CN105907861A - Method and primer for quick detection and classification of mycobacteria - Google Patents

Abstract

The invention belongs to the field of microbial molecular detection, in particular to a primer and a method for rapid detection and typing of mycobacteria. The primers for rapid detection and typing of mycobacteria provided by the invention include 7 pairs of specific primers for mycobacteria. The primers provided by the present invention for the rapid detection and typing of mycobacteria can not only distinguish tuberculosis mycobacteria and non-tuberculous mycobacteria, but also can further distinguish each member of the tuberculosis mycobacterium complex, with specificity With the advantages of high precision and high accuracy, it can effectively assist the clinical diagnosis of tuberculosis and guide the clinical development of early and effective chemotherapy.

Description

A primer and method for rapid detection and typing of mycobacteria

technical field

The invention belongs to the field of microbial molecular detection, in particular to a primer and a method for rapid detection and typing of mycobacteria.

Background technique

At present, tuberculosis is still a serious public health problem, and it is on the rise all over the world. Mycobacterium tuberculosis complex (MTC) is the main pathogenic bacteria causing human tuberculosis. MTC includes Mycobacterium tuberculosis (M.Tuberculosis), Mycobacterium Africa (Type I and Type II) (M.Africanum Type I and Type II), Mycobacterium bovis (M.Bovis), BCG (M.bovis BCG) , Mycobacterium microti (M. Microti), Mycobacterium caprae (M. Caprae) and Mycobacterium Kennedy (M. Canettii).

In the detection of mycobacteria, it is difficult to distinguish non-tuberculous mycobacteria (MOTT) from mycobacterium tuberculosis, but there are great differences in treatment between mycobacterium tuberculosis and non-tuberculosis mycobacteria. Medication can easily lead to the emergence of drug-resistant strains and aggravate the condition. Therefore, it is important to effectively treat or control tuberculosis to establish a rapid and accurate method for detecting tuberculosis and non-tuberculosis mycobacteria strains clinically, and to further distinguish and identify the members of the Mycobacterium tuberculosis complex. in advance.

At present, the methods for detecting mycobacteria mainly include: restriction fragment length polymorphism analysis (IS6110-RFLP), spacer oligonucleotide typing technique (Spoligotyping), variable number of tandem repeats (MIRU), and variable number of tandem repeats (MIRU). Number of tandem repeats typing method (VNTR). IS6110-RFLP has high resolution and good reproducibility, but its discrimination ability is low for strains carrying 1 or less copies of IS6110; it is also found in some non-tuberculous mycobacteria strains There is a copy of IS6110. Spoligotyping does not require a large amount of DNA, and can further type mycobacteria to the subspecies level, but its resolution is low, the cost is high, and the detection efficiency is still far behind expectations. MIRU does not require a large amount of high-quality DNA, and the test results can be digitally coded to facilitate the comparison of results between laboratories. The repeatability, sensitivity, and specificity of VNTR typing are 100%, but they are different from MIRU and VNTR. The polymorphism of points determines the resolution of the method, limiting the use of the above two methods.

In order to solve the above problems, more and more detection methods for mycobacteria have emerged as the times require. Chinese patent CN102304575B discloses a method for rapid identification of Mycobacterium tuberculosis and non-tuberculous mycobacteria, and specifically relates to a loop-mediated isothermal gene amplification technology (LAMP technology). The specificity of Mycobacterium tuberculosis 16SRNA Design 4 specific primers, perform PCR amplification, and judge the result by observing the color change with SYBRGreenI. The identification method has the advantages of convenience, high sensitivity, high specificity, etc., has high reference value for the diagnosis and clinical application of mycobacterium tuberculosis or non-tuberculosis mycobacterium infection, and is suitable for popularization and application. However, this identification method can only distinguish tuberculosis mycobacteria from non-tuberculosis mycobacteria, and cannot further type mycobacterium tuberculosis, which is not conducive to the promotion and application of this identification method.

Contents of the invention

In order to solve the defects of narrow detection range and low specificity in the mycobacteria detection method in the prior art, the purpose of the present invention is to provide a kind of primer and method for rapid detection and typing of mycobacteria to solve the above-mentioned problems. question.

The present invention provides a primer for rapid detection and typing of mycobacteria, including the following 7 pairs of mycobacterium-specific primers:

MycoF1: 5'-GGCAAGGTCACCCCCGAAGGG-3' (SEQ ID NO.1),

MycoR1: 5'-AGCGGCTGCTGGGTGATCATC-3' (SEQ ID NO.2);

MycoF2: 5'-TCGCGTGATCCTTCGAAACG-3' (SEQ ID NO.3),

MycoR2: 5'-GCCGTTGCGCTGATTGGACC-3' (SEQ ID NO.4);

MycoF3: 5'-GACCTGACGCCGCTGACAC-3' (SEQ ID NO.5),

MycoR3: 5'-CACCTACACCGCTTCCTGCC-3' (SEQ ID NO.6);

MycoF4: 5'-GACATGTACGAGAGACGGCATGAG-3' (SEQ ID NO.7),

MycoR4: 5'-AATCCAACACGCAGCAACCAG-3' (SEQ ID NO.8);

MycoF5: 5'-AGGGATTCGGCGTTCGGGATTCCTG-3' (SEQ ID NO.9),

MycoR5: 5'-GATCGCGATCGCCAACGATTCAGTGTATG-3' (SEQ ID NO.10);

MycoF6: 5'-GGGTCTGACGGCCAAACTC-3' (SEQ ID NO.11),

MycoR6: 5'-CTCGGTGGCCGGTTTTTC-3' (SEQ ID NO.12);

MycoF7: 5'-CGATAGACCATGAGTCCGTCTC-3' (SEQ ID NO.13),

MycoR7: 5'-GTGGGCGGATGCCAGAATAG-3' (SEQ ID NO. 14).

Further, the concentration ratio of the upstream primer to the downstream primer of the mycobacterium-specific primer is 1:1.

In addition, the present invention also provides a method for rapid detection and typing of mycobacteria, comprising the following steps:

S1 Use the bacterial genomic DNA extraction solution to extract the bacterial genomic DNA in the specimen to be tested;

S2 uses the bacterial genome DNA in the sample to be tested as a template, and uses 7 pairs of mycobacterium-specific primers according to claim 1 to carry out PCR amplification respectively;

S3 The obtained PCR product is subjected to agarose gel electrophoresis.

Further, the formula of bacterial genomic DNA extract in the step S1 is: Chelex 100 resin 5%; Tris-HCl 10mM, pH8.3; EDTA disodium 0.1mM; sodium azide 0.1%; proteinase K 100 μg/ml.

Further, the PCR amplification reaction conditions in the step S2 include: stage 1: 95°C for 5 minutes; stage 2: 95°C for 1 minute; stage 3: 58°C for 1 minute; stage 4: 72°C for 1 minute; 32 cycles; stage 5 : 72°C for 5min; store at 16°C.

Further, in the step S2, the sequence amplified by the mycobacterium-specific primer 1 is 761bp, the sequence amplified by the mycobacterium-specific primer 2 is 219bp, and the sequence amplified by the mycobacterium-specific primer 3 is 531bp. The sequence amplified by the specific primer 4 is 1031bp, the sequence amplified by the mycobacterium-specific primer 5 is 666bp, the sequence amplified by the mycobacterium-specific primer 6 is 991bp, and the sequence amplified by the mycobacterium-specific primer 7 is 392bp.

Wherein, the amplified sequence of the primer pair 1 is 761bp, and the specific sequence is as follows:

ggcaaggtcaccccgaagggtgagaccgagctgacgccggaggagcggctgctgcgtgccatcttcggtgagaaggcccgcgaggtgcgcgacacttcgctgaaggtgccgcacggcgaatccggcaaggtgatcggcattcgggtgttttcccgcgaggacgaggacgagttgccggccggtgtcaacgagctggtgcgtgtgtatgtggctcagaaacgcaagatctccgacggtgacaagctggccggccggcacggcaacaagggcgtgatcggcaagatcctgccggttgaggacatgccgttccttgccgacggcaccccggtggacattattttgaacacccacggcgtgccgcgacggatgaacatcggccagattttggagacccacctgggttggtgtgcccacagcggctggaaggtcgacgccgccaagggggttccggactgggccgccaggctgcccgacgaactgctcgaggcgcagccgaacgccattgtgtcgacgccggtgttcgacggcgcccaggaggccgagctgcagggcctgttgtcgtgcacgctgcccaaccgcgacggtgacgtgctggtcgacgccgacggcaaggccatgctcttcgacgggcgcagcggcgagccgttcccgtacccggtcacggttggctacatgtacatcatgaagctgcaccacctggtggacgacaagatccacgcccgctccaccgggccgtactcgatgatcacccagcagccgct；

The amplified sequence of the primer pair 2 is 219bp, and the specific sequence is as follows:

cgctcatcgctgcgttcgcggtagcccgccccgcacagggcgtcggcttcagcccccatcaaggcggcgatgaacgtcgagagcagcccgcgcagcagatccgggctcgcctgtgcgagttggtcagccagaagctgctcggtgtcgataagatgagaagaggtcattgcgtcatttccttcgattgacttttgctggtcgtttcgaaggatcacgcga；

The amplified sequence of the primer pair 3 is 531bp, and the specific sequence is as follows:

gacctgacgccgctgacactgccgacgcccgcgcagttggcgttacgtcgatcggtgatccgacgtgccggcgcagtgattgacgcgaaccggtcctggcgtcggttgatgtcgttggcgcggtaggcgttccccgatgtgtggaccgcgttcgccgggtcgttaccgaccgcgacagcggtgctggggcgttggcccgacatccgcttgctggccggcgcaccgacccgccacgttgaccgccgtcatcgccgcgcacacccgcggtgtcgccgacaccccggcccggccgaggccatcaagaccgccgcaaccggctgggccgcgttctgggacgggcacctcgacctggacgcactggccgtcgatgtcaccgagcatctcagcgacctcaccgacgaccgatgcgcgcgttggtgatgccggtgaccaagaaggtgttgatcttgggtgactagtcaatggtggtggccagggtgagcagttcggggatctgcgagtcgatgcgccaggcaggaagcggtgtaggtg；

The amplified sequence of the primer pair 4 is 1031bp, and the specific sequence is as follows:

gacatgtacgagagacggcatgagcgcggaatgtgcgaccgtgccgtcgagatgaccgacgtcggcgctacggcagcccccaccggacctatcgcgcggggcagcgtcgctcgggtcggcgcggcgaccgcgttggccgttgcctgcgtctacacggtcatctatctggcggcccgcgacctacccccggcttgtttttcgatattcgcggtgttttggggggcgctcggcattgccaccggcgccacccacggcctcctgcaagaaacgacccgcgaggtccgctgggtgcgctccacccaaatagttgcgggccatcgtacccatccgctgcgggtggccgggatgattggcaccgtcgcggccgtcgtaattgcgggtagctcaccgctgtggagccgacagctattcgtcgaggggcgctggctgtccgtggggctactcagcgttggggtggccgggttctgcgcgcaggcgaccctgctgggcgcgctggccggcgtcgaccggtggacacagtacgggtcactgatggtgaccgacgcggtcatccggttggcggtcgccgcggcagcggttgtgatcggatggggtctggccgggtacttgtgggccgccaccgcgggagcggtggcgtggctgctcatgctgatggcctcgcccaccgcgcgcagcgcggccagcctgctgacgcccgggggaatcgccacgttcgtgcgcggtgccgctcattcgataaccgccgcgggtgccagcgcgattctggtaatgggtttcccagtgttgctcaaagtgacctccgaccagttaggggcaaagggcggagcggtcatcctggctgtgaccttgacgcgtgcgccgcttctggtcccactgagcgcgatgcaaggcaacctgatcgcgcatttcgtcgaccggcgcacccaacggcttcgggcgctgatcgcaccggcgctggtcgtcggcggcatcggtgcggtcgggatgttggccgcagggc ttaccggtccctggttgctgcgtgttggatt;

The amplified sequence of the primer pair 5 is 666bp, and the specific sequence is as follows:

agggattcggcgttcgggattcctgaagtcaagcggggtctggttgccggcggcgggggattgctgcggttgccggagcgcatcccgtatgcgatagccatggagttggcgctgaccggtgacaacctaccggccgaacgcgcgcacgagctggggctcgtcaacgttttggccgagccggggaccgccctcgatgctgcgatcgcgttggcggagaagatcaccgccaatgggccgctggcggtggtggccaccaagcggattatcaccgagtcgcgtgggtggagtcccgacactatgttcgctgagcagatgaagatcctggtgccggtgttcacctccaacgacgcgaaggaaggtgcgatcgcgttcgccgagaggcgccggccccgttggacgggcacctagcccagctacgcgacggtgtagcccatcggcagcaggacactcttttgctgggtgaagtgttcgacaccctcgggcccgttctcgcggccgattccggagttcttgtagccgccgaagggtgagccgggatcgaaggcgtaccagttgattccgtatgtcccggtgcggatctgctgcgagatcttgatgcctttgggcacgtcggtggtccacacgctgcccgccagcccatacactgaatcgttggcgatcgcgatc；

The amplified sequence of the primer pair 6 is 991bp, and the specific sequence is as follows:

gggtctgacggccaaactcatcatctggtacccgcactatgcctggctgttgttgagcgtctacctcacggtagccctggttgcgctcgtggtggtcgggtcgatggctcacgtccggcgcgtttcaccggtcgtaaaacgaactctggaattgatcgacggcgccatgatcgctgccatcattcccatgctgctgtggatcaccggggtgtacgacacggtccgcaatatccggttctgagccggatcggctgattggcggttcctgacagaacatcgaggacacggcgcaggtttgcataccttcggcgcccgacaaattgctgcgattgagcgtgtggcgcgtccggtaaaatttgctcgatggggaacacgtataggagatccggcaatggctgaaccgttggccgtcgatcccaccggcttgagcgcagcggccgcgaaattggccggcctcgtttttccgcagcctccggcgccgatcgcggtcagcggaacggattcggtggtagcagcaatcaacgagaccatgccaagcatcgaatcgctggtcagtgacgggctgcccggcgtgaaagccgccctgactcgaacagcatccaacatgaacgcggcggcggacgtctatgcgaagaccgatcagtcactgggaaccagtttgagccagtatgcattcggctcgtcgggcgaaggcctggctggcgtcgcctcggtcggtggtcagccaagtcaggctacccagctgctgagcacacccgtgtcacaggtcacgacccagctcggcgagacggccgctgagctggcaccccgtgttgttgcgacggtgccgcaactcgttcagctggctccgcacgccgttcagatgtcgcaaaacgcatcccccatcgctcagacgatcagtcaaaccgcccaacaggccgcccagagcgcgcagggcggcagcggcccaatgcccgcacagcttgccagcgctgaaaaaccggccaccgag；

The amplified sequence of the primer pair 7 is 392bp, and the specific sequence is as follows:

cgatagaccatgagtccgtctccatcggccctgctcgccgaccacccggaccgcattcgttggaacgcgaaatacgagtgcgctgaccccacggaggcggtatttgcgcccatatcctggctcggcgacgtgctgcagttcggggtgccagaagggccggttctggaactggcgtgcggtcggtccggcaccgcgctggggctagccgcggcgggccgctgcgtgactgcgatcgacgtttccgataccgcgttggttcagctcgagctcgaagcgacccgacgggaattggccgatcgcctcacactggtgcacgccgatctctgctcctggcagtcgggggatggacgctttgctctggtactttgccgactattctggcatccgcccac。

Further, the mycobacterium is Mycobacterium tuberculosis or Mycobacterium africanum type II, Mycobacterium Kennedy, Mycobacterium africanum type I, Mycobacterium capricum, Mycobacterium bovis, BCG, Mycobacterium microti and nontuberculous mycobacteria.

In addition, the present invention also provides the application of the primers for rapid detection and typing of mycobacteria in preparing a kit for detecting mycobacteria.

The method for rapid detection and typing of mycobacteria provided by the present invention: utilize different types of mycobacterium strains to contain different specific genes, or the same gene, and the fragments of these same genes are different among different bacterial species Variability, designed 7 pairs of specific primers for different types of strains. The 7 pairs of specific primers were used to perform PCR amplification on the strain to be tested respectively, and the presence or absence and size of the corresponding PCR amplification products obtained by each primer were detected, and finally the strain type of the sample to be tested was determined.

The method for determining the result of the mycobacterium rapid detection and typing method provided by the present invention is: analyze the agarose gel electrophoresis result of the PCR product:

A) If the 7 pairs of primers in the sample are positive amplification results, and the PCR amplification product of primer 1 has a fragment size of 761bp, the PCR amplification product of primer 2 has a fragment size of 219bp, and the PCR amplification product of primer 3 has a fragment size of 219bp. A fragment with a size of 531bp appeared, the PCR amplification product of primer 4 appeared a fragment with a size of 1031bp, the PCR amplification product of primer 5 appeared a fragment with a size of 666bp, and the PCR amplification product of primer 6 appeared a fragment with a size of 991bp. If the PCR amplification product of 7 shows a fragment with a size of 392bp, it indicates that Mycobacterium tuberculosis (M.Tuberculosis) or African mycobacterium type II (M.africanum subtype II) in the Mycobacterium tuberculosis complex has been detected;

B) If the results of PCR amplification products of 7 pairs of primers in the specimen are: primers 1-6 are positive amplification results, and the PCR amplification product of primer 1 has a fragment size of 761bp, and the PCR amplification product of primer 2 has a size of 219bp The PCR amplification product of primer 3 has a fragment of 531bp in size, the PCR amplification product of primer 4 has a fragment of 1031bp in size, the PCR amplification product of primer 5 has a fragment of 666bp in size, and the PCR amplification product of primer 6 has a fragment of 666bp in size. The amplification product has a fragment size of 991bp; and the PCR amplification product of primer 7 does not contain a fragment size of 392bp, indicating that the Mycobacterium Kennedy (M.Canettii) in the Mycobacterium tuberculosis complex has been detected;

C) If the results of 7 pairs of PCR amplification products in the sample are: primers 1-4 and primers 6 and 7 are positive amplification results, and the PCR amplification product of primer 1 has a fragment size of 761bp, the PCR amplification of primer 2 The product has a fragment size of 219bp, the PCR amplification product of primer 3 has a fragment size of 531bp, the PCR amplification product of primer 4 has a fragment size of 1031bp, and the PCR amplification product of primer 6 has a fragment size of 991bp, The PCR amplification product of primer 7 has a fragment of 392bp in size; and the PCR amplification product of primer 5 does not contain a fragment of 666bp in size, which indicates that Mycobacterium africa type I in the Mycobacterium tuberculosis complex ( M. africanum subtype I);

D) If the results of 7 pairs of PCR amplification products in the specimen are: primers 1-4 and primer 6 are positive amplification results, and the PCR amplification product of primer 1 has a fragment size of 761bp, the PCR amplification product of primer 2 A fragment with a size of 219bp appeared, the PCR amplification product of primer 3 had a fragment of 531bp in size, the PCR amplification product of primer 4 had a fragment of 1031bp in size, and the PCR amplification product of primer 6 had a fragment of 991bp in size; and The PCR amplification product of primer 5 does not contain a fragment with a size of 666bp, and the PCR amplification product of primer 7 does not contain a fragment with a size of 392bp, indicating that Mycobacterium capricatus (M. Caprae);

E) If the results of 7 pairs of PCR amplification products in the specimen are: primers 1-3 and primer 6 are positive amplification results, and the PCR amplification product of primer 1 has a fragment size of 761bp, the PCR amplification product of primer 2 A fragment with a size of 219bp appeared, the PCR amplification product of primer 3 had a fragment with a size of 531bp, and the PCR amplification product with primer 6 had a fragment with a size of 991bp; while the PCR amplification product of primer 4 did not contain a fragment with a size of 1031bp , the PCR amplification product of primer 5 does not contain the fragment that size is 666bp, and the PCR amplification product of primer 7 does not contain the fragment that size is 392bp, then explanation has detected the Mycobacterium bovis in the Mycobacterium tuberculosis complex ( M. Bovis);

F) If the results of 7 pairs of PCR amplification products in the specimen are: primers 1-3 are positive amplification results, and the PCR amplification product of primer 1 has a fragment size of 761bp, and the PCR amplification product of primer 2 has a size of For a fragment of 219bp, the PCR amplification product of primer 3 showed a fragment of 531bp in size; while the PCR amplification product of primer 4 did not contain a fragment of 1031bp in size, and the PCR amplification product of primer 5 did not contain a fragment of 666bp in size. The PCR amplification product of 6 does not contain a fragment with a size of 991bp, and the PCR amplification product of primer 7 does not contain a fragment with a size of 392bp, which means that BCG (M.bovis BCG) in the Mycobacterium tuberculosis complex has been detected;

G) If the results of 7 pairs of PCR amplification products in the specimen are: primer 1, primer 2, primer 4, primer 6 and primer 7 are positive amplification results, that is, the PCR amplification product of primer 1 has a fragment size of 761bp, The PCR amplification product of primer 2 has a fragment size of 219bp, the PCR amplification product of primer 4 has a fragment size of 1031bp, the PCR amplification product of primer 6 has a fragment size of 991bp, and the PCR amplification product of primer 7 has a A fragment with a size of 392bp; and the PCR amplification product of primer 3 does not contain a fragment with a size of 531bp, and the PCR amplification product of primer 5 does not contain a fragment with a size of 666bp, indicating that the Mycobacterium tuberculosis complex has been detected Mycobacterium microti (M. Microti);

H) If the results of 7 pairs of PCR amplification products in the specimen are: only primer 1 is a positive amplification result, and the PCR amplification product of primer 1 has a fragment size of 761bp; while the PCR amplification product of primer 2 does not contain a fragment of size The PCR amplification product of primer 3 does not contain a fragment of 531bp in size, the PCR amplification product of primer 4 does not contain a fragment of 1031bp in size, and the PCR amplification product of primer 5 does not contain a fragment of 666bp in size. The PCR amplified product of primer 6 does not contain a fragment with a size of 991 bp, and the PCR amplified product of primer 7 does not contain a fragment with a size of 392 bp, indicating that non-tuberculous mycobacteria (M.Fortuitum) have been detected.

The primers provided by the invention for rapid detection and typing of mycobacteria can not only distinguish tuberculosis mycobacteria and non-tuberculosis mycobacteria, but also can further distinguish each member bacteria in the tuberculosis mycobacterium complex. Using the 7 pairs of primers provided by the present invention under the same amplification reaction conditions, the PCR reactions of 7 different primer pairs can be completed at one time, and the results of gel electrophoresis can be used to quickly identify tuberculosis and non-tuberculous mycobacteria, as well as tuberculosis mycobacteria The members of the Bacillus complex have the advantages of simple and quick operation, good specificity and high accuracy, and can effectively assist the clinical diagnosis of tuberculosis and guide the clinical development of early and effective chemotherapy.

Compared with the prior art, the beneficial effects of the present invention are: the primers provided by the present invention for the rapid detection and typing of mycobacteria can distinguish tuberculosis mycobacteria and non-tuberculous mycobacteria, and can further distinguish Each member of the Mycobacterium tuberculosis complex has the advantages of good specificity and high accuracy, which greatly improves the detection efficiency. In addition, the present invention also provides a method for rapid detection and typing of mycobacteria. By using the specific primers provided by the present invention, the operation is simple and fast, and the specificity is good, which can effectively assist the clinical diagnosis of tuberculosis and guide clinical Carrying out early and effective chemotherapy is an ideal method for rapid detection and typing of mycobacteria.

Description of drawings

Fig. 1 is a graph showing the amplification results of primer pairs 1-7 to Mycobacterium tuberculosis (M.Tuberculosis) or African mycobacterium type II (M.africanum subtype II);

Fig. 2 is the amplification result figure of primer pair 1-7 to Mycobacterium Kennedy (M.Canettii);

Fig. 3 is a graph showing the amplification results of primer pairs 1-7 to African mycobacterium type I (M.africanum subtype I);

Fig. 4 is the amplification result figure of primer pair 1-7 to Mycobacterium capricatus (M.Caprae);

Fig. 5 is the amplification result figure of primer pair 1-7 to Mycobacterium bovis (M.Bovis);

Figure 6 is a diagram showing the amplification results of primer pairs 1-7 to BCG (M.bovis BCG);

Fig. 7 is a graph showing the amplification results of primer pairs 1-7 to Mycobacterium microti (M.Microti);

Fig. 8 is a diagram showing the amplification results of primer pairs 1-7 for non-tuberculous mycobacteria (M.Fortuitum).

detailed description

The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be assumed that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, some simple deduction or replacement can be made, which should be regarded as belonging to the protection scope of the present invention. Among them, Chelex 100 resin was purchased from bio-rad company (article number: 1432832).

Embodiment 1, primer

The invention provides a primer for rapid detection and typing of mycobacteria.

Table 1 Primers provided by the present invention

Embodiment 2, the pairing of primer and its amplified fragment size

The sequence amplified by mycobacterium-specific primer 1 provided by the invention is 761bp, the sequence amplified by mycobacterium-specific primer 2 is 219bp, the sequence amplified by mycobacterium-specific primer 3 is 531bp, and the sequence amplified by mycobacterium-specific primer 4 is 531bp. The sequence amplified by the mycobacterium-specific primer 5 is 1031bp, the sequence amplified by the mycobacterium-specific primer 5 is 666bp, the sequence amplified by the mycobacterium-specific primer 6 is 991bp, and the sequence amplified by the mycobacterium-specific primer 7 is 392bp.

Table 2 The pairing of primers and the size of their amplified fragments

Wherein, the amplified sequence of the primer pair 1 is 761bp, and the specific sequence is as follows:

ggcaaggtcaccccgaagggtgagaccgagctgacgccggaggagcggctgctgcgtgccatcttcggtgagaaggcccgcgaggtgcgcgacacttcgctgaaggtgccgcacggcgaatccggcaaggtgatcggcattcgggtgttttcccgcgaggacgaggacgagttgccggccggtgtcaacgagctggtgcgtgtgtatgtggctcagaaacgcaagatctccgacggtgacaagctggccggccggcacggcaacaagggcgtgatcggcaagatcctgccggttgaggacatgccgttccttgccgacggcaccccggtggacattattttgaacacccacggcgtgccgcgacggatgaacatcggccagattttggagacccacctgggttggtgtgcccacagcggctggaaggtcgacgccgccaagggggttccggactgggccgccaggctgcccgacgaactgctcgaggcgcagccgaacgccattgtgtcgacgccggtgttcgacggcgcccaggaggccgagctgcagggcctgttgtcgtgcacgctgcccaaccgcgacggtgacgtgctggtcgacgccgacggcaaggccatgctcttcgacgggcgcagcggcgagccgttcccgtacccggtcacggttggctacatgtacatcatgaagctgcaccacctggtggacgacaagatccacgcccgctccaccgggccgtactcgatgatcacccagcagccgct；

The amplified sequence of the primer pair 2 is 219bp, and the specific sequence is as follows:

cgctcatcgctgcgttcgcggtagcccgccccgcacagggcgtcggcttcagcccccatcaaggcggcgatgaacgtcgagagcagcccgcgcagcagatccgggctcgcctgtgcgagttggtcagccagaagctgctcggtgtcgataagatgagaagaggtcattgcgtcatttccttcgattgacttttgctggtcgtttcgaaggatcacgcga；

The amplified sequence of the primer pair 3 is 531bp, and the specific sequence is as follows:

gacctgacgccgctgacactgccgacgcccgcgcagttggcgttacgtcgatcggtgatccgacgtgccggcgcagtgattgacgcgaaccggtcctggcgtcggttgatgtcgttggcgcggtaggcgttccccgatgtgtggaccgcgttcgccgggtcgttaccgaccgcgacagcggtgctggggcgttggcccgacatccgcttgctggccggcgcaccgacccgccacgttgaccgccgtcatcgccgcgcacacccgcggtgtcgccgacaccccggcccggccgaggccatcaagaccgccgcaaccggctgggccgcgttctgggacgggcacctcgacctggacgcactggccgtcgatgtcaccgagcatctcagcgacctcaccgacgaccgatgcgcgcgttggtgatgccggtgaccaagaaggtgttgatcttgggtgactagtcaatggtggtggccagggtgagcagttcggggatctgcgagtcgatgcgccaggcaggaagcggtgtaggtg；

The amplified sequence of the primer pair 4 is 1031bp, and the specific sequence is as follows:

gacatgtacgagagacggcatgagcgcggaatgtgcgaccgtgccgtcgagatgaccgacgtcggcgctacggcagcccccaccggacctatcgcgcggggcagcgtcgctcgggtcggcgcggcgaccgcgttggccgttgcctgcgtctacacggtcatctatctggcggcccgcgacctacccccggcttgtttttcgatattcgcggtgttttggggggcgctcggcattgccaccggcgccacccacggcctcctgcaagaaacgacccgcgaggtccgctgggtgcgctccacccaaatagttgcgggccatcgtacccatccgctgcgggtggccgggatgattggcaccgtcgcggccgtcgtaattgcgggtagctcaccgctgtggagccgacagctattcgtcgaggggcgctggctgtccgtggggctactcagcgttggggtggccgggttctgcgcgcaggcgaccctgctgggcgcgctggccggcgtcgaccggtggacacagtacgggtcactgatggtgaccgacgcggtcatccggttggcggtcgccgcggcagcggttgtgatcggatggggtctggccgggtacttgtgggccgccaccgcgggagcggtggcgtggctgctcatgctgatggcctcgcccaccgcgcgcagcgcggccagcctgctgacgcccgggggaatcgccacgttcgtgcgcggtgccgctcattcgataaccgccgcgggtgccagcgcgattctggtaatgggtttcccagtgttgctcaaagtgacctccgaccagttaggggcaaagggcggagcggtcatcctggctgtgaccttgacgcgtgcgccgcttctggtcccactgagcgcgatgcaaggcaacctgatcgcgcatttcgtcgaccggcgcacccaacggcttcgggcgctgatcgcaccggcgctggtcgtcggcggcatcggtgcggtcgggatgttggccgcagggc ttaccggtccctggttgctgcgtgttggatt;

The amplified sequence of the primer pair 5 is 666bp, and the specific sequence is as follows:

agggattcggcgttcgggattcctgaagtcaagcggggtctggttgccggcggcgggggattgctgcggttgccggagcgcatcccgtatgcgatagccatggagttggcgctgaccggtgacaacctaccggccgaacgcgcgcacgagctggggctcgtcaacgttttggccgagccggggaccgccctcgatgctgcgatcgcgttggcggagaagatcaccgccaatgggccgctggcggtggtggccaccaagcggattatcaccgagtcgcgtgggtggagtcccgacactatgttcgctgagcagatgaagatcctggtgccggtgttcacctccaacgacgcgaaggaaggtgcgatcgcgttcgccgagaggcgccggccccgttggacgggcacctagcccagctacgcgacggtgtagcccatcggcagcaggacactcttttgctgggtgaagtgttcgacaccctcgggcccgttctcgcggccgattccggagttcttgtagccgccgaagggtgagccgggatcgaaggcgtaccagttgattccgtatgtcccggtgcggatctgctgcgagatcttgatgcctttgggcacgtcggtggtccacacgctgcccgccagcccatacactgaatcgttggcgatcgcgatc；

The amplified sequence of the primer pair 6 is 991bp, and the specific sequence is as follows:

gggtctgacggccaaactcatcatctggtacccgcactatgcctggctgttgttgagcgtctacctcacggtagccctggttgcgctcgtggtggtcgggtcgatggctcacgtccggcgcgtttcaccggtcgtaaaacgaactctggaattgatcgacggcgccatgatcgctgccatcattcccatgctgctgtggatcaccggggtgtacgacacggtccgcaatatccggttctgagccggatcggctgattggcggttcctgacagaacatcgaggacacggcgcaggtttgcataccttcggcgcccgacaaattgctgcgattgagcgtgtggcgcgtccggtaaaatttgctcgatggggaacacgtataggagatccggcaatggctgaaccgttggccgtcgatcccaccggcttgagcgcagcggccgcgaaattggccggcctcgtttttccgcagcctccggcgccgatcgcggtcagcggaacggattcggtggtagcagcaatcaacgagaccatgccaagcatcgaatcgctggtcagtgacgggctgcccggcgtgaaagccgccctgactcgaacagcatccaacatgaacgcggcggcggacgtctatgcgaagaccgatcagtcactgggaaccagtttgagccagtatgcattcggctcgtcgggcgaaggcctggctggcgtcgcctcggtcggtggtcagccaagtcaggctacccagctgctgagcacacccgtgtcacaggtcacgacccagctcggcgagacggccgctgagctggcaccccgtgttgttgcgacggtgccgcaactcgttcagctggctccgcacgccgttcagatgtcgcaaaacgcatcccccatcgctcagacgatcagtcaaaccgcccaacaggccgcccagagcgcgcagggcggcagcggcccaatgcccgcacagcttgccagcgctgaaaaaccggccaccgag；

The amplified sequence of the primer pair 7 is 392bp, and the specific sequence is as follows:

cgatagaccatgagtccgtctccatcggccctgctcgccgaccacccggaccgcattcgttggaacgcgaaatacgagtgcgctgaccccacggaggcggtatttgcgcccatatcctggctcggcgacgtgctgcagttcggggtgccagaagggccggttctggaactggcgtgcggtcggtccggcaccgcgctggggctagccgcggcgggccgctgcgtgactgcgatcgacgtttccgataccgcgttggttcagctcgagctcgaagcgacccgacgggaattggccgatcgcctcacactggtgcacgccgatctctgctcctggcagtcgggggatggacgctttgctctggtactttgccgactattctggcatccgcccac。

Embodiment 3, a kind of method for rapid detection and typing of mycobacteria

1) Apply the bacterial genomic DNA extraction solution to extract the bacterial genomic DNA in the specimen to be tested: select the specimen for pretreatment, and then use the bacterial genomic DNA extraction solution to extract the bacterial genomic DNA, wherein the composition of the DNA extraction solution is shown in Table 3 :

Table 3 Components of DNA extraction solution

2) taking the bacterial genome DNA in the sample to be extracted as a template, adopting 7 pairs of primers described in Table 1 in Example 1 to carry out PCR amplification respectively, and the components of the PCR reaction mixture are as shown in Table 4:

Table 4 PCR reaction mixture components

PCR reaction mixture Volume(μl) 2×Taq PCR Master Mix 15 Primer Forward (10μM) 0.5 Primer Reverse (10μM) 0.5 dna 0.5 ddH ₂ O 8.5 Total 25

The reaction conditions are: stage 1: 95°C for 5 min; stage 2: 95°C for 1 min; stage 3: 58°C for 1 min; stage 4: 72°C for 1 min; 32 cycles; stage 5: 72°C for 5 min; storage at 16°C.

3) The obtained PCR product is subjected to agarose gel electrophoresis.

4) The gel imaging results are shown in Table 5.

Table 5 PCR agarose gel electrophoresis patterns

A) If the 7 pairs of primers in the sample are positive amplification results, and the PCR amplification product of primer 1 has a fragment size of 761bp, the PCR amplification product of primer 2 has a fragment size of 219bp, and the PCR amplification product of primer 3 has a fragment size of 219bp. A fragment with a size of 531bp appeared, the PCR amplification product of primer 4 appeared a fragment with a size of 1031bp, the PCR amplification product of primer 5 appeared a fragment with a size of 666bp, and the PCR amplification product of primer 6 appeared a fragment with a size of 991bp. If the PCR amplification product of 7 has a fragment with a size of 392bp, it indicates that Mycobacterium tuberculosis (M.Tuberculosis) or African mycobacterium type II (M.africanum subtype II) in the Mycobacterium tuberculosis complex has been detected, The test results are shown in Figure 1;

B) If the results of PCR amplification products of 7 pairs of primers in the specimen are: primers 1-6 are positive amplification results, and the PCR amplification product of primer 1 has a fragment size of 761bp, and the PCR amplification product of primer 2 has a size of 219bp The PCR amplification product of primer 3 has a fragment of 531bp in size, the PCR amplification product of primer 4 has a fragment of 1031bp in size, the PCR amplification product of primer 5 has a fragment of 666bp in size, and the PCR amplification product of primer 6 has a fragment of 666bp in size. The amplification product has a fragment size of 991bp; and the PCR amplification product of primer 7 does not contain a fragment size of 392bp, which indicates that Mycobacterium Kennedy (M.Canettii) in the Mycobacterium tuberculosis complex has been detected. as shown in picture 2;

C) If the results of 7 pairs of PCR amplification products in the sample are: primers 1-4 and primers 6 and 7 are positive amplification results, and the PCR amplification product of primer 1 has a fragment size of 761bp, the PCR amplification of primer 2 The product has a fragment size of 219bp, the PCR amplification product of primer 3 has a fragment size of 531bp, the PCR amplification product of primer 4 has a fragment size of 1031bp, and the PCR amplification product of primer 6 has a fragment size of 991bp, The PCR amplification product of primer 7 has a fragment of 392bp in size; and the PCR amplification product of primer 5 does not contain a fragment of 666bp in size, which indicates that Mycobacterium africa type I in the Mycobacterium tuberculosis complex ( M.africanum subtype I), the test results are as shown in Figure 3;

D) If the results of 7 pairs of PCR amplification products in the specimen are: primers 1-4 and primer 6 are positive amplification results, and the PCR amplification product of primer 1 has a fragment size of 761bp, the PCR amplification product of primer 2 A fragment with a size of 219bp appeared, the PCR amplification product of primer 3 had a fragment of 531bp in size, the PCR amplification product of primer 4 had a fragment of 1031bp in size, and the PCR amplification product of primer 6 had a fragment of 991bp in size; and The PCR amplification product of primer 5 does not contain a fragment with a size of 666bp, and the PCR amplification product of primer 7 does not contain a fragment with a size of 392bp, indicating that Mycobacterium capricatus (M. Caprae), test result as shown in Figure 4;

E) If the results of 7 pairs of PCR amplification products in the specimen are: primers 1-3 and primer 6 are positive amplification results, and the PCR amplification product of primer 1 has a fragment size of 761bp, the PCR amplification product of primer 2 A fragment with a size of 219bp appeared, the PCR amplification product of primer 3 had a fragment with a size of 531bp, and the PCR amplification product with primer 6 had a fragment with a size of 991bp; while the PCR amplification product of primer 4 did not contain a fragment with a size of 1031bp , the PCR amplification product of primer 5 does not contain the fragment that size is 666bp, and the PCR amplification product of primer 7 does not contain the fragment that size is 392bp, then explanation has detected the Mycobacterium bovis in the Mycobacterium tuberculosis complex ( M.Bovis), the test results are shown in Figure 5;

F) If the results of 7 pairs of PCR amplification products in the specimen are: primers 1-3 are positive amplification results, and the PCR amplification product of primer 1 has a fragment size of 761bp, and the PCR amplification product of primer 2 has a size of For a fragment of 219bp, the PCR amplification product of primer 3 showed a fragment of 531bp in size; while the PCR amplification product of primer 4 did not contain a fragment of 1031bp in size, and the PCR amplification product of primer 5 did not contain a fragment of 666bp in size. The PCR amplification product of 6 does not contain the fragment that is 991bp in size, and the PCR amplification product of primer 7 does not contain the fragment that is 392bp in size, which means that BCG (M.bovis BCG) in the Mycobacterium tuberculosis complex has been detected, The test results are shown in Figure 6;

G) If the results of 7 pairs of PCR amplification products in the specimen are: primer 1, primer 2, primer 4, primer 6 and primer 7 are positive amplification results, that is, the PCR amplification product of primer 1 has a fragment size of 761bp, The PCR amplification product of primer 2 has a fragment size of 219bp, the PCR amplification product of primer 4 has a fragment size of 1031bp, the PCR amplification product of primer 6 has a fragment size of 991bp, and the PCR amplification product of primer 7 has a A fragment with a size of 392bp; and the PCR amplification product of primer 3 does not contain a fragment with a size of 531bp, and the PCR amplification product of primer 5 does not contain a fragment with a size of 666bp, indicating that the Mycobacterium tuberculosis complex has been detected Mycobacterium microti (M.Microti), the test results are shown in Figure 7;

H) If the results of 7 pairs of PCR amplification products in the specimen are: only primer 1 is a positive amplification result, and the PCR amplification product of primer 1 has a fragment size of 761bp; while the PCR amplification product of primer 2 does not contain a fragment of size The PCR amplification product of primer 3 does not contain a fragment of 531bp in size, the PCR amplification product of primer 4 does not contain a fragment of 1031bp in size, and the PCR amplification product of primer 5 does not contain a fragment of 666bp in size. The PCR amplification product of primer 6 does not contain a fragment with a size of 991bp, and the PCR amplification product of primer 7 does not contain a fragment with a size of 392bp, which means that non-tuberculous mycobacteria (M.Fortuitum) has been detected, and the test results are shown in the figure 8.

Compared with the prior art, the beneficial effects of the present invention are: the primers provided by the present invention for the rapid detection and typing of mycobacteria can distinguish tuberculosis mycobacteria and non-tuberculous mycobacteria, and can further distinguish Each member of the Mycobacterium tuberculosis complex has the advantages of good specificity and high accuracy, which greatly improves the detection efficiency. In addition, the present invention also provides a method for rapid detection and typing of mycobacteria. By using the specific primers provided by the present invention, the operation is simple and fast, and the specificity is good, which can effectively assist the clinical diagnosis of tuberculosis and guide clinical Carrying out early and effective chemotherapy is an ideal method for rapid detection and typing of mycobacteria.

Claims (8)

1. A primer for rapid detection and typing of mycobacteria, characterized in that it comprises the following 7 pairs of mycobacterium-specific primers: MycoF1: 5'-GGCAAGGTCACCCCCGAAGGG-3', MycoR1: 5'-AGCGGCTGCTGGGTGATCATC-3'; MycoF2: 5'-TCGCGTGATCCTTCGAAACG-3', MycoR2: 5'-GCCGTTGCGCTGATTGGACC-3'; MycoF3: 5'-GACCTGACGCCGCTGACAC-3', MycoR3: 5'-CACCTACACCGCTTCCTGCC-3'; MycoF4: 5'-GACATGTACGAGAGACGGCATGAG-3', MycoR4: 5'-AATCCAACACGCAGCAACCAG-3'; MycoF5: 5'-AGGGATTCGGCGTTCGGGATTCCTG-3', MycoR5: 5'-GATCGCGATCGCCAACGATTCAGTGTATG-3'; MycoF6: 5'-GGGTCTGACGGCCAAACTC-3', MycoR6: 5'-CTCGGTGGCCGGTTTTTC-3'; MycoF7: 5'-CGATAGACCATGAGTCCGTCTC-3', MycoR7: 5'-GTGGGCGGATGCCAGAATAG-3'. 2. The primer for rapid detection and typing of mycobacteria according to claim 1, wherein the concentration ratio of the upstream primer and the downstream primer of the mycobacterium-specific primer is 1:1. 3. A method for rapid detection and typing of mycobacteria, characterized in that, comprising the steps: S1 Use the bacterial genomic DNA extraction solution to extract the bacterial genomic DNA in the specimen to be tested; S2 uses the bacterial genome DNA in the sample to be tested as a template, and uses 7 pairs of mycobacterium-specific primers according to claim 1 to carry out PCR amplification respectively; S3 The obtained PCR product is subjected to agarose gel electrophoresis. 4. the method for rapid detection and typing of mycobacteria as claimed in claim 3, is characterized in that, the prescription of bacterial genomic DNA extracting solution is: Chelex 100resin 5%; Tris-HCl 10mM, pH8.3; disodium edetate 0.1 mM; sodium azide 0.1%; proteinase K 100 μg/ml. 5. The method for rapid detection and typing of mycobacteria according to claim 3, wherein the PCR amplification reaction conditions in the step S2 include: stage 1: 95°C for 5min; stage 2: 95°C 1 min at °C; stage 3: 1 min at 58°C; stage 4: 1 min at 72°C; 32 cycles; stage 5: 5 min at 72°C; store at 16°C. 6. the method for mycobacterium rapid detection and typing as claimed in claim 3, is characterized in that, the sequence that mycobacterium-specific primer 1 amplifies is 761bp in the described step S2, and mycobacterium-specific primer 2 The amplified sequence is 219bp, the sequence amplified by mycobacterium-specific primer 3 is 531bp, the sequence amplified by mycobacterium-specific primer 4 is 1031bp, the sequence amplified by mycobacterium-specific primer 5 is 666bp, and the sequence amplified by mycobacterium-specific primer 5 is 666bp. The sequence amplified by primer 6 is 991bp, and the sequence amplified by the mycobacterium-specific primer 7 is 392bp. 7. the method for mycobacterium rapid detection and typing as claimed in claim 3, is characterized in that, described mycobacterium is Mycobacterium tuberculosis or African mycobacterium type II, Kennedy mycobacterium, African mycobacterium Mycobacterium type I, Mycobacterium capricatus, Mycobacterium bovis, BCG, Mycobacterium microti, and nontuberculous mycobacteria. 8. The application of the primer for rapid detection and typing of mycobacteria as claimed in claim 1 in the preparation of a test kit for detecting mycobacteria.