CN100368559C - A Primer Design Method for Multiplex PCR for Identifying Mycobacterium Tuberculosis Species - Google Patents

Abstract

A method for designing a primer of a multiplex PCR for identifying Mycobacterium tuberculosis strains is characterized by comprising the following steps: a. respectively adding a section of non-bacterial genome sequence to the 5' ends of oligonucleotide primers P1 and P2 capable of being specifically combined with bacterial genome sequence to obtain specific long primers YB1-P1 and YB2-P2 which are used as primer pairs of first-stage polymerase chain reaction in the composite amplification process, namely specific long primer pairs; b. the non-bacterial genome sequence is directly used as a primer pair of the multiplex amplification second-stage polymerase chain reaction, namely an amplification primer pair.

Description

A Primer Design Method for Multiplex PCR for Identifying Mycobacterium Tuberculosis Species

technical field

The invention relates to a multiplex PCR primer design method for amplifying gene fragments of multiple bacteria at one time to identify Mycobacterium tuberculosis strains.

Background technique

Since the 1990s, with the rapid development of molecular biology in the medical field, researchers have also introduced molecular biology techniques into the scope of tuberculosis research. Polymerase chain reaction (PCR) is a molecular biology technique based on nucleic acid biochemistry, and it is one of the most valuable techniques in the field of biology. Since the diagnosis of tuberculosis was introduced in 1989, it has immediately become the focus of much attention in the field of tuberculosis bacteriological diagnosis. After years of scientific research and clinical testing, this method has been continuously improved, and its sensitive, rapid and specific characteristics have been verified in most reports.

This project takes four clinically common Mycobacterium tuberculosis strains (bovine type, human type, avian type, and mouse type) as the research objects, and selects the gene sequences of different types of Mycobacterium tuberculosis to design specific primers. Detect a variety of bacteria, establish a new multiplex PCR detection of Mycobacterium tuberculosis strains, for rapid diagnosis and typing of Mycobacterium tuberculosis. The development of molecular biology technology and the application of PCR technology have opened up a new way of classification and identification. The current gene identification technology has the characteristics of fast, simple and high resolution, and can carry out multi-phase taxonomic identification, and can determine the precise location and species determination according to the relationship of phylogenetic evolution, making the taxonomic identification method more objective and reasonable.

Multiplex PCR is to design multiple pairs of primers to amplify in one PCR reaction system, so as to generate fragments of different specific target regions, and observe each specific amplified fragment by gel electrophoresis, which is used to identify the genus and species of Mycobacterium tuberculosis level. In the article "Mycobacterium tuberculosis strains unidentified using the IS6110 probe canbe detected by oligonucleotides derived from the Mt308sequence" published in "Research in Microbiology" No. 146 in 1995, Thierry et al. designed and amplified the IS6110 sequence fragment (325bp) and the 638kD gene bp ) and the amplified product was analyzed, and good results were obtained. The article "Assessment of genetic markers for species differentiation within the Mycobacterium tuberculosis complex" published by Liebana et al. in "Journal of Clinical Microbiology" No. 34 in 1996 reported the amplification of 16srDNA (1030bp) and the 372bp gene fragment of MAB70. Rapid identification of the Mycobacterium tuberculosis complex and the Non-tuberculosis Mycobacterium complex. The article "Multiplex polymerase chain reaction for the detection of mycobacterial DNA in cases of tuberculosis and sarcoidosis" published in "Moden Pathology" by Ikonomopoulos JA, etc. in the 12th issue of 1999 used mpb64 (243bp), IS6110 (916bp) and 85kD protein 3 ( Gene fragments, analyzing 300 clinical specimens, can quickly detect Mycobacterium tuberculosis, Mycobacterium avium, and Slow-growing Mycobacterium complex.

The above are examples of the use of multiplex PCR technology for the detection of Mycobacterium tuberculosis at home and abroad. They simply mixed multiple different detected Mycobacterium tuberculosis genes, and carried out PCR amplification reaction in the same reaction, so as to achieve the purpose of multiplex PCR. The problems in these methods are: (1) strict PCR reaction conditions are required, such as the concentration of various reaction components, the ratio between the reaction components, the temperature and time of PCR amplification, etc. (2) Since a variety of different primers exist in the same reaction tube, the reaction between the primers is more obvious, such as the formation of primer dimers, etc., which will lead to the loss of a large number of primers, which can easily lead to this multiple reaction. Failure of the PCR reaction. (3) The selection of primers will be very strict, and it must be ensured that primer dimers will not form between them, which will greatly reduce the possibility of multiplex PCR of multiple different types of Mycobacterium tuberculosis.

Invention content:

The purpose of the present invention is to provide a multiplex PCR primer design method for detection of Mycobacterium tuberculosis with high efficiency and high sensitivity.

The basic train of thought of the inventive method is: select a pair of short DNA fragments of non-bacterial genome as heavy amplification primer pair YB1 and YB2, and between the oligonucleotide primers (P1, P2) that can specifically combine with bacterial genome sequence The primer pair YB1 and YB2 were added to the 5' end respectively to obtain specific long primers YB1-P1 and YB2-P2.

Here, the oligonucleotide primers P1 and P2 capable of specifically binding to the bacterial genome sequence are original primers capable of binding to the bacterial genome of the locus to be amplified. Since the multiplex PCR reaction is the PCR amplification carried out to multiple loci at the same time, relative to different loci, the oligonucleotide primers P1 and P2 that can specifically bind to the bacterial genome sequence of the locus are different, Correspondingly, the specific long primers YB1-P1 and YB2-P2 of each locus to be amplified are also different due to differences in P1 and P2.

When carrying out multiple PCR reactions, add the above-mentioned high-volume amplification primers and specific long primers for different loci to be amplified in the PCR reaction system at the same time (of course, heat-resistant polymerase and mononuclear enzymes required for conventional PCR reactions need to be added. glycosides, etc.). Since the bacterial genome sequence of each locus to be amplified does not contain the sequence corresponding to the large-scale amplification primers YB1 and YB2, when the reaction system carries out the first round of PCR reaction (through a cycle of denaturation, annealing, and extension) ), the primer pair YB1 and YB2 will not combine with the bacterial genome sequence, and the amplified DNA fragment is still determined by the original primer pair P1 and P2 contained in the specific long primer YB1-P1 and YB2-P2. However, after the first round of reaction finishes, the amplified product with both the target gene fragment and the non-bacterial genome sequence YB1 YB2 will be produced at the locus to be amplified. When the PCR reaction system carries out the second round of PCR reaction (i.e. undergoing the second cycle of denaturation, annealing, and extension), the above-mentioned products having both the target gene fragment and the non-bacterial genome sequence YB1, YB2 are further amplified. The primers used are still specific long primers YB1-P1 and YB2-P2. After the reaction is completed, the obtained product has both the target gene fragment and the non-bacterial genome sequence paired with the primer pair YB1 and YB2. In the present invention, we refer to the above-mentioned first-round and second-round PCR reactions using specific long primers as reaction primers as the first-stage reaction, that is, the first-stage polymerase chain reaction in the multiplex PCR process. In other words, the polymerase chain reaction of the first stage in the multiplex amplification process includes the above-mentioned first and second rounds of reaction. The amplified product obtained in this stage of reaction has both the target gene fragment and the non-bacterial genome sequence paired with the primer pair YB1 and YB2, which is referred to as the product of the first stage of multiplex PCR herein. From the third round of PCR reaction, since each gene locus has the product of the above-mentioned first stage, like this, the heavy-duty amplification primers YB1 and YB2 can be used as primers for each of the above-mentioned amplified loci, and the PCR reaction can be performed in the above-mentioned first stage. The products of the first stage are used as templates for amplification. Similar to this, in each round of PCR reaction after the third round, the heavy amplification primers YB1 and YB2 can be used as primers for each amplified locus. After going through multiple rounds (eg 30 rounds) of PCR cycle reactions, the product of the above-mentioned first stage will be amplified to millions of times. In the present invention, we refer to the above-mentioned PCR reaction using high-volume amplification primers as reaction primers as the second-stage reaction, that is, the second-stage polymerase chain reaction in the multiplex PCR process. It should be noted that, in the above design idea of the present invention, the reactions of the first stage and the second stage are carried out in the same reaction system, rather than being divided into two reactions. In fact, even in the third round and after the third round of PCR reactions, the above-mentioned first-stage reactions still exist, but the proportion thereof is extremely low. In addition, when carrying out the PCR amplification reaction of the first stage, many pairs of primers participate in the reaction at the same time, that is, many pairs of specific long primers with different sequences relative to multiple loci to be amplified participate in the reaction at the same time (as mentioned above, The specific long primers YB1-P1 and YB2-P2 of each locus to be amplified are not the same), and the reaction and competition between primers are inevitable, thus greatly reducing the efficiency of PCR amplification. Already mentioned when discussing the prior art. But, with regard to the present invention, the purpose of the PCR amplification of the first stage is not wishing to obtain a large amount of amplified fragments, but only makes the product of the non-bacterial genome sequence with target gene fragment and paired with YB1, YB2 simultaneously (the product of the first stage) can be produced, so the impact of the above drawbacks on the entire multiplex PCR reaction process is extremely weak. After the first stage of PCR amplification, the 5' end of each target gene fragment to be amplified has a non-bacterial genome sequence paired with YB1 and YB2, so a pair of heavy-duty amplification primers (YB1, YB2) are added. Multiple loci can then be amplified simultaneously. Since there is only one pair of primers participating in the reaction in the second-stage amplification carried out thereafter, that is, the heavy amplification primer pair YB1 and YB2, there is no competition and reaction between the primers, and there is no need to adjust the concentration between the primers, thereby greatly improving The amplification efficiency of each gene locus was shown. Therefore, it can be said that only a small amount of templates need to be amplified in the first-stage PCR reaction, while the second-stage amplification is to amplify all loci with high efficiency. As far as its essence is concerned, in the PCR reaction of the second stage, multiple pairs of primers in the existing compound amplification method are simultaneously amplified multiple loci, and transformed into a pair of primers (YB1, YB2) to simultaneously amplify multiple loci. at the same time, YB1 and YB2 are not specific primers relative to each specific DNA fragment being amplified, therefore, the above-mentioned process is also amplified simultaneously by many pairs of specific primers in the existing multiplex amplification method Multiple specific gene fragments are transformed into simultaneous amplification of multiple specific gene fragments by a pair of non-specific primers.

In the present invention, the selection of the above-mentioned high-volume amplification primers YB1 and YB2 is extremely critical, and its design needs to consider the following three elements: 1. It must be a non-bacterial genome sequence, that is, its sequence will not be identical to the bacterial genome sequence; ②. Select an appropriate primer length. An oligonucleotide chain composed of 18 to 24 bases is a relatively optimized primer length; ③. Select the content of base GC in the primer and the annealing temperature Tm of the primer.

The temperature that the present invention selects when designing the Tm value of primer is 55 ℃, because at this temperature, most of the PCR reactions in the multiplex PCR can be carried out, and this temperature will cause a large amount of non-specific reactions after the first PCR reaction simultaneously. The heterogeneous bands, we must reduce the heterogeneous bands by reducing the concentration of specific long primers. After reducing the concentration of long primers, the interaction between each primer is reduced, and the non-specific products are reduced. At the same time, the specific products we need are also reduced, but this does not affect the overall amplification efficiency. As mentioned above, the first two rounds of PCR reactions are only the beginning of the above-mentioned compound amplification process. The products of this round of PCR reactions do not require a large amount, and only need to produce a small amount of DNA with both non-bacterial genome sequences and target genes. Fragments, that is, generate a small amount of templates for the second-stage PCR reaction, and the templates can be amplified in large quantities through subsequent PCR reactions. After the PCR reaction in the first stage, the primers participating in the reaction are no longer specific long primer pairs (YB1-P1, YB2-P2), but heavy amplification primer pairs (YB1, YB2).

However, the primer pair that simply satisfies the above three elements cannot serve as the high-volume amplification primer pair YB1 and YB2 in the present invention. Although theoretically speaking, the primer pair YB1 and YB2 can be designed and obtained according to a certain principle, it can be found in practice that the primers designed according to the primer design principle can be practically applied and can achieve better results. not much. That is to say, the "primer pair" that can be used in practice must be obtained through a large number of arduous experiments, which is a process of optimizing primers.

For example, based on the limitations of the above three elements, we have designed the following 7 pairs of primers (YB3-YB16):

$\left\{\begin{array}{c}\mathrm{<span\; class="notranslate">\; YB</span>}\mathrm{<span\; class="notranslate">\; 3</span>}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; 5</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; CACCATTAGCACCCAAAGCT</span>}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; 3</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span>\\ \mathrm{<span\; class="notranslate">\; YB</span>}\mathrm{<span\; class="notranslate">\; 4</span>}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; 5</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; TGATTTCACGGAGGATGGTG</span>}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; 3</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span>\end{array}\right.$

$\left\{\begin{array}{c}\mathrm{<span\; class="notranslate">\; YB</span>}\mathrm{<span\; class="notranslate">\; 5</span>}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; 5</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; GGTCTATCACCCCTATTAACCAC</span>}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; 3</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span>\\ \mathrm{<span\; class="notranslate">\; YB</span>}\mathrm{<span\; class="notranslate">\; 6</span>}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; 5</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; CTGTTAAAAGTGCATACCGCCA</span>}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; 3</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span>\end{array}\right.$

$\left\{\begin{array}{c}\mathrm{<span\; class="notranslate">\; YB</span>}\mathrm{<span\; class="notranslate">\; 7</span>}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; 5</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; GGTGTTGATGATGACATGGCG</span>}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; 3</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span>\\ \mathrm{<span\; class="notranslate">\; YB</span>}\mathrm{<span\; class="notranslate">\; 8</span>}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; 5</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; GTACCCCTCTGCAGCATGAGAGTAG</span>}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; 3</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span>\end{array}\right.$

$\left\{\begin{array}{c}\mathrm{<span\; class="notranslate">\; YB</span>}\mathrm{<span\; class="notranslate">\; 9</span>}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; 5</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; TGGAGAAATCTGGCACCAC</span>}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; 3</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span>\\ \mathrm{<span\; class="notranslate">\; YB</span>}\mathrm{<span\; class="notranslate">\; 10</span>}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; 5</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; GAGGCGTACAGGGATAGCAC</span>}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; 3</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span>\end{array}\right.$

$\left\{\begin{array}{c}\mathrm{<span\; class="notranslate">\; YB</span>}\mathrm{<span\; class="notranslate">\; 11</span>}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; 5</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; TTGTCAACTTCAGATACCACTGGAG</span>}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; 3</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span>\\ \mathrm{<span\; class="notranslate">\; YB</span>}\mathrm{<span\; class="notranslate">\; 12</span>}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; 5</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; CAAGGCAGATTTAACCACAGGTG</span>}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; 3</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span>\end{array}\right.$

$\left\{\begin{array}{c}\mathrm{<span\; class="notranslate">\; YB</span>}\mathrm{<span\; class="notranslate">\; 13</span>}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; 5</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; AGGGAAGAGGAGAGAGAAAGAGC</span>}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; 3</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span>\\ \mathrm{<span\; class="notranslate">\; YB</span>}\mathrm{<span\; class="notranslate">\; 14</span>}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; 5</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; CGGCAGTTAGTAGACTATCCAAGG</span>}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; 3</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span>\end{array}\right.$

$\left\{\begin{array}{c}\mathrm{<span\; class="notranslate">\; YB</span>}\mathrm{<span\; class="notranslate">\; 15</span>}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; 5</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; GATTGCTCAACAACCATG</span>}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; 3</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span>\\ \mathrm{<span\; class="notranslate">\; YB</span>}\mathrm{<span\; class="notranslate">\; 16</span>}<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; 5</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; -</span>\mathrm{<span\; class="notranslate">\; TTTCACTCTAGACCAAGCTTTG</span>}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; 3</span>}}^{<span\; class="notranslate">\; \&prime;</span>}<span\; class="notranslate">\; )</span>\end{array}\right.$

By searching the bacterial gene database, there is no sequence corresponding to the above-mentioned primers YB3-YB16 in the bacterial genome, thereby ensuring that these primers do not combine with the bacterial genome sequence.

However, it was found in subsequent experiments that the above-mentioned 7 pairs of primers could not successfully carry out the second stage of high-volume amplification.

In addition, similar to the above example, in the process of forming the technical solution of the present invention, we also screened, researched and experimented a large number of primer pairs satisfying the above three elements according to the characteristics of the object to be amplified, in order to find a primer that can A "primer pair" that is practically applied and can achieve better results.

It is through the above-mentioned a large number of screening, research and experiments that the method of the present invention has optimized the primer pair YB1 and YB2:

YB1 5′-CGCCTGTTTAACAAAAACAT-3′

YB2 5′-CCGGTCTGAACTCAGATCACGT-3′

By searching the bacterial gene database, there is no sequence corresponding to the above-mentioned primers YB1 and YB2 in the bacterial genome, thereby ensuring that the above-mentioned primers do not combine with the bacterial genome sequence. At the same time, it is more important that experimental studies show that the primer pair (YB1, YB2) can successfully carry out the second stage of high-volume amplification, thereby constituting the high-volume amplification primers mentioned in the present invention.

The above-mentioned high-volume amplification primers are added to the 5' ends of the oligonucleotide primers (P1, P2) that can specifically bind to the bacterial genome sequence to obtain specific long primers YB1-P1 and YB2-P2.

In summary, the primer design method of the multiplex PCR of identification Mycobacterium tuberculosis strain of the present invention is:

a. Oligonucleotide primers capable of specifically binding to the bacterial genome sequence

Add a non-bacterial genome sequence to the 5' end of P1 and P2:

YB1 5′-CGCCTGTTTAACAAAAACAT-3′

YB2 5'-CCGGTCTGAACTCAGATCACGT-3',

Thus, the specific long primers YB1-P1 and YB2-P2 are obtained as the primer pair for the first stage of polymerase chain reaction in the compound amplification process, that is, the specific long primer pair;

b. Directly use the non-bacterial genome sequence:

YB1 5′-CGCCTGTTTAACAAAAACAT-3′

YB2 5′-CCGGTCTGAACTCAGATCACGT-3′

As a pair of primers for the second stage of polymerase chain reaction of multiplex amplification, that is, a pair of primers for heavy-duty amplification.

Compared with the aforementioned prior art, the present invention designs primers for multiplex PCR reactions, so that a large number of target gene fragments can be amplified with high efficiency, and the reproducibility of the experimental results is extremely high. The concentration of each pair of primers can be adjusted accurately, which simplifies the whole experimental process. In addition, after using the primers designed by the method of the present invention to carry out multiple PCR reactions, silver nitrate staining can be used to detect the results, and the cost requirements for experimental equipment are not high. The majority of molecular hospitals require bacterial testing.

Detailed ways

In this example, the designed primers were used to simultaneously perform multiple PCR reactions on four genes hsp65 gene, 32-KDa protein gene, IS6110 gene and mtp40 gene in the same PCR reaction system.

The designed high-volume amplification primers are:

YB1 5′-CGCCTGTTTAACAAAAACAT-3′

YB2 5′-CCGGTCTGAACTCAGATCACGT-3′

Corresponding to the above-mentioned high-volume amplification primers, the specific long primers YB1-P1 and YB2-P2 designed for each locus are:

hsp65 gene:

YB1-P1: 5′-CGCCTGTTTAACAAAAACAT ACCAACGATGGTGTGTCCAT-3′

YB2-P2: 5′-CCGGTCTGAACTCAGATCACGT CTTGTCGAACCGCATACCCT-3′

32-KDa protein gene:

YB1-P1: 5′-CGCCTGTTTAACAAAAACAT CGGCAACGCGCCGTCGGTGG-3′

YB2-P2: 5′-CCGGTCTGAACTCAGATCACGT CCCCCCACGGCACCGCCGGG-3′

IS6110 gene:

YB1-P1: 5′-CGCCTGTTTAACAAAAACAT CGGAGACGGTGCGTAAGTGG-3′

YB2-P2: 5′-CCGGTCTGAACTCAGATCACGT GATGGACCGCCAGGGCTTGC-3′

mtp40 gene:

YB1-P1: 5′-CGCCTGTTTAACAAAAACAT TTCCTGACCAGCGAGCTGCCG-3′

YB2-P2: 5′-CCGGTCTGAACTCAGATCACGT CCCCAGTACTCCCAGCTGTGC-3′

It should be noted that the primers P1 and P2 specifically binding to the bacterial genome sequence are:

hsp65 gene:

P1: 5′-ACCAACGATGGTGTGTCCAT-3′

P2: 5′-CTTGTCGAACCGCATACCCT-3′

32-KDa protein gene:

P1: 5′-CGGCAACGCGCCGTCGGTGG-3′

P2: 5′-CCCCCCACGGCACCGCCGGG-3′

IS6110 gene:

P1: 5′-CGGAGACGGTGCGTAAGTGG-3′

P2: 5′-GATGGACCGCCAGGGCTTGC-3′

mtp40 gene:

P1: 5′-TTCCTGACCAGCGAGCTGCCG-3′

P2: 5′-CCCCAGTACTCCCCACCTGTGC-3′

In order to further illustrate the use method and effect of the primers designed in this example, the specific process of the primers designed in this example for identifying Mycobacterium tuberculosis species in multiple PCR reactions will be described below.

Multiplex PCR reactions were carried out in a PE-9600 amplification instrument. The components in the PCR reaction system mainly include: template DNA (bacterial genome sequence), specific long primer pair, high-volume amplification primer pair, heat-resistant DNA polymerase, MgCl2, deoxynucleoside triphosphate dNTP, BSA (fetal bovine serum albumin ), 1×Buffer (buffer) and double distilled water DDH2O. Wherein, DNA thermostable polymerase, Mgl2 and 10×Buffer (buffer) are directly provided by a commercially available PCR kit (manufactured by Huamei Company, China, product name is thermostable Taq DNA polymerase).

The concentration of each component in the PCR reaction system is as follows:

Reagent Single tube metering DDH₂O 13.5μl dNTP 7.5μl (200μM) 10□buffer 3.75μl Primer high-volume amplification primers hsp65 gene specific long primer 32-KDa protein gene specific long primer IS6110 gene-specific long primer mtp40 gene specific long primer 0.3μl (400nM) 0.3μl (40nM) 0.3μl (40nM) 0.3μl (40nM) 0.3μl (40nM) 0.3μl (400nM) 0.3μl (40nM) 0.3μl (40nM) 0.3μl (40nM) 0.3μl (40nM) Taq enzyme 1μl (3u) BSA 3.75μl Mgcl₂ 3μL (2.25mM) Sample 2μl

The amplification process is as follows:

a. The first round and the second round of PCR reaction, that is, the first stage of amplification (small amount of amplification), the primers for the reaction are long primers.

① Denaturation: heating to 94°C to unravel the double-stranded DNA;

② Annealing: Cool down to 55°C, at this temperature, the long primers will combine with the template DNA;

③. Extension: raise the temperature to 72°C, which is the most suitable temperature for DNA polymerase reaction. Under the action of Mgcl2, the polymerase adds dNTP at the 5' end of the long primer along the 5'→3' direction of the template.

After the first round and the second round of PCR reactions, a product (product of the first stage) with the target gene fragment and the non-bacterial genome sequence paired with YB1 and YB2 is obtained.

b. In the third and subsequent rounds of PCR reactions, that is, the second stage of amplification, the reacting primers are high-volume amplification primers. The PCR reaction mechanism of denaturation, annealing and extension is similar to the previous rounds of amplification, but in the annealing process, the heavy amplification primer is combined with the template, and the 5' end of the template has a non Bacterial genome sequence; at the same time, during the extension process, dNTPs are added at the 5' end of the high-volume amplification primer along the 5'→3' direction of the above template. A total of 36 rounds of multiple amplification cycles were performed, and the cycle parameters were as follows:

Pre-denaturation: 94°C for 3 minutes

Denaturation: 94°C for 50 seconds

Renaturation: 55°C for 50 seconds

Extension: 72℃ for 50 seconds

Number of cycles: 36

Extension: 72°C for 10 minutes

In addition, in view of the different molecular weights and fragment lengths of the amplified products, the electrophoresis detection method is used to separate and detect them, so as to verify the amplification results. When performing separation and detection, firstly mix the amplified product with the loading buffer in a certain ratio, add it into the polyacrylamide gel hole in the electrophoresis tank, and apply voltage to both ends of it for electrophoresis. Since the molecular size of each amplification product is different, under the action of the same electric field force, the distance it travels in the same medium per unit time is also different. Therefore, after being processed in the electrophoresis apparatus for a period of time, the gaps between the amplification products gradually widen. After that, the gel is taken out and stained (silver staining method is used in this example), and finally the amplification results can be seen visually.

The electrophoresis detection raw materials, equipment and conditions used are as follows:

Polyacrylamide gel composition:

30% polyacrylamide (PAG) solution 9.5ml

5×TBE solution 7ml

DDH2O 18.5ml

10% ammonium persulfate (PA) 300μl

TEMED 30μl

Electrophoresis tank: DYY-III electrophoresis tank (Beijing Liuyi Instrument Factory)

Electrophoresis instrument: pharmacia1000 multifunctional electrophoresis instrument

Electrophoresis conditions:

1. Loading volume: sample 3μl Gel loading buffer: 2μl

Electrode buffer: 1×TBE solution

2. Using constant current electrophoresis Current: 80mA Time: 1 hour and 30 minutes

The dyeing process is as follows:

Using silver staining method:

1. Fix with 10% ethanol for 10 minutes

2. Wash twice with DD H2O

3. Fix with 1% nitric acid for 3 minutes

4. Wash twice with DD H2O

5. 1% silver nitrate staining for 20 minutes

6. Wash 3 times with DD H2O

7. 3% Na2CO3, formaldehyde solution for color development

8. Wash with DD H2O to stop color development

The results obtained by using the primers designed in this embodiment for multi-locus amplification are compared with the fragment sizes obtained by using the original specific primers (i.e. P1, P2) for multi-locus amplification as follows:

hsp65 gene: the size of the fragment amplified with the original specific primers: 443bp

The fragment size after compound amplification with the primers of the examples is: 485bp

32-KDa protein gene: the size of the fragment amplified with the original specific primers: 396bp

The fragment size after compound amplification with the primers of the examples is: 438bp

IS6110 gene: the size of the fragment amplified with the original specific primers: 986bp

The fragment size after composite amplification with the primers of the examples is: 1028bp

Mtp40 locus: the size of the fragment amplified with the original specific primers: 506bp

The fragment size after compound amplification with the primers of the examples is: 548bp

After electrophoresis and staining, it can be seen that after the primers designed in this example are used for multi-locus amplification, the amplification efficiency is high, the amount of amplified product is large, and the bands of the gel are clear and easy to distinguish after staining; After the obtained amplified product is sequenced by a sequencer, it is confirmed that the DNA sequence of the amplified product is consistent with the sequence of the target DNA fragment. However, when using the original specific primers (P1, P2) for multi-locus amplification, due to the influence of the reaction and competition between primers, the amount of amplified products generated is very small, and the gel is stained with bands. Totally unrecognizable, poorly amplified.

Using our primer design method and a new multiplex PCR identification method for Mycobacterium tuberculosis species, we have successfully detected more than 100 clinical samples of different types (human, bovine, and avian) and compared normal samples And standard typing samples, it is confirmed that our method can be successfully used for typing, and the reproducibility of the experimental results is extremely high.

Claims (1)

1. a kind of primer design method of the multiplex PCR of identification Mycobacterium tuberculosis strain, it is characterized in that carry out in the following manner: a. Add a non-bacterial genome sequence to the 5' end of the oligonucleotide primers P1 and P2 that can specifically bind to the Mycobacterium tuberculosis genome sequence: YB1 5′-CGCCTGTTTAACAAAAACAT-3′ YB2 5'-CCGGTCTGAACTCAGATCACGT-3', Thus, the specific long primers YB1-P1 and YB2-P2 are obtained as the primer pair for the first stage of polymerase chain reaction in the compound amplification process, that is, the specific long primer pair; b. Directly use the non-bacterial genome sequence: YB1 5′-CGCCTGTTTAACAAAAACAT-3′ YB2 5′-CCGGTCTGAACTCAGATCACGT-3′ As a pair of primers for the second stage of polymerase chain reaction of multiplex amplification, that is, a pair of primers for heavy-duty amplification.