycobacterium tuberculosis, 6-escherichia coli, 7-baumannii, 8-Klebsiella pneumoniae, 9-staphylococcus aureus, 10-pseudomonas aeruginosa, 11-distilled water negative control.
Detailed Description
The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings, but the scope of the present invention is not limited to the following.
Example 1
A kit for rapidly detecting and diagnosing Mycobacterium tuberculosis complex comprises a primer pair and a probe, wherein the primer pair comprises: the sequences of the upstream primer IS1081-F and the downstream primer IS1081-R are shown in SEQ ID No. 1-2; the corresponding probe IS a probe IS1081-P, and the sequence of the probe IS shown in SEQ ID NO. 3.
In order to further realize the purpose of high precision, the primer pair IS designed according to the nucleotide sequence of a specific target gene IS1081 gene of the Mycobacterium tuberculosis complex known from the Pub-Med gene bank; the mycobacterium tuberculosis complex comprises a mixed complex of standard mycobacterium tuberculosis, bovine mycobacterium tuberculosis, african mycobacterium tuberculosis, hamster mycobacterium and goat mycobacterium; by defining the species in the mycobacterium tuberculosis complex, the common mycobacterium tuberculosis IS taken as the basis, the target gene IS1081 IS taken as the reference for design, the obtained primer pair and the corresponding probe can provide better accuracy, and the detection specificity can cover the common mycobacterium tuberculosis, so that certain representativeness IS achieved, and the purpose of high precision IS further achieved.
The invention also provides a preparation method of the kit for rapidly detecting and diagnosing the mycobacterium tuberculosis complex, which comprises the following steps:
s1, according to the gene sequence of the Mycobacterium tuberculosis complex provided by the Pub-Med gene library, carrying out multi-sequence comparison by using the H37Rv whole genome of the Mycobacterium tuberculosis complex, selecting a conserved region, and designing an upstream primer IS1081-F, a downstream primer IS1081-R and a probe IS1081-P shown as SEQ ID No. 1-21 aiming at a specific target gene IS1081 of the Mycobacterium tuberculosis complex;
s2, selecting a qPCR MIX reaction system by using the primer and the probe to form a mixed system;
s3, adding a standard sample into the mixed system, selecting a PCR amplification system, uniformly mixing, and placing the mixture in a fluorescent quantitative PCR instrument for PCR amplification to obtain an amplification result;
s4, respectively testing the sensitivity and the specificity of the obtained amplification result, and judging the sensitivity and the specificity of the designed probe;
s4 for the sensitivity and specificity test results, with 25618D standard DNA of Mycobacterium tuberculosis standard gene as reference, judging whether the designed probe site is reasonable, if reasonable, executing the next step, if not, returning to S1 for redesign, designing each designed primer pair and probe as shown in Table 1,
table 1 shows the conditions of the respective primer pairs and probes
Wherein, HEX is a HEX fluorescent group marked at the 5 'end, BHQ1 is a BHQ1 quenching group marked at the 3' end, F is an upstream primer, R is a downstream primer, and P is a probe;
the obtained results were analyzed by Design and analysis as shown in FIG. 1, in which the upstream primer IS1081-F corresponding to reference numeral 11And the downstream primer IS1081-R1Composed primer pair and probe IS1081-P1The design curve of (2) IS more ideal, so that the upstream primer IS1081-F as shown in SEQ ID NO. 1-SEQ ID NO.3 IS selected1And the downstream primer IS1081-R1The primer pair and the probe IS1081-P1；
S5 IS screened to obtain a primer pair of an upstream primer IS1081-F1 and a downstream primer IS1081-R1 and a probe IS1081-P1, namely the preparation method.
To further achieve high sensitivity, the sensitivity test was performed by 10-fold gradient dilution of the M.tuberculosis H37Rv whole genome DNA strain, 10 times each0-106Taking 2 mu L of each concentration into the mixed system, taking ultrapure water as a negative control, carrying out sensitivity test, and establishing and applying a micro-droplet digital PCR detection system for Mycobacterium tuberculosis IS6110 and IS1081 in the existing literature (Leyahui; Lulisna; Doboping et al) [ J]Beijing medicine 2018,40(4): 314-; by comparing the method and the primers disclosed in the literature, the sensitivity is accurately compared with the existing sensitivity, so that the sensitivity data is accurately obtained, and the reliability of the sensitivity is improved.
In order to further realize the purpose of high precision, the specificity test IS that the mycobacterium tuberculosis of the whole genome DNA of the mycobacterium tuberculosis H37Rv IS used as a positive control, the DNA of the mycobacterium bovis, the mycobacterium africanum, the mycobacterium microti, the mycobacterium caprine, the escherichia coli, the mycobacterium baumannii, the klebsiella pneumoniae, the staphylococcus aureus and the pseudomonas aeruginosa IS used as a sample for specificity detection, ultrapure water IS used as a negative control, and the primer pair consisting of the upstream primer IS1081-F and the downstream primer IS1081-R and the probe IS1081-P are respectively subjected to PCR amplification to judge the specificity of various floras; the mycobacterium bovis is selected from mycobacterium bovis with BAA-935D-2 standard DNA, the mycobacterium africanum is selected from mycobacterium africanum with 25420DQ standard DNA, the mycobacterium microti is selected from mycobacterium microti with 19422DQ standard DNA, and the mycobacterium caprine is selected from mycobacterium caprine with BAA-824D-2 standard DNA; as a result of Design and analysis, as shown in FIG. 4, the specific curves of the Mycobacterium tuberculosis were shown in FIGS. 1 to 5, but the specific curves of the infectious microbes of 6 to 10 were not shown, so that it can be considered that the primer pair consisting of the upstream primer IS1081-F and the downstream primer IS1081-R and the probe IS1081-P both exhibited specificity for Mycobacterium tuberculosis;
the qPCR amplification system and the program are provided by a Vazyme qPCR premix kit, and the amplification system specifically comprises the following components: 10 μ L of qPCR MIX, 0.4 μ L of 10 μmol/L primer pair, 0.2 μ L of 10 μmol/L probe IS1081-P, 2 μ L of DNA template, 7 μ L of ddH2O; the amplification procedure is specifically as follows: carrying out pollution digestion at 37 ℃ for 2 min, pre-denaturation at 95 ℃ for 5 min, denaturation at 95 ℃ for 10 s, annealing at 60 ℃ for 30s, and carrying out 45 cycles of denaturation and annealing; the specific amplification system and the specific amplification program are utilized to cooperate with different bacteria detection to determine the specificity of the whole amplification system and the mixed system, thereby realizing the purpose of high precision.
Example 2
The Mycobacterium tuberculosis complex includes a mixed complex of standard Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium africanum, and the rest of the steps and the formula are the same as those in example 1.
Example 3
The Mycobacterium tuberculosis complex includes a mixed complex of standard Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium microti, and the rest of the steps and the formula are the same as those in example 1.
Example 4
The Mycobacterium tuberculosis complex includes a mixed complex of standard Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium caprine, and the rest of the steps and the formula are the same as those in example 1.
The fold of the sensitivity of each example with respect to that of the document and the specificity in the specific detection were counted separately, wherein,
comprehensive statistics results Table 2
TABLE 2 Table of the sensitivity fold and specificity of each example
As can be seen from table 1, the detection sensitivity of the kit obtained by using the mixed complex group including standard mycobacterium tuberculosis, mycobacterium bovis, mycobacterium africanum, mycobacterium microti and mycobacterium caprine, and the system and the procedure of qPCR amplification using the primer pair and the probe designed in the present application is10 times higher than that described in the existing document, and the kit can specifically identify most of the mycobacterium tuberculosis, thereby embodying the superiority of the present invention.
The foregoing is illustrative of the preferred embodiments of this invention, and it is to be understood that the invention is not limited to the precise form disclosed herein and that various other combinations, modifications, and environments may be resorted to, falling within the scope of the concept as disclosed herein, either as described above or as apparent to those skilled in the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (8)
1. A diagnostic kit for rapid detection of Mycobacterium tuberculosis complex is characterized in that: the kit comprises a primer pair and a probe, wherein the primer pair comprises: upstream primer IS1081-F1And downstream leadingSubstance IS1081-R1The sequences of the two are shown in SEQ ID NO. 1-2; the probe IS a probe IS1081-P1The sequence is shown in SEQ ID NO. 3.
2. The kit for rapid detection and diagnosis of Mycobacterium tuberculosis complex as claimed in claim 1, wherein: the primer pair IS obtained by designing according to the known nucleotide sequence of the specific target gene IS1081 gene of the Mycobacterium tuberculosis complex in the Pub-Med gene bank.
3. The kit for rapid detection and diagnosis of Mycobacterium tuberculosis complex as claimed in claim 2, wherein: the mycobacterium tuberculosis complex comprises one or more of standard mycobacterium tuberculosis, bovine mycobacterium tuberculosis, african mycobacterium, hamster mycobacterium and goat mycobacterium.
4. The method for preparing a diagnostic kit for rapid detection of Mycobacterium tuberculosis complex according to any one of claims 1-3, wherein the kit comprises: the method comprises the following steps:
s1, according to the gene sequence of the Mycobacterium tuberculosis complex provided by the Pub-Med gene library, carrying out multi-sequence comparison by using the H37Rv whole genome of the Mycobacterium tuberculosis complex, selecting a conserved region, and designing an upstream primer IS1081-F, a downstream primer IS1081-R and a probe IS1081-P shown as SEQ ID No. 1-21 aiming at a specific target gene IS1081 of the Mycobacterium tuberculosis complex;
s2, selecting a qPCR MIX reaction system by using the primer and the probe to form a mixed system;
s3, adding a standard sample into the mixed system, selecting a PCR amplification system, uniformly mixing, and placing the mixture in a fluorescent quantitative PCR instrument for PCR amplification to obtain an amplification result;
s4, respectively testing the sensitivity and the specificity of the obtained amplification result, and judging the sensitivity and the specificity of the designed probe;
s4, judging whether the designed probe site is reasonable or not by taking the standard gene as a reference according to the result of the sensitivity and specificity test, executing the next step if the designed probe site is reasonable, and returning to S1 for redesign if the designed probe site is not reasonable;
s5 screening to obtain an upstream primer IS1081-F1And the downstream primer IS1081-R1The primer set and the probe IS1081-P1Namely the preparation method.
5. The method for preparing a diagnostic kit for rapid detection of Mycobacterium tuberculosis complex according to claim 4, wherein the kit comprises: the sensitivity test is that the mycobacterium tuberculosis H37Rv whole genome DNA strain is subjected to 10-fold gradient dilution, and the dilution is respectively 10 times0-106And taking 2 mu L of each concentration into the mixed system, and carrying out the sensitivity test of the kit by using ultrapure water as a negative control.
6. The method for preparing a diagnostic kit for rapid detection of Mycobacterium tuberculosis complex according to claim 4, wherein the kit comprises: the specificity test IS that the mycobacterium tuberculosis of the whole genome DNA of the mycobacterium tuberculosis H37Rv IS used as a positive control, the DNA of the mycobacterium tuberculosis of cattle, the mycobacterium africanum, the mycobacterium microti, the mycobacterium caprine, the escherichia coli, the baumannii, the klebsiella pneumoniae, the staphylococcus aureus and the pseudomonas aeruginosa IS used as a sample for specificity detection, ultrapure water IS used as a negative control, and PCR amplification IS respectively carried out on a primer pair consisting of an upstream primer IS1081-F and a downstream primer IS1081-R and a probe IS1081-P, so as to judge the specificity of the mycobacterium tuberculosis to various floras.
7. The method for preparing a diagnostic kit for rapid detection of Mycobacterium tuberculosis complex according to claim 6, wherein: the mycobacterium bovis is selected from mycobacterium bovis with BAA-935D-2 standard DNA, the mycobacterium africanum is selected from mycobacterium africanum with 25420DQ standard DNA, the mycobacterium microti is selected from mycobacterium microti with 19422DQ standard DNA, and the mycobacterium caprine is selected from mycobacterium caprine with BAA-824D-2 standard DNA.
8. The method for preparing the kit for rapid detection and diagnosis of Mycobacterium tuberculosis complex according to claim 7, wherein the kit comprises: the qPCR amplification system and the program are provided by a Vazyme qPCR premix kit, and the amplification system specifically comprises the following components: 10 μ L of qPCR MIX, 0.4 μ L of 10 μmol/L of the primer pair, 0.2 μ L of 10 μmol/L of the probe IS1081-P, 2 μ L of DNA template, 7 μ L of ddH2O；
The amplification procedure is specifically as follows: digestion of the contamination at 37 ℃ for 2 min, pre-denaturation at 95 ℃ for 5 min, denaturation at 95 ℃ for 10 s, annealing at 60 ℃ for 30s, and 45 cycles of denaturation and annealing.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113249502A (en) * | 2021-05-08 | 2021-08-13 | 上海康黎诊断技术有限公司 | Related gene, method, primer group and kit for mycobacterium tuberculosis complex flora identification and drug resistance detection |
| CN114657271A (en) * | 2022-03-15 | 2022-06-24 | 中国动物卫生与流行病学中心 | Method for rapidly detecting bovine tuberculosis |
| CN114813671A (en) * | 2022-04-06 | 2022-07-29 | 四川大学华西医院 | Analysis method for selectively regulating and controlling QDs and calcein fluorescence signals based on metal ions and nanoparticles thereof and application |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100304371A1 (en) * | 2009-05-29 | 2010-12-02 | Ana Maria Zarraga | Compositions and methods for detecting mycobacteria |
| CN102409102A (en) * | 2011-11-30 | 2012-04-11 | 中国农业大学 | PCR(Polymerase Chain Reaction) primers and method for identifying mycobacterium bovis |
| CN103882130A (en) * | 2014-03-24 | 2014-06-25 | 中国科学院微生物研究所 | Primer pair for detecting mycobacterium tuberculosis complex group and application of primer pair |
| CN107002148A (en) * | 2014-10-10 | 2017-08-01 | 新泽西鲁特格斯州立大学 | The chain reaction primer of polymerase and probe of mycobacterium tuberculosis |
| CN109680084A (en) * | 2019-01-23 | 2019-04-26 | 北京岱美仪器有限公司 | A kind of primed probe and method for the compound group of Fluorescence quantitative PCR detection mycobacterium tuberculosis |
| CN111269996A (en) * | 2020-02-10 | 2020-06-12 | 四川大学华西医院 | Mycobacterium tuberculosis complex rapid detection kit and preparation method thereof |
| CN112725475A (en) * | 2021-01-05 | 2021-04-30 | 四川大学华西医院 | Mycobacterium tuberculosis detection primer, probe composition, kit and application |
-
2021
- 2021-02-03 CN CN202110149207.5A patent/CN112725486A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100304371A1 (en) * | 2009-05-29 | 2010-12-02 | Ana Maria Zarraga | Compositions and methods for detecting mycobacteria |
| CN102409102A (en) * | 2011-11-30 | 2012-04-11 | 中国农业大学 | PCR(Polymerase Chain Reaction) primers and method for identifying mycobacterium bovis |
| CN103882130A (en) * | 2014-03-24 | 2014-06-25 | 中国科学院微生物研究所 | Primer pair for detecting mycobacterium tuberculosis complex group and application of primer pair |
| CN107002148A (en) * | 2014-10-10 | 2017-08-01 | 新泽西鲁特格斯州立大学 | The chain reaction primer of polymerase and probe of mycobacterium tuberculosis |
| CN109680084A (en) * | 2019-01-23 | 2019-04-26 | 北京岱美仪器有限公司 | A kind of primed probe and method for the compound group of Fluorescence quantitative PCR detection mycobacterium tuberculosis |
| CN111269996A (en) * | 2020-02-10 | 2020-06-12 | 四川大学华西医院 | Mycobacterium tuberculosis complex rapid detection kit and preparation method thereof |
| CN112725475A (en) * | 2021-01-05 | 2021-04-30 | 四川大学华西医院 | Mycobacterium tuberculosis detection primer, probe composition, kit and application |
Non-Patent Citations (6)
| Title |
|---|
| LINGNA LYU: "Evaluation of digital PCR assay in detection", 《BMC INFECTIOUS DISEASES》 * |
| NIYAZ AHMED: "PCR-Based Rapid Detection of Mycobacterium tuberculosis", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
| 中华人民共和国国家质量监督检疫总局 中国国家标准化管理委员会: "GB_T 27639-2011 结核病病原菌实时荧光PCR检测方法", 《中华人民共和国国家标准》 * |
| 李国利: "应用双重PCR技术快速鉴定结核与非结核分支杆菌", 《中古现代医学杂志》 * |
| 李自慧: "结核分枝杆菌IS6110 和IS1081 微滴数字PCR", 《北京医学》 * |
| 贾广乐: "牛结核TaqMan荧光定量PCR方法的建立", 《动物检疫学分会2010年年会论文集》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113249502A (en) * | 2021-05-08 | 2021-08-13 | 上海康黎诊断技术有限公司 | Related gene, method, primer group and kit for mycobacterium tuberculosis complex flora identification and drug resistance detection |
| CN114657271A (en) * | 2022-03-15 | 2022-06-24 | 中国动物卫生与流行病学中心 | Method for rapidly detecting bovine tuberculosis |
| CN114813671A (en) * | 2022-04-06 | 2022-07-29 | 四川大学华西医院 | Analysis method for selectively regulating and controlling QDs and calcein fluorescence signals based on metal ions and nanoparticles thereof and application |
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