CN100368559C - Primer design method of multiple PCR for discriminating vaccine of tubercle branch bacillus - Google Patents

Abstract

The present invention relates to a design method for a primer for identifying multiplex PCR of tubercle branch bacillus strains. The present invention is characterized in that the method has the steps that a, a section of abacterial genome sequence is added on the 5' end of oligonucleotide primers P1, P2 which can be in specific binding with a bacterial genome sequence to obtain specificity long primers YB1-P1 and YB2-P2, and the specificity long primers YB1-P1 and YB2-P2 are used as a primer pair, namely a specificity long primer pair for the first stage polymerase chain reaction in the multiplex amplification process; b, the abacterial genome sequence is directly used as a primer pair, namely a discharge amplification primer pair for the second stage polymerase chain reaction in the multiplex amplification process.

Description

Multiple PCR primer design method for identifying mycobacterium tuberculosis strain

Technical Field

The invention relates to a primer design method for multiplex PCR (polymerase chain reaction) for identifying mycobacterium tuberculosis strains by amplifying gene segments of a plurality of bacteria at one time.

Background

With the rapid development of molecular biology in the medical field since the 90 s of the 20 th century, researchers have also introduced molecular biology techniques into the field of tuberculosis research. Polymerase Chain Reaction (PCR) is a molecular biology technique based on nucleic acid biochemistry and is one of the most valuable techniques in the field of biology. Since the introduction of the diagnosis of tuberculosis in 1989, it has become the focus of much attention in the field of the diagnosis of tuberculosis bacteria. Through years of scientific research and clinical examination, the method is continuously perfected, and the response is sensitive, quick and specific characteristics are verified in most reports.

The project takes 4 clinically common mycobacterium tuberculosis strains (bovine, human, avian and murine) as research objects, selects gene sequences of different types of mycobacterium tuberculosis respectively, designs specific primers, simultaneously detects multiple bacteria in the same system, establishes a brand-new multiple PCR detection mycobacterium tuberculosis strain, and is used for rapid diagnosis and typing of the mycobacterium tuberculosis. The development of molecular biology technology and the application of PCR technology open up a new way for classification and identification. The existing gene identification technology has the characteristics of rapidness, simplicity, convenience and high resolution, can perform multi-phase classification identification, and can determine accurate position and seed according to phylogenetic relation so that the classification identification method is more objective and reasonable.

In the multiplex PCR, a plurality of pairs of primers are designed to be amplified in a PCR reaction system, so that different specific target region fragments are generated, and each specific amplified fragment is observed through gel electrophoresis to be used for identifying the level of the mycobacterium tuberculosis to the genus species. The amplification of IS6110 sequence fragment (325 bp) and 65kD protein gene fragment (383 bp) was designed and analyzed in Mycobacterium tuberculosis strains under identified use, published by Thierry et al in Research in Microbiology 1995, 146. A372 bp gene fragment for amplifying 16srDNA (1030 bp) and MAB70 was reported in an Association of genetic markers for specificity differentiation and in the Mycobacterium tuberculosis complex published by Liebana et al, journal of Clinical Microbiology, 1996, and both of the Mycobacterium tuberculosis complex and the nontuberculous Mycobacterium complex were rapidly identified by agarose gel electrophoresis. Ikonomopoulos JA et al in "model Pathology" 1999 12 published the article "Multiplex polymerase chain reaction for the detection of mycobacteriol DNA in cassettes of tuberculosis and sarcoidosis" applied the gene fragments of mpb64 (243 bp), IS6110 (916 bp) and 65kD protein (383 bp) to analyze 300 clinical specimens, which can rapidly detect Mycobacterium tuberculosis, mycobacterium avium, and Mycobacterium lentum complex.

The above is an example of the use of multiplex PCR technology for detection of tubercle bacillus at home and abroad. They simply mix multiple different tubercle bacillus genes to perform PCR amplification reaction in the same reaction, thereby achieving the purpose of multiple PCR. These methods have problems in that: (1) Stringent PCR reaction conditions are required, such as concentrations of various reaction components, ratios between reaction components, temperature, time of PCR amplification, and the like. (2) Since a plurality of different primers exist in the same reaction tube, the reactions between the primers are obvious, such as the formation of primer dimer, etc., which results in the loss of a great amount of primers, and thus the failure of such multiplex PCR reaction is easily caused. (3) The choice of primers will be very strict and it must be ensured that no primer dimers will form between them, which will greatly reduce the possibility of multiplex PCR with many different types of Mycobacterium tuberculosis.

The invention content is as follows:

the invention aims to provide a primer design method of multiplex PCR for tubercle bacillus detection, which has high efficiency and high sensitivity.

The basic idea of the method is as follows: a pair of short DNA fragments of non-bacterial genomes are selected as amplification primer pairs YB1 and YB2, and the primer pairs YB1 and YB2 are respectively added to the 5' ends of oligonucleotide primers (P1 and P2) which can be specifically combined with bacterial genome sequences, so as to obtain specific long primers YB1-P1 and YB2-P2.

Here, the oligonucleotide primers P1, P2 capable of binding specifically to a bacterial genomic sequence are the original primers which can bind to the bacterial genome of the locus to be amplified. Because the multiplex PCR reaction is simultaneously used for carrying out PCR amplification on a plurality of loci, compared with different loci, the oligonucleotide primers P1 and P2 which can be specifically combined with the bacterial genome sequence of the locus are different, and correspondingly, the specific long primers YB1-P1 and YB2-P2 of each locus to be amplified are also different due to the difference of P1 and P2.

When performing multiplex PCR reaction, adding the amplification primers and specific long primers for different loci to be amplified into the PCR reaction system at the same time (naturally, adding heat-resistant polymerase and mononucleotide required by conventional PCR reaction). Since the bacterial genome sequence of each locus to be amplified does not contain the sequence corresponding to the amplification primers YB1 and YB2, when the reaction system carries out the first round of PCR reaction (undergoes a cycle process of denaturation, annealing and extension), the primer pair YB1 and YB2 can not be combined with the bacterial genome sequence, and the amplified DNA fragment is determined by the original primer pair P1 and P2 contained in the specific long primers YB1-P1 and YB2-P2. However, after the first round of reaction is finished, an amplification product with both the target gene fragment and the non-bacterial genomic sequence YB1 YB2 is generated at the locus to be amplified. When the PCR reaction system is subjected to a second round of PCR reaction (namely, the second round of PCR reaction is subjected to denaturation, annealing and extension), the product with the target gene fragment and the non-bacterial genome sequences YB1 and YB2 is further amplified, primers used for amplification are still specific long primers YB1-P1 and YB2-P2, and after the reaction is finished, the obtained product simultaneously has the target gene fragment and the non-bacterial genome sequences matched with the primer pairs YB1 and YB2. In the present invention, the first and second PCR reactions using the specific long primers as the reaction primers are referred to as a first-stage reaction, i.e., a polymerase chain reaction in the first stage of the multiplex PCR process. In other words, the polymerase chain reaction at the first stage of the multiplex amplification process includes the first and second rounds of reaction described above. The amplification product obtained from this stage reaction has both the target gene fragment and the non-bacterial genomic sequence paired with the primer pair YB1 and YB2, which is referred to herein as the product of the first stage of multiplex PCR. Since the third PCR reaction has already been performed with the first-stage product for each locus, the amplification primers YB1 and YB2 are put in the amplification primers for each locus, and the PCR reaction is performed using the first-stage product as a template. Similarly, in each PCR reaction after the third round, the amplification primers YB1, YB2 can be used as primers for each amplification locus. After multiple (e.g., 30) PCR cycles, the first stage product is amplified to millions of times. In the present invention, the PCR reaction using the amplimer primers as the reaction primers is referred to as a second stage reaction, i.e., a second stage PCR in the multiplex PCR process. It should be noted that, in the above design concept of the present invention, 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 and subsequent PCR reactions, the reaction in the first stage still remains, but the ratio thereof is extremely low. In addition, when the first stage of PCR amplification reaction is performed, a plurality of primers simultaneously participate in the reaction, i.e., a plurality of pairs of specific long primers with different sequences relative to a plurality of loci to be amplified participate in the reaction (as mentioned above, the specific long primers YB1-P1 and YB2-P2 of each locus to be amplified are not the same), the reaction and competition between the primers are difficult to avoid, thereby greatly reducing the efficiency of PCR amplification, which is a disadvantage that has been mentioned in the discussion of the prior art. However, in the present invention, the objective of the first stage PCR amplification is not to obtain a large amount of amplified fragments, but only to produce a product (first stage product) having both the objective gene fragments and non-bacterial genome sequences that pair with YB1 and YB2, and therefore the above-mentioned adverse end effects on the whole multiplex PCR reaction process are extremely weak. After the first stage of PCR amplification, the 5' end of each target gene segment to be amplified has a non-bacterial genome sequence paired with YB1 and YB2, so that a plurality of loci can be simultaneously amplified after a pair of amplification primers (YB 1 and YB 2) is added. In the second stage amplification, only one pair of primers participating in the reaction, namely the amplification primer pair YB1 and YB2, does not have competition and reaction among the primers and does not need to adjust the concentration among the primers, thereby greatly improving the amplification efficiency of each locus. Thus, it can be said that the first stage of PCR reaction only needs to amplify a small amount of template, while the second stage of amplification is to amplify all loci efficiently. In essence, in the second stage of PCR reaction, a plurality of gene loci are simultaneously amplified by a plurality of pairs of primers in the existing composite amplification method, and are converted into a plurality of gene loci simultaneously amplified by a pair of primers (YB 1 and YB 2); meanwhile, YB1 and YB2 are not specific primers relative to each specific DNA fragment to be amplified, so that the process is changed from the process of simultaneously amplifying a plurality of specific gene fragments by a plurality of pairs of specific primers in the existing composite amplification method into the process of simultaneously amplifying a plurality of specific gene fragments by a pair of non-specific primers.

In the present invention, the selection of the amplification primers YB1 and YB2 is very critical, and the design needs to consider the following three factors: (1) it must be a non-bacterial genomic sequence, i.e., its sequence will not be identical to a bacterial genomic sequence; (2) selecting proper primer length, wherein the oligonucleotide chain consisting of 18-24 bases is the primer length which is relatively optimized; (3) selecting the content of base GC in the primer and the annealing temperature Tm of the primer.

The temperature selected in the present invention is 55 ℃ for designing Tm value of primer, because most PCR reactions in multiplex PCR can be performed at this temperature, and this temperature will cause a lot of non-specific bands after the first PCR reaction, we must reduce the bands by reducing the concentration of specific long primer. After reducing the concentration of the long primer, the interaction between the primers is reduced, the non-specific products are reduced, and at the same time, the specific products required by us are also reduced, but the whole amplification efficiency is not influenced. As mentioned above, the first two PCR reactions are only the beginning of the multiplex amplification process, the products of the PCR reaction do not need to have a large amount, and only a small amount of DNA fragments having both the non-bacterial genome sequence and the target gene are generated, i.e., a small amount of template for the second PCR reaction is generated, so that the template can be amplified by the subsequent PCR reaction. After the first stage of PCR reaction, the primers participating in the reaction are no longer specific long primer pairs (YB 1-P1, YB 2-P2), but amplification primer pairs (YB 1, YB 2).

However, a primer pair that simply satisfies the above three elements can serve as the amplification primer pair YB1, YB2 described in the present invention. Although theoretically, the primer pair YB1 and YB2 can be designed and obtained according to a certain principle, in practice, it can be found that the primer designed according to the principle of primer design can be practically used and can obtain a good effect. That is, a "primer pair" that is actually practical must be obtained through a large number of laborious tests, which is a preferred procedure for primer pair.

For example, based on the above three element limitations, we have designed the following 7 references (YB 3-YB 16):

by searching the bacterial gene database, sequences corresponding to the above primers YB3 to YB16 do not exist in the bacterial genome, thereby ensuring that these primers do not bind to the bacterial genome sequence.

However, in subsequent experiments, it was found that the above 7 pairs of primers did not successfully perform the second amplification step.

In addition, similar to the above examples, in the process of forming the technical scheme of the present invention, based on the characteristics of the object to be amplified, we have screened, studied and experimented a large number of primer pairs satisfying the above three elements, in order to find "primer pairs" that can be practically used and can achieve better effects.

Through the screening, research and experiment, the method of the invention preferably selects the primer pairs YB1 and YB2:

YB1 5′-CGCCTGTTTAACAAAAACAT-3′

YB2 5′-CCGGTCTGAACTCAGATCACGT-3′

by searching the bacterial gene database, the sequences corresponding to the primers YB1 and YB2 do not exist in the bacterial genome, thereby ensuring that the primers do not bind to the bacterial genome sequence. Meanwhile, more importantly: experimental studies show that the primer pair (YB 1, YB 2) can successfully carry out amplification in the second stage, thereby forming the amplification primer of the invention.

The amplification primers are added to the 5' ends of oligonucleotide primers (P1, P2) capable of specifically binding with bacterial genome sequences to obtain specific long primers YB1-P1 and YB2-P2.

In summary, the primer design method of the multiple PCR for identifying Mycobacterium tuberculosis strains comprises the following steps:

a. oligonucleotide primers capable of specifically binding to bacterial genomic sequences respectively

The 5' end of P1 and P2 is added with a non-bacterial genome sequence:

YB1 5′-CGCCTGTTTAACAAAAACAT-3′

YB2 5′-CCGGTCTGAACTCAGATCACGT-3′，

thereby obtaining specific long primers YB1-P1 and YB2-P2, which are used as a primer pair of a first-stage polymerase chain reaction in the composite amplification process, namely a specific long primer pair;

b. directly with the non-bacterial genomic sequence:

YB1 5′-CGCCTGTTTAACAAAAACAT-3′

YB2 5′-CCGGTCTGAACTCAGATCACGT-3′

the primer pair is used as the primer pair of the multiplex amplification second stage polymerase chain reaction, namely the amplification primer pair.

Compared with the prior art, the primers of the multiplex PCR reaction are designed, so that target gene fragments can be amplified in a large amount with high efficiency, the reproducibility of an experimental result is extremely high, meanwhile, the concentration of each pair of primers does not need to be adjusted in a complicated experimental process, and the whole experimental process is simplified. In addition, after the primers designed by the method are used for multiple PCR reaction, the detection result can be detected by adopting a silver nitrate dyeing mode, and the cost requirement on experimental equipment is low, so that the method has more advantages of Chinese characteristics and meets the requirement of bacterial inspection in most of current Chinese molecular hospitals.

Detailed Description

In this example, primers were designed for multiplex PCR reaction of four genes, hsp65 gene, 32-KDa protein gene, IS6110 gene and mtp40 gene, simultaneously in the same PCR reaction system.

The designed amplification primers are as follows:

YB1 5′-CGCCTGTTTAACAAAAACAT-3′

YB2 5′-CCGGTCTGAACTCAGATCACGT-3′

corresponding to the amplification primers, the specific long primers YB1-P1 and YB2-P2 designed for each gene locus are as follows:

the 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′

the primers P1 and P2 specifically binding to the bacterial genome sequence are:

the 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′-CCCCAGTACTCCCACCTGTGC-3′

to further illustrate the application method and effect of the primers designed in this example, the following description will proceed to the specific process of the primers designed in this example for multiplex PCR reaction to identify Mycobacterium tuberculosis species.

The multiplex PCR reaction was performed in a PE-9600 amplification apparatus. The PCR reaction system mainly comprises the following components: template DNA (bacterial genome sequence), specific long primer pair, amplimer pair, thermostable DNA polymerase, mgCl2, dNTP, BSA (calf serum albumin), 1 × Buffer, and double distilled water DDH2O. Wherein, the DNA thermostable polymerase, mgl2 and 10 xbuffer (Buffer) are directly provided by a PCR kit (the manufacturer is Huamei company, china, the name of which is thermostable TaqDNA polymerase).

The concentrations of the components in the PCR reaction system are as follows:

reagent	Single tube metering
						DDH 2 O	13.5μl
dNTP	7.5μl(200μM)
						10□buffer	3.75μl
Primer and method for producing the same	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
						Mgcl 2	3μL(2.25mM)
Sample	2μl

The amplification process is as follows:

a. the first and second PCR reactions, i.e., the first stage of amplification (small amount of amplification), are performed using long primers.

(1) And (3) denaturation: heating to 94 ℃ to untwist the double-stranded DNA;

(2) and annealing: cooling to 55 ℃, and combining the long primer with the template DNA at the temperature;

(3) and extending: the temperature is raised to 72 ℃, which is the most suitable temperature for the DNA polymerase reaction, and the polymerase adds dNTP along the 5' → 3' direction of the template at the 5' end of the long primer under the action of Mgcl2 and the like.

After the first round and the second round of PCR reaction, a product (product of the first stage) which simultaneously has target gene segments and non-bacterial genome sequences matched with YB1 and YB2 is obtained.

b. The third and subsequent PCR reactions, i.e., the second stage of amplification, are initiated with amplification primers. The PCR reaction mechanism of denaturation, annealing and extension is similar to that of the previous rounds of amplification, but in the annealing process, an amplification primer is combined with a template, and the 5' end of the template has a non-bacterial genome sequence matched with YB1 and YB 2; meanwhile, dNTPs are added to the 5' end of the amplification primer in the 5' → 3' direction of the template during the extension process. The whole composite amplification is cycled for 36 times, and the cycling parameters are as follows:

pre-denaturation: 3 minutes at 94 DEG C

Denaturation: 94 ℃ for 50 seconds

Renaturation: 55 ℃ for 50 seconds

Extension: 72 ℃ for 50 seconds

Cycle number: 36

Extension: 10 minutes at 72 DEG C

In addition, in view of the difference in molecular weight and fragment length of the amplified product, it is particularly detected by separation using an electrophoresis detection method to examine the amplification result. When separation detection is carried out, the amplification product and the sample loading buffer solution are mixed uniformly according to a certain proportion, added into the polyacrylamide gel hole in the electrophoresis tank, and voltage is applied to two ends of the polyacrylamide gel hole for electrophoresis. Because the molecules of the amplification products have different sizes, the distance traveled in the same medium per unit time under the action of the same electric field force is also different. Therefore, after a period of treatment in the electrophoresis apparatus, the difference between the amplification products is gradually opened, and then the gel is taken out and dyed (in this case, silver dyeing is adopted), and finally the amplification result can be visually seen.

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

polyacrylamide gel composition:

9.5ml of 30% Polyacrylamide (PAG) solution

5 XTBE solution 7ml

DDH2O 18.5ml

Ammonium Persulfate (PA) 10% 300. Mu.l

TEMED 30μl

Electrophoresis tank: DYY-III electrophoresis tank (Beijing six instrument factory)

Electrophoresis apparatus: pharmacia1000 type multifunctional electrophoresis apparatus

Electrophoresis conditions:

1. sample loading amount: sample 3 μ l gel loading buffer: 2 μ l

Electrode buffer solution: 1 XTBE solution

2. Electrophoresis current is performed in a constant current mode: 80mA time: 1 hour and 30 minutes

The dyeing process is as follows:

adopting a silver dyeing method:

1. fixing with 10% ethanol for 10 min

2. DD H2O Wash 2 times

3. Fixing with 1% nitric acid for 3 min

4. DD H2O Wash 2 times

5. 1% silver nitrate for 20 min

6. DD H2O Wash 3 times

7. 3% of Na2CO3, color development in a formaldehyde solution

8. DD H2O wash-stop color development

The results of the multi-locus amplification using the primers designed in this example were compared to the sizes of the fragments amplified using the original specific primers (i.e., P1, P2) as follows:

the hsp65 gene: the fragment amplified with the original specific primers had the following sizes: 443bp (bp)

The fragment size after multiplex amplification with the example primers was: 485bp

32-kDa protein gene: the fragment amplified with the original specific primers had the following sizes: 396bp

The fragment sizes after multiplex amplification with the example primers were: 438bp

IS6110 gene: the fragment amplified with the original specific primers had the following sizes: 986bp

The fragment size after multiplex amplification with the example primers was: 1028bp

Mtp40 locus: the fragment amplified with the original specific primers had the following sizes: 506bp

The fragment sizes after multiplex amplification with the example primers were: 548bp

After electrophoresis and dyeing treatment, the primers designed by the embodiment are adopted to carry out multi-locus amplification, the amplification efficiency is high, the generation amount of amplification products is large, and the bands of the gel after dyeing are clear and easy to distinguish; meanwhile, after sequencing the amplification product by a sequencer, the DNA sequence of the amplification product is confirmed to be consistent with the sequence of the target DNA fragment. When the original specific primers (P1, P2) are used for multi-locus amplification, the generation amount of amplification products is very small because of the influence of reaction and competition between the primers, bands are completely unidentifiable after the gel is dyed, and the amplification effect is very poor.

By using the primer design method and the brand-new multiple PCR identification method of the mycobacterium tuberculosis strains, more than 100 clinical samples of different types (human type, bovine type and bird type) are successfully detected, and by comparing with a normal sample and a standard typing sample, the successful typing can be realized by using the method, and the reproducibility of the experimental result is extremely high.

Claims (1)

1. A method for designing a primer of multiple 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 which can be specifically combined with the genome sequence of the mycobacterium tuberculosis:

YB1 5′-CGCCTGTTTAACAAAAACAT-3′

YB2 5′-CCGGTCTGAACTCAGATCACGT-3′，

thereby obtaining specific long primers YB1-P1 and YB2-P2, which are used as a primer pair of a first-stage polymerase chain reaction in the composite amplification process, namely a specific long primer pair;

b. directly with the non-bacterial genomic sequence:

YB1 5′-CGCCTGTTTAACAAAAACAT-3′

YB2 5′-CCGGTCTGAACTCAGATCACGT-3′

the primer pair is used as the primer pair of the multiplex amplification second stage polymerase chain reaction, namely the amplification primer pair.