CN100389206C - A PCR method with multiple primers, its reaction solution and its application in the preparation of detection reagents - Google Patents

Abstract

The invention relates to a polymerase chain reaction method with multiple primers and its reaction solution and its application in the preparation of microorganism detection reagents, in particular to two denaturation temperatures of 92-97°C and 65-87°C in the PCR process. The first 2 or 3 cycles and other subsequent cycles overcome the defect of non-specific pairing and amplification between forward and reverse primers in the existing multiplex PCR for the same DNA chain, resulting in the formation of non-specific products. Through appropriate product design and two denaturation temperature settings, the possibility of non-target products synthesized in the early stage of PCR participating in subsequent cycles is significantly reduced, and it is suitable for the preparation of single-tube multiplex PCR rapid detection reagents, which are used in hepatitis B virus and hepatitis C Hepatitis virus, human immunodeficiency virus, human papillomavirus and other virus variants, serotype, genotype or its subtype detection, and bacterial drug resistance gene detection such as Mycobacterium tuberculosis have good application prospects.

Description

A PCR method with multiple primers, its reaction solution and its application in the preparation of detection reagents

technical field

The invention relates to molecular biology technology, in particular to a polymerase chain reaction method, its reaction solution and its application in the preparation of detection reagents.

technical background

The classic polymerase chain reaction method was invented in the mid-1980s. It uses a forward primer and a reverse primer to amplify a specific gene segment. Soon, a multiplex PCR (Multiplex-PCR) method was developed in which two or more pairs of primers were used to simultaneously amplify several products in one reaction tube. Several primer pairs of current multiplex PCR are usually complementary to several different target gene sequences carried on separate cDNAs, or carried on the same genomic DNA but separated by a considerable distance from each other. The current multiplex PCR is often used to compare the expression levels of different genes. In clinical testing, multiplex PCR can be used to detect two or more pathogens at the same time, such as the simultaneous detection of HAV, HBV and HCV. In addition to using several pairs of primers at the same time, the current multiplex PCR is almost completely consistent with the standard PCR in terms of primer design and reaction conditions. In the standard PCR method, the specificity of the amplified product is mainly controlled by the annealing temperature, so it is not specific. The formation of a product is often difficult to avoid in the current multiplex primer PCR, which makes the number of primer pairs in multiplex PCR not too many, otherwise, multiple pairs of target products and non-specific products formed by each pair of primers will make the PCR reaction itself tend to Due to the complexity, the final product is also difficult to be confirmed from the electrophoresis strip. The current multiplex PCR method is even more unsuitable for amplifying the same gene, because in this case, in addition to the target product and non-specific products, amplification products between non-paired primers of the target gene are often formed. However, using multiplex PCR to amplify the same target gene has great application value in clinical testing, especially in reducing false negatives in virus gene detection.

PCR has been widely used in the detection of microorganisms in the human body, including the detection of infectious disease pathogens. The principle is that the gene sequence of viruses, bacteria or other microorganisms is different from that of the human body. Highly specific primers can be designed according to a certain gene sequence of the target microorganism. If a person is infected with the microorganism, DNA or RNA can be extracted from some parts of the human body such as blood, and a specific gene fragment can be amplified by PCR method, and vice versa. However, the practice of clinical testing shows that there are serious false negative problems when using PCR method to detect microorganisms, especially viruses. For example, dozens of papers have pointed out that patients who have been proved to be positive for hepatitis B according to clinical and immunological methods are often tested negative by PCR methods. In addition to technical reasons such as nucleic acid purification errors and samples containing substances that inhibit PCR reactions, false negatives are mainly caused by the variability of viral genes. There are so many variants of viral genes such as HBV, HCV, and HIV whose sequences have been discovered and elucidated that it is almost impossible for any pair of primers to match all variants exactly, even in the most conserved regions, as in Annealing at very stringent temperatures can cause false negatives. It is therefore impossible for standard PCR methods using a pair of primers to overcome false negatives due to variant sequence diversity.

We selected six positive primers and six reactants from the 35 pairs of primers reported in the literature to detect HBV, and used the primer design software Oligo to analyze their complementarity with the sequence of representative strains of each of the seven HBV genotypes. The results are shown in Table 1.

Table 1. Analysis of complementarity between 12 HBV primers and the sequences of 7 representative strains

The results in Table 1 show that most of the primers reported in the literature are only completely or highly matched with a few or individual genotypes, and some primers have a large number of mismatches with all representative strains. Among the 12 selected primers, only S8(-) is compatible with all The representative strain of genotype is completely matched. However, the homology analysis of S8 (1) with software BLAST shows that it also has serious mismatches with a considerable number of variant sequences. These facts show that: even in the conserved regions of viral genes, it is extremely difficult to find A pair of primers can completely match the variants of all known sequences. Moreover, although the gene bank has a large number of variant sequences, it does not cover all variants. Obviously, multiple pairs of primers are designed in different regions of a viral gene in a reaction tube Multiplex PCR can effectively reduce or eliminate false negatives caused by variant sequence diversity without increasing detection steps. Assuming that the designed 3 pairs of primers have serious mismatches with 20%, 30% or 40% of the variants, Then there are 20%, 30% or 40% false negatives respectively when applying the standard PCR method, if make multiple PCR false negatives with three pairs of primers, there must be reduction in various degrees. Further assume that variation is random, then false negatives can be reduced to 2.4%.

In the multiplex PCR method of the present invention, in the design of each pair of primers, the melting temperature of the amplification product is lower as one of the consideration criteria, and each pair of primers can complete the amplification at a lower subsequent denaturation temperature, thereby passing The dual screening mechanism of annealing temperature and denaturation temperature effectively controls the non-specific amplification of each pair of primers. When the multiplex PCR with ultra-low denaturation temperature is used to amplify the same gene, it can effectively prevent the amplification between non-paired primers, so that the use of multiplex PCR to overcome the false negative defect of microbial detection can be realized. Applying the same principle, one or more pairs of common primers can be used to detect viruses, while additional typing primers can be used to type viruses, or additional (drug-resistant) sequence-specific primers can be used to identify certain Clinically valuable mutant strains such as drug-resistant strains.

Contents of the invention

technical problem to be solved

The technical problem to be solved by the present invention is to provide a polymerase chain reaction method with multiple primers and its reaction solution and its application in the preparation of detection reagents, so as to break through the limitations of multiple primer design in the existing multiple PCR method and overcome the limitations of multiple primers in a single tube. The defect that a PCR reaction cannot amplify multiple fragments of the same or different DNA sequences independently and simultaneously without affecting each other.

Invention idea

The multiple primer PCR method of the present invention also uses more than two pairs of primers, but it is quite different from the existing multiple PCR in terms of primer design principles, product characteristics and PCR reaction process. Each pair of primers in the multiplex primers of the present invention is designed according to the order of the target templates, taking the denaturation temperature characteristics of the amplified products as the main consideration standard, and the number of primers is greatly increased compared with conventional multiplex PCR. It can be adjacent, and can make the non-specific amplification product synthesized in the previous stage lose its template function in the subsequent cycle after 2 or 3 cycles because it cannot be melted, thereby reducing or eliminating the due to The complexity of the PCR product caused by the interaction of unpaired primers increases the specificity and sensitivity of the reaction.

Technical solutions

The polymerase chain reaction method of the present invention is to carry out PCR reaction to template DNA strand simultaneously by more than 2 pairs of primers, and wherein template denaturation temperature is 92-97 ℃ in the first 2 or 3 cycles, and is 65-87 ℃ in subsequent cycles. °C, preferably 75-83 °C. There are 2-10 pairs of primers in the reaction, designed on different DNA strands, or all primers are designed on the same template DNA strand.

For the above-mentioned multiple primer PCR reaction solution, the optimal concentration of each primer in the reaction solution is 0.02-0.2 μM.

The polymerase chain reaction method of above-mentioned multiple primers is suitable for preparing the detection reagent of microorganisms such as various viruses, bacterium, chlamydia, mycoplasma, is used for reducing and eliminating the false negative of detection, and said virus can be hepatitis B virus, Hepatitis C virus human immunodeficiency virus, papillomavirus, etc., said bacteria can be Mycobacterium tuberculosis, Neisseria gonorrhoeae, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, Streptococcus pneumoniae, Escherichia coli, etc., said chlamydia It can be Chlamydia trachomatis etc., and said mycoplasma can be Ureaplasma urealyticum etc.

The polymerase chain reaction method of the above-mentioned multiple primers is suitable for preparing the detection reagents of serotypes, genotypes and subtypes thereof or drug resistance of various microorganisms such as the above viruses, bacteria, chlamydia, mycoplasma, and the method is to combine the multiple primers One is designed on the specific sequence of serotype, genotype and its subtype or drug resistance, and at the same time, the melting temperature of the amplified product is 65-87°C.

The primer and product design method is as follows: first, a typical strain of the target gene is selected, which includes conserved regions and subtype characteristic regions and sites in different regions of its sequence, and is searched with primer software, or with the help of primer software according to the amplified product. Preliminary construction of the regions and lengths of multiple PCR primer pairs that meet the requirements of the above-mentioned technical scheme for characteristics such as melting temperature; then, in the typical strain and 2-5 or more mutant strains with different subtype characteristics or other characteristics For one or several regions, including conserved regions and subtype characteristic regions, a pair or several pairs of primers are initially selected. Finally, considering factors such as the complementarity between the primer pair and the sequences of various mutant strains, the 3' end complementarity between the primer pair, and other factors, the primer pair for multiplex PCR was selected.

The PCR reactions of the present invention all adopt the Advantage 2 kit of Clontech Company.

Beneficial effects (2 and 4 repeat more)

1. When the multiplex PCR of the present invention is used to amplify different templates, the melting temperature of the amplified product designed according to each template sequence is very low, and the amplification is controlled by the double mechanism of rigorous annealing temperature and ultra-low denaturation temperature Specificity, the number of PCR electrophoresis bands is consistent with the number of primer pairs, and the number of multiple primer pairs is greatly improved compared with the current multiplex PCR.

2. Under the above circumstances, if the annealing temperature is 3 to 12°C lower than the maximum allowable annealing temperature, even if there are 1 to 4 mismatches between the primer and the template sequence, the amplification can still be completed smoothly, which reduces or False negatives due to template sequence variation are eliminated. In the case of very low annealing temperature, the non-specific products formed in the first 2 to 3 cycles of PCR can be aborted in subsequent cycles by controlling the denaturation temperature, thereby ensuring the specificity of the amplified product. However, in standard PCR and current multiplex PCR, lowering the annealing temperature leads to non-specific products and false negatives caused by strict annealing temperature cannot achieve the best of both worlds.

3. When the multiplex PCR of the present invention is used to amplify the same template, if each pair of primers matches the template, each target sequence can be effectively and specifically amplified under stringent annealing temperature and ultra-low denaturation temperature, and the target sequence from the same template can be obtained. Electrophoretic bands of target genes. By comparing different bands of the same sample, the amplification efficiency of each pair of primers can be judged; by comparing the bands of different samples, several sets of data can be obtained at the same time through one tube test, and the virus quantity or gene expression of different samples can be analyzed. semi-quantitative comparison.

4. Under the above circumstances, if the annealing temperature is 3 to 12°C lower than the maximum allowable annealing temperature, each pair of primers can complete the amplification even if there are about 1 to 4 mismatches with the template sequence without appearing Non-specific band. Because as long as one pair can amplify a target amplification band among several pairs of primers in multiplex PCR, it indicates that the target gene exists. Therefore, multiplex PCR with ultra-low denaturation temperature and low annealing temperature provides a very broad space for detecting various mutant strains of any virus. It is not difficult to analyze, if about 3 pairs of primers are selected in several relatively conserved regions of a virus gene, so that the melting temperature of each amplified product is quite low, then, no matter how complicated the variation of the virus sequence is, the order of the primers will False negatives caused by sequence mismatches with the target gene can be completely overcome and avoided. In other words, well-designed multiplex PCR with ultra-low denaturation temperature can detect various mutant strains without worrying about false negatives.

5. Multiplex PCR with ultra-low denaturation temperature for the same template can also be used to simultaneously detect target genes and identify their allelic mutations. In this case, an ultra-low denaturation temperature multiplex PCR with two or more pairs of primers can be designed, wherein one pair of primers is common to the two alleles, and one primer or two primers of the other pair The 3' base or several bases adjacent to the 3' end are unique to the wild (or mutant) allele, under stringent annealing temperature and ultra-low denaturation temperature. The wild (or mutant) allele is amplified to obtain two bands, while the mutant (or wild) allele is only amplified to obtain one band, thus simplifying the PCR reaction of the positive control. Conventional PCR methods can also identify alleles, but a positive control test must be done when a negative result occurs to show that the negative result is not caused by PCR technology.

6. Viruses and bacteria can often be divided into different genotypes or serotypes, which are of great significance in clinical diagnosis and treatment. The existing multiplex PCR is limited in the detection of the same virus sequence with a small genome. Design primers for detection It is also difficult to identify the subtype-specific genes of the virus at the same time. The present invention solves this problem. If the characteristic sequences different from other subtypes can be found for various subtypes, the method of point 5 can be used to simultaneously detect the virus and identify the subtype of the virus.

Detailed ways

Example 1.

Comparison of ultra-low denaturing temperature multiplex PCR with conventional multiplex PCR.

Three pairs of primers were designed on the human actomyosin gene (GenBank No. BC016045), and their sequences and properties are listed in Tables 2 and 3, respectively.

Table 2. The primer sequence of Example 1

Table 3. Primer properties of Example 1

Since the three pairs of genes target the same target gene and are relatively close to each other, unpaired amplification products will be formed under conventional multiplex PCR conditions, and their lengths and melting temperatures are shown in Table 4.

Table 4. Embodiment 1 Unpaired primer amplification product characteristics

unpaired primers Amplified product length (bp) Amplification product melting temperature (°C) A01 forward primer and A02 reverse primer 360 87.9 A01 forward primer and A03 reverse primer 701 85.8 A02 forward primer and A03 reverse primer 444 82.4

The PCR reaction of Example 1 adopts the Advantage-2 kit of Clontech, the reaction volume of each tube is 5 microliters, the final concentration of each primer is 0.1uM, and each reaction tube contains 1 microliter of cDNA, and the cDNA is prepared from human tissue total RNA with Poly (dT)18 is a primer synthesized according to its regulations using the reverse transcription kit of Clontech.

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, cycle denaturation at 95°C for 30 seconds, annealing at 62°C for 30 seconds, and extension at 72°C for 1 minute, a total of 28 cycles. Final extension at 72°C for 3 minutes. The second group is an ultra-low denaturation temperature multiplex PCR method. The conditions for the first denaturation, cycle annealing, cycle extension and final extension are the same as those of the first group. Change the cycle of denaturation to 80°C for 30 seconds. The test results are shown in Table 5.

Table 5. Embodiment 1 comparative test result

+ indicates one or several bands, 0 means no bands, and the number represents the number of bands

Example 2.

Multiplex PCR with 6 pairs of primers.

The primer design is shown in Table 6.

The primer properties are listed in Table 7.

The characteristics of the amplification products with unpaired primers are shown in Table 8.

Table 6. Example 2 primer sequence

Table 7. Example 2 Primer Characteristics

The base length of human actomyosin is less than 2000 bases, and 6 pairs of primers have the possibility of 15 kinds of unpaired combination amplification. The measured length and melting temperature of the amplified products are shown in Table 8.

Table 8. Embodiment 2 Unpaired primer amplification product characteristics

The melting temperatures of the six paired amplification products were all lower than 76.8°C, while the melting temperatures of the 15 unpaired amplification products were all higher than 80.9°C. Example 2 The reaction reagents and conditions are the same as in Example 1, but the subsequent cycle of denaturation is 79°C for 30 seconds. As a result, any combination of A04-A09 primer pairs including multiple PCR with 6 pairs of primers can obtain amplification products without non-pairing products and non-specific amplification products.

Example 3.

Design scheme and implementation conditions of ultra-low denaturation temperature multiplex PCR with 10 pairs of primers.

The Cyclin-D gene (GenBank No. NC_053056) was analyzed with the primer design software Oligo, and 10 pairs of primers with high stringency and the melting temperature of the amplified products were all lower than 81°C were searched. The positions and characteristics of the 10 pairs of primers and the characteristics of the 45 unpaired interference amplification products are listed in Table 9 and Table 10.

Table 10. 10 pairs of primers obtained by Cyclin D gene test

Primer pair number product location length Amplified product Tm 1 672--734 62 79.8 2 732-781 49 79.7 3 1397-1745 348 80.1 4 1901-2010 109 76.7 5 2065-2145 80 77.0 6 2327-2724 397 79.8 7 3061-3317 256 79.3 8 3635-3821 186 80.3 9 3824-3863 39 76.6 10 3988-4016 28 78.8

Table 11. Test results of mutual interference products between each pair of primers

Primer pair number Interfering primer pair sequence number Interference product location 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 2 4 732-2010 1288 87.0 2 5 732-2145 1413 87.0 2 6 732-2724 1992 85.9 2 7 732-3317 2585 85.7 2 8 732-3821 3089 86.0 2 9 732-3863 3131 86.0 2 10 732-4016 3284 86.5 3 4 1397-2010 613 83.1 3 5 1397-2145 748 83.7 3 6 1397-2724 1327 83.6 3 7 1397-3317 1920 83.9 3 8 1397-3821 2424 84.7 3 9 1397-3863 2466 84.8 3 10 1397-4016 2620 85.6 4 5 1901-2145 244 82.1 4 6 1901-2724 823 82.8 4 7 1901-3317 1416 83.7 4 8 1901-3821 1900 84.7 4 9 1901-3863 1962 84.9 4 10 1901-4016 2115 85.7

5 6 2065-2724 659 82.4 5 7 2065-3317 1252 83.6 5 8 2065-3821 1767 84.8 5 9 2065-3863 1798 84.9 5 10 2065-4016 1951 85.8 6 7 2327-3317 990 82.9 6 8 2327-3821 1494 84.6 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°C, and most of them are between 83-87°C, which is obviously not in the same temperature range as the normal product, that is to say, it is possible to obtain in the 4300-base sequence of the gene 10 pairs of primers meet the design requirement that the melting temperature of the amplification product and the interference product are not in the same range. If the subsequent cycle denaturation temperature is 81°C, the denaturation of interfering products can be excluded and only the target amplification product can be obtained.

Example 4.

Multiple primer PCR reduces or eliminates the false positive design scheme and implementation conditions of hepatitis B virus detection.

The commonly used primers for detecting HBV (Table 1) were modified in length and order according to the design concept of the present invention, and re-coordinated to form three pairs of primers, the order and characteristics of which are shown in Tables 12 and 13. Bold bold letters represent modified nucleotides, and the primer positions are based on the sequence of the virus variant with the gene bank number E00010.

The characteristics of the three unpaired interference products of the three pairs of primers are listed in Table 14.

Table 12. Example 4 primer sequences

Table 13. Example 4 Primer Sequence Characteristics

Table 14. Embodiment 4 Unpaired interference amplification product characteristics

unpaired primers Amplified product length Amplification product melting temperature (°C) HBV01 forward primer and HBV02 reverse primer 1564 87.5 HBV01 forward primer and HBV03 reverse primer 1900 87.2 HBV02 forward primer and HBV03 reverse primer 414 84.6

It can be seen that the melting temperatures of paired products are all lower than 79°C, while the melting temperatures of unpaired interference amplification products are all higher 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. Amplify the target product in one place.

Example 5.

The design scheme and implementation conditions for the simultaneous detection of combined mutations in the detection of hepatitis B virus by multiple primers.

(Background note: More than 1,000 hepatitis B virus gene sequences stored in the gene bank and clinical testing and analysis at home and abroad show that about a quarter of the hepatitis B virus simultaneously occurs at positions 1762 and 1764 of the C gene promoter. The so-called joint mutation that changes to T and G to A reduces the transcription and synthesis speed of HBV mRNA, so it has a certain clinical relationship with the severity and treatment of the disease. Usually PCR-RFLP, that is, PCR-limited The polymorphism method of endonuclease fragments can be used to identify wild strains and combined mutants of the C gene promoter (see Chinese Journal of Experimental and Clinical Virology, 2000, 14 (2) 163p; Chinese Journal of Laboratory Medicine 1999, 22 (5) ). This method needs to carry out secondary PCR, and wherein one artificially introduces T to A mutation at position 1767, so that the PCR product of the mutant strain contains a Bcl-I restriction site sequence TGATCA, while the wild strain PCR product sequence is AGTTCA, not cut by Bcl-I enzyme, enzymatically digests the amplified product and then electrophoresis can identify wild type and mutant type. The complicated operation process limits its application in clinical diagnosis. In addition, some variants are in Mutations also occurred at positions 1763, 1765 and 1766, so that after the mutation at position 1767 was artificially introduced, the joint mutant strain at positions 1762 and 1764 still could not obtain the TGATCA sequence, so it was not cut by the enzyme and a false negative judgment occurred.)

In this example, a pair of common primers is designed to check the existence of HBV virus. Two sets of primers were designed to identify the wild type and the joint mutant type, and one set of positive primers had two, both of which had position 1764 as their 3' ends. And another group of anti-primers has two, all with 1762 as its 3' end, the sequence and characteristics of common primers and primers for detecting joint mutations are listed in Table 15 and Table 16.

Table 15. Primer sequence of Example 5

Note: The position of the HBV04 primers is based on the variant sequence of the gene expansion number E00010; the position of the HBV05 and HBV06 primers is based on the variant sequence of the gene bank number AF479684.

Table 16. Characteristics of the primers of Example 5

The melting temperature of the target amplified products of the common primer HBV04 for detecting HBV and the combined mutation primers HBV05 and HBV06 for detecting HBV was lower than 82°C, and the denaturation step could be completed when the subsequent denaturation temperature was 83°C. Both the "wild-type" anti-primer of HBV05 and the "wild-type" forward primer of HBV06 could anneal to the wild-type template at 65°C, but could not anneal to the co-mutant template at the same temperature to complete amplification. Similarly, the "mutant" anti-primer of HBV05 and the "mutant" forward primer of HBV06 can anneal to the co-mutant template at 65°C, but cannot anneal to the wild-type template at the same temperature. During detection, each sample was divided into two tubes, and each tube contained HBV04 primer pair, HBV05 forward primer and HBV06 reverse primer. Wherein tube A also contains two "wild type" primers, while tube B also contains two "mutant" primers. If there are three bands of 119, 46 and 68 in the PCR results of tube A, it means that the sample is wild type, and if there are three bands in tube B, it means that the sample is a combined mutant of 1762 and 1764. If there are no bands in tubes A and B, it means that the sample is negative for HBV.

Example 6.

Design scheme and implementation conditions for multiple primer PCR to reduce or eliminate false positives in hepatitis C virus detection.

The non-coding region of the upstream of the HCV gene is quite conservative, and a large number of primers reported in the literature are taken from this region. According to the design concept of the present invention, 4 of the 4 pairs of primers are selected, and after slightly adjusting the length of the primers, etc., they are reconfigured. Form 2 pairs of primers, the order and characteristics of which are listed in Table 17 and Table 18.

Table 17. Example 6 Primer Sequence

^＊ The sequence of HCV variants with the gene bank number 177039 shall prevail

Table 18. Example 6 Primer Characteristics

The length of the amplification product between the HCV01 forward primer and the HCV02 reverse primer was 284 bases, and the melting temperature of the amplification product was 89.7°C. Therefore, when the annealing temperature is controlled at 55-64°C and the subsequent denaturation temperature is 84°C, the synthesis of the target amplification product can be completed. The unpaired interference amplification product formed at the beginning of the reaction was aborted at the subsequent denaturation temperature of 84°C.

Example 7.

Design scheme and implementation conditions of multiple primer PCR to reduce and eliminate false negative detection of human immunodeficiency virus.

Two pairs of primers were designed respectively in the conserved regions of HIV virus gag and env genes, wherein HIV01 was selected from the primer pairs reported in the literature, and the length of the primers was only slightly modified. The positive primers of the other three pairs of primers were all selected from the primers reported in the literature, and their lengths were slightly modified. The sequence and properties of the 4 pairs of primers are listed in Tables 19 and 20.

Table 19. Example 7 Primer Sequence

^＊ The sequence position is based on the HIV variant with the gene bank number U23487

Table 20. Example 7 Primer Characteristics

The melting temperatures of the target amplification products of the 4 pairs of primers are all lower than 78°C. When the annealing temperature is 55-68°C and the subsequent denaturation temperature is 80°C, the amplification can be completed exclusively. The distance between the gag gene and the env gene is more than 5000 bases, so the HIV01 and HIV02 primers will not cause unpaired interference amplification with the HIV03 and HIV04 primers, while the HIV01 forward primer and HIV02 reverse primer and HIV03 forward primer and HIV04 The length and melting temperature of the non-paired interference amplification products of the anti-primers were calculated to be 280 bases, 82.3°C and 557 bases, 83.3°C, respectively. increase.

Example 8.

Design scheme and implementation conditions of simultaneous detection of different types of human papillomaviruses by multiplex primer PCR.

About 70 types of human papillomaviruses have been identified, and the sequence within each type is relatively conservative, while the differences between types are relatively large. Among them, types 6, 11 and 16 are more common or related to malignant tumors. Example 8 A pair of primers were designed to match Type 16, which were obtained from literature reports and only adjusted in length, named HPV16-01. Two pairs of primers were designed to match types 11 and 6, named HPV11-01 and HPV11-02. The forward primer of HPV11-01 and the reverse primer of HPV11-02 were slightly adjusted in length according to literature reports. The reverse primer of HPV11-01 is connected with the forward primer of HPV11-02, and the sequence of this part is used as a probe in the literature report. The sequences and properties of the three pairs of primers are shown in Table 21 and Table 22.

Table 21. Example 8 Primer Sequence

^* The sequence and position of HPV16 primers are based on HPV type 16 with gene bank number NC_001526, and the order and position of primers for HPV11 are based on HPV type 11 with gene bank number M14119

Table 22. Example 8 Primer Characteristics

The melting temperatures of the target amplification products of the above three pairs of primers are all lower than 78.1°C. When the annealing temperature is controlled at 50-64°C and the follow-up temperature is 80°C, the target product can be amplified exclusively. If a 210bp band appears after electrophoresis staining in the PCR reaction solution, it indicates that it is HPV16; if a 43 or/and 64bp band appears, it indicates that the sample is HPV11 or 6; if there is no band, it indicates that the above three types of HPV do not exist. If the subsequent denaturation temperature is increased to 82°C, a 107bp band will also appear, which is the amplification product of the HPV11-01 forward primer and HPV11-02 reverse primer, which also indicates that the sample contains HPV11 or HPV6 viruses.

Example 9.

Design scheme and implementation conditions of simultaneous detection of hepatitis B virus HBV, hepatitis C virus HCV and human immunodeficiency virus HIV by multiple primer PCR.

The primer pair HBV04 in the above table 16, the primer pair HCV01 in the table 17 and the primer pair HIV03 in the table 19 were used for multiplex PCR. Under the condition that the annealing temperature was 50-64°C and the subsequent denaturation temperature was 84°C, all three pairs of primers could Specific amplification of target products. The 119bp, 70bp and 146bp bands after electrophoresis staining indicated the presence of HBV, HCV and HIV, respectively.

Claims (7)

1. a polymerase chain reaction method of multiple primers, the following reaction is carried out at the template DNA strand by 2 pairs of above primers simultaneously: (1) template denaturation; (2) primer annealing; (3) DNA polymerase catalyzed extension synthesis of complementary DNA strands; (4) Carry out the amplification reaction in cycles according to steps (1)-(3); It is characterized in that the template denaturation temperature is 92-97° C. in the first 2 or 3 cycles, and 65-87° C. in the subsequent cycles. 2. The polymerase chain reaction method according to claim 1, characterized in that primer pairs are 2-10 pairs in the reaction. 3. The polymerase chain reaction method according to claim 1, characterized in that all primers are designed on different templates or the same template DNA strand. 4. The polymerase chain reaction method according to any one of claims 1, 2 or 3, characterized in that the denaturation temperature in subsequent cycles is 75-83°C. 5. The polymerase chain reaction method according to claim 1, characterized in that the concentration of each primer in the reaction solution is 0.02-0.2 μM. 6. the application of the polymerase chain reaction method of multiple primers as claimed in claim 1 in the mutant strain, serotype, genotype and subtype thereof or drug-resistant strain detection reagent of preparation virus, bacterium, chlamydia, mycoplasma, its It is characterized in that multiple primers are designed on the specific sequences of mutant strains, serotypes, genotypes and their subtypes or drug-resistant strains, and the melting temperature of each product is 65-87°C. 7. the application of reaction method according to claim 6 is characterized in that said virus, bacterium, chlamydia, mycoplasma comprise hepatitis B virus, hepatitis C virus, human immunodeficiency virus, human papillomavirus, Mycobacterium tuberculosis , Neisseria gonorrhoeae, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, Streptococcus pneumoniae, Escherichia coli, Chlamydia trachomatis and Ureaplasma urealyticum.