WO2004090150A2

WO2004090150A2 - Pcr method using multiple primer pairs and the solution used in it and the use of the method in the detection reagents

Info

Publication number: WO2004090150A2
Application number: PCT/CN2004/000329
Authority: WO
Inventors: Dingbang Xu; Wenhui Xu; Defen Zhu; Wenkai Xie
Original assignee: Dingbang Xu; Wenhui Xu; Defen Zhu; Wenkai Xie
Priority date: 2003-04-11
Filing date: 2004-04-09
Publication date: 2004-10-21
Also published as: WO2004090150A3

Abstract

The invention relates to a method of polymerase chain reaction which use multiple primer pairs and also relates to the solution is used in it. Specifically, the PCR reaction was divided into three phases. During the first phase's 2-25 cycles, annealing temperature varies from 60-80°C. During the third phase's 5-25 cycles; denaturing temperature varies from 65-87°C. The second phase, which consists of 2-4 cycles, exists between the first and the third phase. The annealing temperature of this phase is same with that of the first phase. The method of the invention has overcome the disadvantage in the art that non-specific amplification between the sense and antisence primers and the nonspecific amplification product thus obtained in the multiplex PCR. The method is suitable for the preparation of the rapid detect agents that used in single-tube multiplex PCR and it also has good application future in the detection of the virus, such as HBV, HCV, HIV, HPV and so on, and the variants, stereotype, genotype or the subtype thereof, and in the detection of drug resistant gene of the bacterium such as tubercle bacillus.

Description

PCR method for multiple pairs of primers, reaction solution and application thereof in detection reagent

The invention relates to molecular biology technology, in particular to a method for polymerase chain reaction of multiple pairs of primers, a reaction solution thereof, and application in preparing a microorganism detection reagent. technical background

The polymerase chain reaction (PCR) technology was invented in the mid-1980s. It is a method for amplifying specific DNA. It is widely used in molecular biology and medicine. In the past two decades, improved methods with different characteristics and uses have been developed. Among them, the most widely used is nested PCR (Nested PCR, Nested PCR) with multiple primers and multiple primers. PCR (multiplex PCR).

Several primer pairs of current multiplex PCR are usually complementary to several different target gene sequences. These target gene sequences are carried on separate cDNAs or on the same genomic DNA but are separated by a considerable distance. The current multiplex PCR is often used to compare the expression levels of different genes. In clinical tests, multiplex PCR can be used to detect two or more pathogens at the same time, such as the simultaneous detection of hepatitis A virus (HAV) and hepatitis B virus (HBV). And hepatitis C virus (HCV). Except for the use of several pairs of primers, the current multiplex PCR is almost identical to the standard PCR in the design and reaction conditions of the primers. The specificity of the amplification products in the standard PCR method is mainly controlled by the annealing temperature. One product is often difficult to avoid in the current multi-pair primer PCR, which makes the number of pairs of primers not too many, otherwise the non-specific product formed by multiple pairs of target products and each pair of primers will complicate the PCR reaction itself The final product is difficult to confirm from the electrophoresis band. The current multiplex PCR method is more unsuitable for amplifying the same gene, because in this case, in addition to the target product and the non-specific product, an amplification product between unpaired primers of the target gene is often formed. Although, multiplex PCR amplification of the same target gene has great application value in clinical testing, especially in reducing false negatives of viral gene detection. However, clinical testing practice has shown that there is a serious problem of false negatives when using PCR methods to detect microorganisms, especially when detecting viruses. Standard PCR methods using a pair of primers and current multiplex PCR are unlikely to overcome false negatives due to the variety of variant sequences.

Nested PCR uses outer and inner primers to perform PCR, which can increase the specificity of amplification products and greatly improve the detection sensitivity. However, the current nested PCR has serious defects, which hinders the application and promotion of this method in nucleic acid detection. First, the current nested PCR requires two reaction tubes to perform the reaction in two rounds. The first step is to use jacketed primers to amplify about 20 cycles. Then, a small amount of the reaction solution containing the jacketed product is added to the jacketed primer and The second reaction tube of the fresh reaction solution was amplified with inner primers for about 30 cycles. Obviously, the two-step operation is a major obstacle to clinical high-throughput detection. Second, the operation of the two-step reaction significantly increases the chance of contamination of the jacket amplification products, which is a serious disadvantage for clinical testing. Third, in order to dilute the coat primer so that it stops working in the second round of reaction, usually only 5% or less of the coat amplification reaction solution is added to the second round of reaction solution, and more than 95% can be used as the second reaction solution. The coat amplification products of the round reaction template were not fully utilized, which is equivalent to about 5-10 PCR cycles wasted. Fourth, although the coat primer is diluted in the second round of reaction, it is still possible to continue to play a role in amplifying the coat product.

In order to overcome the above defects, some so-called single-tube one-step nested PCR methods have appeared, but the results are not satisfactory. First, the design principle of this single-tube one-step method is that the melting temperature of the inner primer is low. The annealing temperature is adjusted to control the stop and start of the inner primer. In the first stage, the inner primer cannot be used at higher temperatures. Annealed, but the jacket primers were still annealed and amplified at lower temperatures in the second stage. Second, in order to stop the effect of the coat primer at a later stage, the coat primer concentration must be determined empirically or experimentally. If the coat primer concentration is too high, the amplification will continue at a later stage, and the non-specific amplification of the coat primer will continue; if the coat primer concentration is too low, it will seriously affect the annealing speed of the initial primer and the template. It is often difficult to strike a good balance. Another single-tube method has been patented ES P9801642 (July, 31, 1998). The paper was published in Nucleic Acid Research (1999) 27 (6): 1564. This method Place the first round of reaction solution containing the outer primer, including Taq enzyme, in a normal PCR reaction tube, and the second round of reaction solution containing the inner primer, but not including Taq enzyme, in a thin tube of a specific specification. Place this thin tube in a PCR reaction tube. Due to the capillary phenomenon, the reaction solution in the thin tube is not mixed with the reaction solution outside the tube containing the Taq enzyme. After the first round of reaction is completed, the reaction tube is centrifuged, and the liquid in the thin tube is mixed with the external liquid under the centrifugal force, and then the second round of reaction is started. The melting temperature of the outer primer is lower than that of the inner primer. The second round reaction uses a higher annealing temperature, and the outer primer stops working. This design is painstakingly unique, but as the author himself pointed out, the quality and operation of the thin tube must be carefully controlled to avoid failure due to the escape of liquid in the thin tube. In addition, centrifugation is required between the first round reaction and the second round reaction. This operation step at normal temperature makes it difficult to achieve the hot start operation of the inner primer, and it is easy to form non-specific amplification products. Summary of the invention

The purpose of the present invention is to provide a polymerase chain reaction method of multiple pairs of primers, a reaction solution thereof, and application thereof in preparing a microbial detection reagent, so as to break through the limitation of the design of multiple pairs of primers in the existing multiplex PCR method and overcome a single tube. The PCR reaction cannot independently amplify the defects of multiple fragments of the same DNA sequence at the same time without affecting each other; at the same time, it overcomes the cumbersome steps of various current nested PCR methods, easy to contaminate, the amplification of the inner and outer primers affect each other or require special reactions Disadvantages of containers.

The present invention uses more than two pairs of primers, but the principles of primer design, product characteristics, and PCR reaction process are very different from the existing multiplex PCR and nested PCR. Each pair of primers is based on the order of the target template. The denaturation temperature characteristics of the product are designed as the main consideration.

Outer primers designed for nested PCR have extremely high melting temperatures, while inner primers have relatively low melting temperatures. In the first half of the PCR, conventional denaturation temperature and high annealing temperature are used. At this stage, only outer primers can The annealing completes the amplification, and the inner primer cannot be amplified because it does not meet the annealing conditions. At the same time, the designed amplification product of the outer primer has a higher melting temperature, while the amplified product of the inner primer has a higher melting temperature. The degree is relatively low. In the second half of the PCR, a low denaturation temperature and a low annealing temperature are used. At this stage, the inner primer can use the amplification product of the outer primer as a template to complete the amplification of the inner product. Although the jacket primer can also be annealed at a lower annealing temperature, it cannot be denatured at a lower denaturation temperature, so the jacket primer cannot be amplified in the second half of the stage. Therefore, one-step nested PCR can be achieved through primer design and adjustment of annealing and denaturation temperatures.

The number of primers used in the multiplex PCR of the present invention is greatly increased compared to the conventional multiplex PCR. The target templates can be adjacent to each other, and the PCR can make the non-specific amplification products synthesized in the previous stage unsolvable after 2 or 3 cycles Strands and lose their template function in subsequent cycles, thereby reducing or eliminating the complexity of PCR products due to unpaired primer interactions during multiplex PCR amplification, increasing the specificity and sensitivity of the reaction.

The invention provides a polymerase chain reaction method for multiple pairs of primers. The following reactions are simultaneously performed on the template DNA strand from 2-10 pairs of primers:

(1) template denaturation;

(2) primer annealing;

(3) DNA polymerase catalyzes the synthesis of complementary DNA strands;

(4) Perform steps (1)-(3) to cycle the amplification reaction;

The whole cycle reaction is divided into three stages. In the 2-25 cycles of the first stage, the annealing temperature ranges from 60 to 80. C. In the 5-25 cycles of the third stage, the denaturation temperature range is 65-87 ° C. There are 2-4 cycles of the second stage between the first and third stages. The first stage is the same and the annealing temperature is the same as the third stage.

A preferred method of the above polymerase chain reaction method for multiple primer pairs is that the template denaturation temperature is 92-97'C in the first 2-6 cycles of the first stage, and 65-87 in the subsequent cycles. ° C, preferably 75-83 ° C. Another solution of the polymerase chain reaction method of the plurality of pairs of primers of the present invention is that two pairs of primers nested with each other simultaneously perform an amplification reaction on the same template DNA strand.

A preferred method of the above polymerase chain reaction method of multiple primers is that the first-stage annealing temperature is 72-78 ° F, and is higher than the melting temperature of the inner primer by more than 3 ° C.

Another preferred method of the above polymerase chain reaction method of multiple primers is that the denaturation temperature range of the third stage is 75-82 ° C, which is higher than the melting temperature of the inner amplification product by 1 Ό or more.

Another preferred method of the above polymerase chain reaction method of multiple primers is that the melting temperature range of the outer primer is 82-90 ° C, the melting temperature range of the inner primer is 55-68 ° C, and the outer primer is The difference from the melting temperature of the inner primer is 15-25 ° C.

Another preferred method of the above polymerase chain reaction method for multiple primer pairs is that the melting temperature range of the outer amplification product is 85-90 ° C, and the melting temperature range of the inner amplification product is 74-80 ° C. The difference between the melting temperature of the outer jacket and the inner jacket is 4-15 ° C.

The present invention also provides a reaction solution for the polymerase chain reaction of the above-mentioned multiple pairs of primers, wherein the concentration of each primer in the reaction solution is 0.02-0.2 μM.

The invention also provides the polymerase chain reaction method of the above-mentioned multiple pairs of primers for preparing mutants, serotypes, genotypes and subtypes or drug-resistant strain detection reagents of various microorganisms such as viruses, bacteria, chlamydia, and mycoplasma. In the application, multiple pairs of primers for the PCR reaction are designed for specific sequences of mutant strains, serotypes, genotypes and subtypes or drug-resistant strains, and the melting temperatures of each product are 65-87 ° C.

An optimized scheme for the application of the above polymerase chain reaction method of multiple pairs of primers in the preparation of virus, bacteria, chlamydia, mycoplasma and other microorganisms, serotypes, genotypes and their subtypes or detection reagents for drug-resistant strains The various microorganisms include Hepatitis B virus, Hepatitis C virus, Human immunodeficiency virus, Human papilloma virus, Mycobacterium tuberculosis, Gonorrhea, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, Pneumococcus pneumoniae , E. coli, Chlamydia trachomatis and Mycoplasma urealyticum. In the specific implementation process, the primer and product design method is as follows: First, a typical strain of the target gene is selected, and different regions of the sequence include a conserved region and a subtype characteristic region and site, and the primer software is used to find the primer, or the primer software is used. According to the melting temperature and other characteristics of the amplified product, the regions and lengths of primer pairs for multiplex PCR are preliminarily planned. Then, in this typical strain and 2-5 or more mutant strains with different subtype characteristics or other characteristics, One or several regions include a conserved region and a subtype characteristic region. Then, after considering the complementarity of the primer pair with the sequence of various mutant strains, the 3 'end complementarity between the primer pairs, and other factors, a primer pair for multiplex PCR is selected.

When the nested polymerase chain reaction method of the present invention containing two or more pairs of primers is used, two pairs of mutually nested primers are simultaneously reacted against the template DNA strand. No need to open the cover.

Based on the same principle, a single-tube triple-nested PCR consisting of a pair of outer jackets, a pair of middle jackets, and a pair of inner jacket primers can be designed in three stages. That is, three pairs of nested primers are used to simultaneously perform a PCR reaction on the template DNA strand:

The first stage: 5-20 cycles, annealing temperature range 65-80Ό;

Second stage: 5-20 cycles, denaturation temperature range 65-87 ° C, annealing temperature range 60-75

Ό;

The third stage: 5-20 cycles, the denaturation temperature range is 65-87 ° C;

Moreover, there are 2 to 4 cycle transition stages between the first stage and the second stage, and between the second stage and the third stage, respectively. the same.

The nested polymerase chain reaction method of the present invention can also perform two sets of nested PCR reactions in a single reaction system, that is, the nested PCR of the above 2 pairs of primers or 3 pairs of primers is combined to perform 2 pairs and 2 pairs Primers, or multiplex nested PCR with 2 and 3 primers. That is, there are 2 sets of 4 pairs of nested primers in the reaction and the sequences of the 2 sets of coat products do not overlap, or there are only 3 pairs of primers, 2 of which are Outer jacket, 1 pair of inner jackets and 2 pairs of outer jackets nested together, or only 3 pairs of primers, 1 of which is outer jacket and 2 pairs of inner jackets, and the relative positions of the 2 inner jackets are separated. Multiple nested PCR can be designed into various forms as required, such as two independent nested PCRs for different target genes, two independent nested PCRs for the same target gene, and two coats for the same target gene And nested PCR with one inner nest, nested PCR with one inner sheath and two inner nests for the same target gene, and nested PCR with two separate inner nests within two overlapping coats for the same target gene.

The various nested polymerase chain reaction methods described in the present invention can be applied to nucleic acid detection, and the template DNA is derived from human or pathogenic microorganisms.

Beneficial effects,

The advantages of performing multiplex PCR with the method of the invention are:

1. When used to amplify different templates, the melting temperature of the amplified products designed according to the sequence of each template is very low, and the specificity of the amplification is controlled through the dual calculation of rigorous annealing temperature and ultra-low denaturation temperature. The number of PCR electrophoresis bands is consistent with the number of primer pairs. The number of pairs of multiple primer pairs is greatly increased compared to the current multiplex PCR.

2. In the above case, if the temperature is about 3 to 12 ° C lower than the maximum allowable annealing temperature, the amplification can be successfully completed even if there is about 1 to 4 mismatches between the primer and the template sequence, which reduces or Eliminate false negatives due to template sequence variation.

3. When used to amplify the same template, if each pair of primers matches the template, each target sequence can be specifically and efficiently amplified under stringent annealing temperature and ultra-low denaturation temperature to obtain electrophoresis from the same target gene Bands. By comparing different bands of the same sample, the amplification efficiency of each pair of primers can be judged. By comparing each band between different samples, several sets of data can be obtained at the same time through a single tube experiment. Semi-quantitative comparison.

4. In the above case, if annealing is performed at a temperature about 3 to 12 ° C lower than the maximum allowable annealing temperature, each pair of primers can complete the amplification even if there is about 1 to 4 mismatches with the template sequence and does not A non-exclusive band appears. Because only one of several pairs of primers in the multiplex PCR can be amplified to obtain an entry Marking the amplified band indicates that the target gene is present. Therefore, the ultra-low denaturation temperature and low annealing temperature are multiple

PCR provides ample scope for reducing and overcoming false negatives of various variants of any virus.

5. Ultra-low denaturation temperature multiplex PCR for the same template can also be used to detect the target gene and identify allele mutations at the same time.

6. If different subtypes can be found in a characteristic sequence that is different from other subtypes, the virus can be detected at the same time and the virus subtypes can be identified.

The advantages of using the multiplex PCR method of the present invention to perform two pairs of primers, namely nested PCR, are:

1. In the present invention, one-step nested PCR can be realized in the true sense through primer design and adjustment of annealing and denaturation temperatures.

2. Compared with ordinary nested PCR, the detection sensitivity is significantly improved, and the specificity is enhanced. At the same time, it overcomes the trouble that the ordinary nested PCR needs to be performed in two tubes or two steps, and it is easy to cause product contamination.

3. Triple nested PCR performed in a single tube further improves detection specificity and sensitivity.

4. One outer jacket with two inner jackets, one inner jacket with two jackets, or two independent sets of multiple nested PCR can also reduce false negative results due to template sequence variation. detailed description

The present invention is further described below with reference to specific embodiments, but the present invention is not limited.

The template DNA used in the examples of the present invention is cDNA. It is prepared by human muscle tissue total RA (Clontech) using a cDNA preparation kit (Clontech Advantage RT for PCR kit). The primers used are Oligo (dT) provided by the kit. ) ₁₈ , PCR volume 10 μl_, each tube add 1 μl_i Mg / RNA. The PCR reaction of the present invention uses the Advantage 2 kit of Clontech Company, and the final primer concentration is 0.02-0.2 μM. The primer design software Oligo 6.0 used in the present invention is produced by Molecular Biology Insightsjnc. The number of the gene bank of the present invention is from GenBank of the National Center for Biotechnology Information.

Example 1: Comparison of conventional and ultra-low denaturation temperature multiple primer PCR

Three pairs of primers were designed on the human actin gene (gene bank number BC016045), and their order and characteristics are shown in Tables 1 and 2, respectively. Table 1.Example 1 primer sequence

Table 2. Primer characteristics of Example 1

The results are shown in Table 3 as unpaired amplification products. Table 3. Characteristics of unpaired primer amplification products in Example 1.

Example 1 The PCR reaction volume is 5 microliters, the final concentration of each primer is 0.1 uM, each reaction tube. Contains 1 microliter of CDNA, cDNA from human tissue total RNA with Poly (dT) 18 using Clontech's reverse transcription kit Synthesized according to its regulations. The PCR reaction conditions are divided into two groups. The first group is the conventional multiplex PCR method, that is, the first denaturation at 95 ° C for 1 minute, the cyclic denaturation at 95Ό30 seconds, the annealing at 62 ° C for 30 seconds, and the extension at 72 ° C for 1 minute, a total of 28 cycles. Finally extended at 72 ° C for 3 minutes. The second group is an ultra-low denaturation temperature multiplex PCR method. The conditions for the first denaturation, cyclic annealing, cyclic extension and final extension are the same as the first group. The first three cycles of denaturation are also 95 ° C for 30 seconds, but the next 25 Cyclic denaturation was changed to 80 ° C for 30 seconds. The test results are shown in Table 4. Table 4. Test results of Example 1

+ Indicates one or more bands, 0 indicates no bands, and numbers indicate the number of bands

It can be seen that compared to ultra-low denaturation temperature multiplex PCR, the non-paired amplification products and non-specific amplification products of conventional multiplex PCR are more serious. Example 2. Extending the number of primer pairs for multiplex PCR to six pairs. Table 5. Primer sequence for Example 2

Table 6. Primer characteristics of Example 2

Primer extension i product long melting temperature strong binding melting temperature name itself 3, complementary to each other 3, complementary length

Degree Degree. C degrees. c

18 67.1 376 <2 <2

A04 37 74.9

19 66.3 387 2 / -1.5 2 / -1.5

16 67.2 383 2 / -1.6 2 / -1.6

A05 47 76.8

22 66.5 362 2 / -1.9 2 / -1.9

22 67.5 398 3 / -3.8 <2

A06 90 76.4

23 66.1 384 <2 2 / -1.9

19 66.9 383 2 / -1.9 3 / -3.2

A07 79 76.1

22 67.0 391 2 / -1.9 2 / -1.9

20 68.4 421 2 / -1.5 2 / -1.5

A08 108 74.9

24 66.2 400 <2 2 / -1.3

21 69.0 418 2 / -1.6 2 / -1.6

A09 63 76.3

27 68.4 408 <2 <2 The above six pairs of primers designed on the human actin gene of less than 2000 bases have the possibility of amplifying 15 kinds of unpaired combinations. Table 7. Amplification product characteristics of non-paired primers for implementation 2

The melting temperatures of six paired amplification products were all lower than 76.8 ° C, while the melting temperatures of 15 unpaired amplification products were all higher than 80.9 ° C. The reaction reagents and conditions were the same as in Example 1, but the subsequent cycle was denatured to 79 ° C for 30 seconds. As a result, any combination of A04-A09 primer pairs, including multiplex PCR of 6 primers, resulted in amplification products without unpaired products and non-paired products. Specific amplification products. Example 3: Design protocol and implementation conditions of ultra-low denaturation temperature multiplex PCR with 10 primer pairs The primer design software Qligo was used to analyze the Cyclin-D gene (gene bank number NC-053056), and 10 pairs of primers with high stringency and melting temperature lower than 81 C were found. The positions and characteristics of the 10 primer pairs and the characteristics of the 45 unpaired interference amplification products are listed in Tables 8 and 9. Table 8. 10 pairs of primers obtained from Cydin D gene test

Table 9: Test results of mutual interference between each pair of primers Primer sequence number Interference primer pair sequence Number of interference product position Interference product length Interference product Tm

1 2 672-781 109 84.6

1 3 672-1745 1073 87.1

1 4 672-2010 1338 87.1

1 5 672-2145 1473 87.0

1 6 672-2724 2052 85.9

1 7 672-3317 2645 85.7

1 8 672-3821 3149 86.0

1 9 672-3863 3191 86.1

1 10 672-4016 3344 86.5

2 3 732-1745 1013 87.0

6 9 2327-3863 1536 84.7

6 10 2327-4016 1689 85.7

7 8 3061-3821 760 84.6

7 9 3061-3863 802 85.5

7 10 3061-4016 955. 85.7

8 9 3635-3863 228 82.1

8 10 3635-4016 381 87.7

9 10 3824-4016 192 92.1 The results show that the Tm value of the interference product is between 82.1-92.1 Ό, and most of it is between 83-87 ° C, which is significantly different from the normal product, that is, at 4300 It is possible to obtain 10 pairs of primers in the base sequence, which meets the design requirements that the melting temperatures of the amplification products and the interference products are not in the same range. If the subsequent cycle denaturation temperature is 81 Ό, the interference product denaturation can be ruled out and only the target amplification product can be obtained. Example 4: Multiple primer pairs PCR reduces or eliminates false positives for hepatitis B virus detection.

The length and sequence of HBV primers were modified according to the design idea of the present invention, and the primers were recombined to form three pairs of primers. The sequences and characteristics are shown in Tables 10 and 11. The bold underlined letters represent the modified nucleotides, and the primer positions are based on the sequence of the virus variants with the gene bank number E00010. The characteristics of the three unpaired interference products of the three primer pairs are shown in Table 12.

Table 10.Example 4 primer sequence.

Primer name Primer position Primer sequence (5 'to 3,)

416-436 CTG CAG CTA TGC CTC ATC TTC

HBV01

461 -479 GAC AAA CGG GCA ACA TAC C

1902-1921 GCA TGG ACA TTG ACC CGT ATA A

HBV02

1958-1979 AAG GAA AGA AGT CAG AAG GCA A

2271-2298 GAG TGT GGA TTC CCA CTC

HBV03

2300-2315 GGG CAT TTG GTG GTG T Table 11. Primer characteristics of Example 4

Table 12. Characteristics of unpaired interference amplification products of Example 4

It can be seen that the temperature of the alkali chain of the paired products is less than 79 ° C, and the melting temperature of the unpaired interference amplification products is greater than 84 ° C. According to these data and the results of Examples 1 and 2, the annealing temperature is controlled at 50-63 ° C, and the subsequent denaturation temperature is controlled at 80-83 ° C. Each primer pair can be an exact or highly matched variant of it as The template specifically amplifies the target product. Example 5: Detection of hepatitis B virus with multiple primers and simultaneous detection of combined mutations

The sequence of more than 1,000 hepatitis B virus genes stored in the gene bank and clinical analysis at home and abroad show that about a quarter of hepatitis B viruses undergo A-to-T and T-total changes simultaneously at positions 1762 and 1764 of the C gene promoter. The so-called joint mutation in which G is transformed into A reduces the rate of HBV mRNA transduction and synthesis, and therefore has a certain clinical relationship with the severity of the disease and treatment. PCR-RFLP, or PCR-restriction endonuclease fragment polymorphism, is commonly used to identify wild-type strains and C gene promoters. Combined mutant strains, but the complicated operation process has limited its popularization and application in clinical diagnosis. In addition, some variants also mutated at positions 1763, 1765, and 1766, so that after artificially introducing the mutation at position 1767, the combined mutants at positions 1762 and 1764 still could not get the TGATCA sequence, so they were not cut by enzymes and appeared false negatives. Judge. In this embodiment, a pair of shared primers is designed to check the presence of HBV virus. Two sets of primers were designed to identify wild-type and joint mutants, one of which had two positive primers, each with 1764 as its 3 'end. The other set of reverse primers has two, each with its 1762 position as its 3 'end. The sequence and characteristics of the common primers and primers used to detect joint mutations are listed in Table 13. Table 13. Primer sequence of Example 5

* HBV04 primer positions are based on the order of variants with gene extension number E00010; HBV05 and HBV06 primer positions are based on the gene bank number as AF479684 variant order Table 14. Characteristics of the primers of Example 5

Bold bold indicates that the binding strength is too low to anneal to the template to complete the amplification

Both the HBV common primer HBV04 and the target amplification products of the combined mutation primers HBV05 and HBV06 have melting temperatures below 82 ° C, and the denaturation step can be completed when the subsequent denaturation temperature is 83 ° C. Both the "wild-type" reverse primers of HBV05 and the "wild-type" positive primers of HBV06 can anneal to the wild-type template at 65 ° C, and neither can be annealed with the combined mutant template at the same temperature to complete the amplification. Similarly, the HBV05 "mutant" reverse and HBV06 "mutant" positive primers can anneal to the combined mutant template at 65 ° C, but cannot anneal to the wild-type template at the same temperature. Each sample was divided into two tubes during the test, and each tube contained HBV04 primer pairs, HBV05 positive primers, and HBV06 reverse primers. Tube A also contains two "wild-type" primers, and tube B also contains two "mutant" primers. For example, three bands of 119, 46, and 68 appear in the PCR results of tube A, indicating that the sample is wild-type, and three bands of tube B indicate that the sample is a combined mutation of 1762 and 1764. If there are no bands in tubes A and B, the sample is HBV negative. Example 6: Multiple pairs of primers reduce or eliminate false negative detection of hepatitis C virus by PCR

The non-coding region upstream of the HCV gene is quite conserved. A large number of primers reported in the literature were taken from this region. Four of our four pairs of primers were selected. After slightly adjusting the primer length, etc., the primers were re-arranged into pairs. And characteristics are listed with Tables 15 and 16. Table 15.Example 6 primer sequences

Based on the sequence of HCV variants with gene bank number AF177039 Table 16. Primer sequence of Example 6

The number of 3 'terminal bases complementary between the HCV01 positive primer and the HCV02 reverse primer is less than 2, the amplification product is 284 bases long, and the melting temperature of the amplification product is 89.7 ° C. Therefore, when the annealing temperature is controlled at 55-64 ° C and the subsequent denaturation temperature is 84 ° C, the target amplification product can be synthesized. The unpaired interference amplification products formed at the beginning of the reaction are aborted at a subsequent denaturation temperature of 84 ° C. Example 7: Multiple pairs of primers for PCR reduce and eliminate human immunodeficiency virus detection false negatives

Two pairs of primers were designed for the gag and env genes in the conserved regions of the HIV virus. Among them, HIV01 was selected from the primer pairs reported in the literature, and only the primer length was slightly modified. The positive primers of the other three pairs of primers were selected from the literature guide, and their lengths were slightly modified. The order and characteristics of the four pairs of primers are listed in Tables 17 and 18. Table 17.Example 7 primer sequences

'Sequence position is based on the HIV variant with gene bank number U23487 Table 18. Primer characteristics of Example 7

The melting temperatures of the target amplification products of the four pairs of primers are all lower than 78 ° C. When the annealing temperature is 55 to 68 ° C and the subsequent denaturation temperature is 80 ° C, the amplification can be specifically performed. The gag and env genes are separated by more than 5000 bases, so the primers of HIV01 and HIV02 will not undergo unpaired interference amplification with the primers of HIV03 and HIV04, while the positive primers of HIV01 and the reverse primers of HIV02 and the positive primers of HIV03 and

The length and melting temperature of the unpaired interference amplification product of HIV04 reverse primers were calculated to be 280 bases, 82.3C and 557 bases, and 83.3Ό, respectively. When the subsequent denaturation temperature was 8CTC, the expansion could not be completed due to the denaturation step being blocked. increase. Example 8: Multiple pairs of primers for the simultaneous detection of different types of human papilloma virus.

About 70 types of human papillomavirus have been identified. The order within each type is more conservative, and the differences between the types are larger. Among them, types 6, 11 and 16 are more common or related to malignant tumors. In Example 8, a pair of primers was designed to match type 16, and the primers taken from the literature were adjusted only in length, and named HPV16-01. In addition, two pairs of primers were designed to match type 11 and 6 and named HPV11-01 and HPV11-02o. HPV11-01 positive primers and HPV11-02 reverse primers were slightly adjusted in length according to literature reports. The reverse primer of HPV11-01 and the positive primer of HPV11-02 are connected, and this site sequence is used as a probe in the literature report. The order and characteristics of the three primer pairs are shown in Table 19 and Table 20. Table 19.Example 8 primer sequences

* HPV16 primer order and position are based on type 16 HPV with gene bank number NC-001526, HPV11 primer order and position are based on type 11 HPV with gene bank number M14119 Table 20. Primer characteristics of Example 8

The melting temperature of the target amplification products of the above three pairs of primers are all lower than 78.1 ° C. When the controlled annealing temperature is 50-64 ° C and the subsequent temperature is 8 CTC, the target products can be specifically amplified. A 210-base band after PCR reverse primer electrophoresis staining indicates HPV16 type, and a 43 or / and 64-base band indicates that the sample is HPV11 or 6 type. If there is no band, the HPV type 3 is not present. If the subsequent denaturation temperature is increased to 82 ° C, a 107 base band will also appear, which is an amplification product of HPV11-01 positive primer and HPV11-02 reverse primer, which also indicates that HPV11 or HPV6 virus is present in the sample. Example 9: Multiple pairs of primers for the simultaneous detection of hepatitis B virus HBV, hepatitis C virus HCV and human immunodeficiency virus HIV design protocol and implementation conditions.

Multiplex PCR was performed on the primers of Table 16 above for HBV, the primers of Table 17 for HCV01, and the primers of Table 19 for HIV. The annealing temperature was 50-64 ° C and the subsequent denaturation temperature was 84 ° C. Specific amplification of the target product. The 119, 70, and 144 base long strips after electrophoresis indicate the presence of HBV, HCV, and HIV, respectively. Example 10:

Single-tube nested PCR with ultra-low denaturation temperature improves PCR specificity. Two pairs of primers were designed on the human glyceraldehyde-3 -phosphate dehydrogenase gene, and their sequence and characteristics are shown in Tables 21 and 22. Table 21. Primer sequence for Example 10

Table 22. Example 10 Primer and Amplification Product Characteristics

The melting temperature of the outer primer is greater than 88.9 ° C, and it can be annealed at a high temperature of 72-78 ° C. The melting temperature of the inner primer is 64.1 ° C, the maximum allowed annealing temperature is 67.1 ° C, and it cannot be annealed at a high temperature of 72-78 ° C. . The melting temperature of the inner primer amplification product is 80.9 ° C, and the denaturation temperature can be fully denatured above 83 ° C, while the melting temperature of the outer primer amplification product is 90.2 °, and it cannot be denatured at 83 °. The procedure of the PCR reaction is shown in Table 23. Multiple coats of G coat and G inner coat were performed under normal conditions. In addition to the two bands of 388 and 69 bases and the non-specific band, 321 · and 136 bases with lengths of 321 and 136 bases caused by amplification between unpaired primers appeared Interference bands. Table 23. Reaction conditions of Example 10 with both outer and inner primers

The expected 69-base long band was obtained without any non-specific amplification bands. In the control experiment, G coat primers and G inner primers were amplified using the same kit according to standard PCR methods, with denaturation of 95 to 30 seconds, annealing at 55 ° C for 30 seconds, and extension of 72 to 60 seconds. The results were 388 bases and 69 base bands, but all non-specific bands appeared. Example 11 1: Two sets of double nested PCR were performed simultaneously.

A total of 4 pairs of primers were designed in two groups of human actin globulin genes. Primer sequence, primer and target amplification product characteristics, primer unpaired product characteristics, and reaction conditions are shown in Tables 26, 27, 28, and 29, respectively. The conditions such as the reaction volume per tube were the same as in Example 1.

Table 24. Example 11 primer sequence

Table 25. Example 11 Primer and amplification product characteristics Primer amplification products

Maximum allowable withdrawal name Melting temperature Melting temperature Fire temperature (° c) Length Length Bit

(° c) (° c) S

35 88.5

A1 coat 1 386 88.5 200-585> 72

33 88.0

19 68.7

A2 inner sleeve 1 46 76.1 306-351 69

20 66.0

29 87.3

A3 coat 2 498 83.3 1173-1670> 72

32 86.1

19 67.7

A4 inner sleeve 2 79 76.1 1412-1490 70

22 67.0 Table 26. Characteristics of various possible unpaired amplification products in Example 11. Primer 3 'ends of the amplification product primers are paired with each other.

(° C) Degrees (° c) Calorie

A1 positive primer and A2 35 88.5 2 / -1.5

152 87.3

Reverse primer 20 66.0 4 / -8.1 A2 forward primer and A1 19 68.7 3 / -3.2

280 90.0

Reverse primer 33 88.0 2 / -1.3

A3 positive primer and A4 29 87.3 2 / -1.6

318 82.7

2 Anti-primer 22 67.0 3 / -3.4 A4 positive primer and A3 19 67.7 <2 259 81.9

Reverse primer 32 86.1 2 / -1.9

A1 positive primer with inner A4 35 88.5 2 / -1.5

1291 89.5

Reverse primer 22 67.0 3 / -3.4

1 A11 positive primer with A3 35 88.5 31-2.5 and 1471 88.9

Reverse primer 32 86.1 2 / -1.9

2 A2 positive primer and A4 19 68.7 2 / -1.6

1185 89.2

Reverse primer 22 67.0 3 / -3.4

A2 positive primer and A3 19 68.7 3A3.2

1365 88.6

Reverse primer 32 86.1 <2

Table 27. Example 11 reaction conditions

Example 12 _:

For multiple nested PCR, outer jacket 2 is the inner jacket of outer jacket 1. The final product is DNA amplified by inner jacket primers. In addition to the primer pair A3 (as outer jacket 2) and A4 (as inner jacket) regions of Example 11, another pair of human actin globin primers A5 was designed as another nested primer (as outer jacket 1) . The order of outer jacket 1, outer jacket 2 and inner primers, target amplification product characteristics, primer unpaired product characteristics, and reaction conditions are shown in Tables 26, 30, 31, 32, and 33, respectively. The conditions such as the reaction volume per tube were the same as in Example 10. Table 28. Example 12 Bow Sequence

Primer position order (5, to 3 ')

GCT CCT CCT GAG CGC AAG TAC TCC GTG

1066- 1095

TGG

A5 coat 1

CAC TCC CAG GGA GAC CAA AAG CCT TCA

1692- 1730

TAC ATC TCA AGT Table 29. Example 12 primer and amplification product characteristics

Table 30. Characteristics of various possible non-matched amplification products in Example 12 Primer reaction between unpaired primers Amplification products Melting temperature Melting temperature 3 ′ complementary length

Degree (...) CC) bases

30 86.1 <2

A5 forward primer and A3 reverse primer 605 85.0

32 86.1 <2

29 87.3 3Z-3.2

A3 forward primer and A5 reverse primer 558 83.8

39 85.9 2 / -1 .3

30 86.1 2 / -3.1

A5 forward primer and A4 reverse primer 425 85.2

22 67.0 2 / -1.9

29 87.3 2 / -1.6

A3 forward primer and A4 reverse primer 318 82.7

22 67.0 3 / -3A

19 67.7 2 / -1.3

A4 forward primer and A5 reverse primer 319 83.0

39 85.9 4 / -4.8

19 67.7 <2

A4 forward primer and A3 reverse primer 259 81 .9

32 86.1 2 / -1 .9 Table 31. Example 12 reaction conditions

Example 13.

Multiple nested PCR. Two pairs of inner primers are used to separate the phases. The final product is two DNA strands.

The outer primer pair of Example 13 is A5, and the sequence is shown in Table 28. The primer set of inner sleeve 1 is A4, and its sequence is shown in Table 24. The order of the inner 2 primer pairs is shown in Table 32. The target amplification product characteristics, primer unpaired product characteristics, and reaction conditions are shown in Tables 33, 34 and 35, respectively. The conditions such as the reaction volume per tube were the same as in Example 1. Table 32. Example 13 primer sequence

Primer position order (5, to 3,)

1553- 1571 ACA GGG GAG GTG ATA GCA GCA T

A6 inner sleeve 2

1636- 1658 AGG CTC ATC ATT CAA AAT AAA AC

Table 33. Example 13 primer and amplification product characteristics

Table 34. Characteristics of various possible non-matched amplification products in Example 13. Primer reaction between unpaired primers of amplification products

Length Position Degree (° c) Degree Degree (° C)

19 67.7

A4 forward primer and A6 reverse primer 247 80.9 1412-1658

23 64.9

Between

30 86.1

A5 forward primer and A4 reverse primer 425 85.2 1066-1490

22 67.0

Outside

30 86.1

A5 forward primer and A6 reverse primer 593 84.6 1066-1658 and 23 64.9

Inside

19 67.7

A4 forward primer and A5 reverse primer 319 83.0 1412-1730 of 39 85.9

Room 19 65.4

A6 forward primer and A5 reverse primer 178 80.9 1553-1730

39 85.9 Table 35. Example 13 reaction conditions

Example 14.

The order of triple-nested PCR, outer primer, middle primer, and inner primer is shown in Tables 28 and 36. The characteristics of target amplification products, the characteristics of primer unpaired products and reaction conditions are shown in Tables 37, 38, and 40, respectively. The conditions such as the reaction volume per tube were the same as in Example 1. Table 36. Example 14 primer sequence

Bow position order (5, to 3,)

1388- 1407 CAT CCC CCA AAG TTC ACA AT

A7 Medium ¾

1470- 1491 GCA AGG GAC TTC CTG TAA CAA T

1424- 1445 ATT GCA CAT TGT TGT T

A8 inner sleeve

1457-1474 ACA ATG CAT CTC ATA TTT

Table 37. Example 14 primer and amplification product characteristics

Table 38. Characteristics of various possible non-matched amplification products of Example 14

Table 39. Example 14 reaction conditions

Results In the third-stage reaction annealing temperatures of 40, 42.9, 45.0, 47.6, 50.6, 53.1, 55.2, and 56.6 ° C, 51-base-long inner jacket amplification products were obtained without any non-specific product bands. Example 15. Design and implementation of ultra-low denaturation temperature nested PCR detection of HBV.

The HBV primers reported in the literature were selected, and their lengths were adjusted in the 5 'region as outer and inner primers, respectively. The order and measurement characteristics are shown in Tables 40 and 41. '' Table 40. Example 15 primer sequence

* Based on the sequence of HBV variants with gene bank number E00010, bold underlined letters represent modified nucleotides Table 41. Example 15 primer and amplification product characteristics

The length and melting temperature of the unpaired amplification product of the outer positive primer and inner reverse primer and the outer positive primer and outer reverse primer were determined to be 310 bases, 85.6 ° C and 292 bases, 85.6 ° C, respectively. According to these data, the first-stage annealing temperature was set to 78 ° C. At this temperature, only the outer primer could anneal to complete the amplification, and the inner primer amplification and unpaired amplification were blocked. Set the third-stage annealing temperature to 65 ° C and the denaturation temperature to 80 ° C. Under this condition, the inner primer can continue to use the first-stage amplification product as a template to carry out the amplification, and the coat primer and unpaired primers can be amplified. Although it can pass the annealing step, it is aborted because the denaturation step cannot be completed. Table 42 shows the approximate melting temperatures of the bow I compounds and the template with several mismatched bases calculated by the nearest neighbor base method.

Table 42. Approximate melting temperatures of the primers of Example 15

In order to detect HBV variants that may have several mismatches with the primers, the annealing temperature in the first stage can be reduced to 71 ° C. At this temperature, the jacket primers can be annealed with templates with 1 to 4 mismatches, thereby enabling detection. The false negatives were significantly reduced or completely eliminated; the inner primers were still unable to complete annealing at this temperature. Second The annealing temperature at this stage can be reduced to about 45 ° C. At this temperature, the inner primer can anneal to the template with 1 to 3 mismatches. Inner primers may have non-specific amplification at 45 ° F annealing temperature, but after the first stage of amplification, the number of target templates of inner primers is several orders of magnitude higher than various interference templates, which will not interfere with the assay. . Example 16. Design and implementation of triple nested PCR detection of HCV.

Design two sets of coat primers to reduce and eliminate false negatives. The outer and inner primers are located in the more conserved 5'-end non-coding region and C gene region of the HCV genome. Both are adjusted in length and melting temperature and regrouped according to the primers reported in the literature. The order and characteristics of the three primer pairs are shown in Tables 43 and 44.

Table 43. Example 16 primer sequence

Based on the sequence of HCV variants with gene bank number AF177039 Table 45. Example 16 primer and amplification product characteristics

Set the denaturation temperature in the first stage to 94 ° C and the annealing temperature to 75 ° C. Under these conditions, the two coat primers can be successfully amplified. The length and melting temperature of the amplified products of the outer primer 1 and the outer primer 2 and the outer primer 2 and the outer primer 1 were calculated to be 720, 91.4 ° C and 679, 91.6 ° C, respectively. The amplification was successful, and the four amplification products were the templates for the second-stage inner primer amplification. In the second stage, the denaturation temperature was set to 85 ° C, and the annealing temperature was 55-65 ° C. The inner primer can continue to amplify using the jacket amplification product as a template to obtain the target band. All four jacket amplification products could not be completed. Denaturation step to stop amplification. Example 17. Design and implementation of two independent nested PCR tests for HIV.

The first set of primers is in the gag gene region of the HIV virus, the second set of primers is in the env gene region, and the two pairs of coat primers are modified in length according to the literature guide. Two pairs of primers were selected among the respective coat amplification products. The order and characteristics of the four primer pairs are listed in Tables 46 and 47. Table 46. Example 17 primer sequences

Table 47. Example 17 primer and amplification product characteristics

Bowed product

Maximum allowable melting temperature

Name Length Length Location Fire temperature (° C) Degrees (° c) Degrees (° c)

32 86.6

HIV1 coat 1 319 83.1 1302-1620> 72

44 85.6

22 65.9

HIV2 inner sleeve 1 92 75.3 1491-1582 68.9

27 66.1

32 86.4

HIV3 coat 2 151 85.2 7759-7909> 72

32 86.8

18 65.5

HIV4 inner sleeve 2 42 75.0 7797-7838 68.5

23 66.7 In the first stage, the annealing temperature was set to 75 ° C. At this temperature, neither of the two inner primer pairs could be annealed to complete the amplification. In the second stage, the denaturation temperature is set to TTC and the annealing temperature is 55-65 ° C. At this temperature, the inner primer can continue to amplify using the jacket amplification product as a template, and the jacket primer amplification product cannot be denatured and aborted. The gag and env genes are more than 5000 bases apart, and the two nested PCR reactions do not interfere with each other. The melting temperatures of the first two unpaired primers are 83.5 ° C and 78.3 ° C; the melting temperatures of the second two unpaired primers are 81.5 ° C and 83.6, respectively. ° C, can not be denatured in the second stage.

Claims

Rights request

1. A polymerase chain reaction method for multiple pairs of primers. The following reactions are simultaneously performed on the template DNA strand from 2-10 pairs of primers:

'(1) template denaturation;

(2) primer annealing;

(3) DNA polymerase catalyzes the synthesis of complementary DNA strands;

(4) Perform steps (1)-(3) to cycle the amplification reaction;

It is characterized in that the entire reaction is divided into three stages. In the first 25 to 25 cycles, the annealing temperature ranges from 60 to 80 ° C, and in the third to 5 to 25 cycles, the denaturation temperature ranges from 65 to 60. At 87 ° C, there are 2 to 4 cycles of the second stage between the first and third stages. This stage has the same denaturation temperature as the first stage and the annealing temperature is the same as the third stage.

The polymerase chain reaction method for multiple primer pairs according to claim 1, characterized in that the template denaturation temperature is 92-97 ° C in the first 2-6 cycles of the first stage, and all are in the subsequent cycles 65—87 ° C.

The polymerase chain reaction method of multiple primers according to claim 2, characterized in that the denaturation temperature in the subsequent cycle is 75-83 ° C.

The method for polymerase chain reaction of multiple primers according to claim 1, characterized in that two pairs of nested primers are simultaneously used to perform amplification reaction on the same template DNA strand.

The method for polymerase chain reaction of multiple primers according to claim 4, characterized in that the first-stage annealing temperature is 72-78 ° C, and is higher than the melting temperature of the inner primer by more than 3 ° C.

The method for polymerase chain reaction of multiple pairs of primers according to claim 4, characterized in that the denaturation temperature range in the third stage is 75-82 ° C, which is higher than the melting temperature of the inner sleeve amplification product 1 Above ° C.

The polymerase chain reaction method of multiple primers according to claim 4, characterized in that the melting temperature range of the outer primer is 82-90 ° C, and the melting temperature range of the inner primer is 55-68 ° C. And the difference between the melting temperature of the outer primer and the inner primer is 15-25 ° C.

The polymerase chain reaction method according to claim 4, wherein the melting temperature range of the outer amplification product is 85-90 ° C, and the melting temperature range of the inner amplification product is 74-80 ° C, and the difference between the melting temperature of the outer and inner jacket amplification products is 4-15 ° C.

-9. The polymerase chain reaction method for multiple pairs of primers according to claim 1, characterized in that a single tube consisting of one pair of outer sleeves, one pair of middle sleeves and one pair of inner sleeve primers is designed in three stages. Triple nested PCR, with 3 pairs of nested primers to perform PCR reaction on the template DNA strand at the same time:

The first stage: 5-20 cycles, annealing temperature range 65-80 ° C;

. C;

The third stage: 5-20 cycles, the denaturation temperature range is 65-87Ό;

Moreover, there are 2 to 4 cycle transition phases between the first and second phases, and between the second and third phases, respectively. The denaturation temperature at this stage is the same as the previous stage and the annealing temperature is the same as the next stage. the same.

10. The reaction solution for polymerase chain reaction of a plurality of pairs of primers according to claim 1, characterized in that the concentration of each primer in the reaction solution is 0.02-0.2 μM.

11. The polymerase chain reaction method of multiple pairs of primers according to claim 1 for preparing mutants, serotypes, genotypes and subtypes or drug-resistant strains of various microorganisms such as viruses, bacteria, chlamydia, and mycoplasma. It is characterized in that multiple pairs of primers are designed for the specific sequences of mutant strains, serotypes, genotypes and subtypes or drug-resistant strains, and the melting temperature of each product is 65-87 ° C.

12. The polymerase chain reaction method of multiple pairs of primers according to claim 11, for preparing mutant strains, serotypes, genotypes, and subtypes or drug resistance of various microorganisms such as viruses, bacteria, chlamydia, and mycoplasma. Strain detection reagents are used, which are characterized in that the various microorganisms include hepatitis B virus, hepatitis C virus, human immunodeficiency virus, human papilloma virus, tuberculosis bacillus, gonorrhea, staphylococcus aureus, and pyu Bacillus, Salmonella, Pneumococcus, E. coli, Chlamydia trachomatis and Mycoplasma urealyticum.