CN100389206C - PCR method of multiple primer, its reaction liquor and application for preparing detection reagent - Google Patents

Abstract

The present invention relates to a polymerase chain reaction (PRC) method of multiple primers, reaction liquid thereof and applications thereof in preparing microbiological detection reagents, particularly to a method using two denaturation temperatures of 92 to 97 DEG C and 65 to 87 DEG C to respectively carry out initial 2 or 3 circulations and other subsequent circulations. The method overcomes the disadvantages that a non-specific product is formed by the non-specific pairing amplification of positive and negative primers of the existing multiple PCR aiming at the same DNA chain. The proper design of the products and the arrangement of the two denaturation temperatures can obviously lower the possibility that a non-target product synthesized in the earlier stage of PCR participates the subsequent circulations. The present invention is suitable for preparing single-tube type multiple PCR quick-detecting reagents, and has good application prospects at the aspects of the detection of various variants, serological types, genotypes or subtypes of viruses, such as hepatitis B viruses, hepatitis C viruses, human immunodeficiency viruses, human papilloma viruses, etc., the detection of bacterium drug-resistance genes, such as tubercle bacilli, etc.

Description

A kind of PCR method of multi-primers and reaction solution thereof and the application in the preparation detection reagent

Technical field

The present invention relates to Protocols in Molecular Biology, particularly relate to a kind of method and reaction solution and the application in the preparation detection reagent of polymerase chain reaction.

Technical background

Classical polymerase chain reaction method was invented the eighties mid-term, and it adopts each special genes fragment that increases of positive and negative primer.Soon, developed again in a reaction tubes with increase simultaneously multiplex PCR (Multiplex-PCR) method of several products of primer more than two pairs or two pairs.Several primers of existing multiplex PCR to usually with the complementation of several different target genes order, these target genes are carried on separately independently on the cDNA in proper order, or are carried on the same genomic dna but apart distance is quite far away.Existing multiplex PCR is usually used in more heterogeneic expression level, and then available multiplex PCR detects two or more pathogeny body simultaneously on clinical detection, as detecting HAV, HBV and HCV simultaneously.Existing multiplex PCR is several to the primer except using simultaneously, almost completely consistent with Standard PC R on primer design and reaction conditions, the specificity of amplified production is mainly controlled by annealing temperature in standard pcr, thereby being formed among the existing multi-primers PCR of non-single-minded product often is difficult to avoid, this makes that the primer logarithm of multiplex PCR can not be too many, otherwise, manyly make the PCR reaction itself be tending towards complicated to target product and each to the formed non-single-minded product of primer, final product also is difficult to obtain confirming from electrophoretic band.Existing multiple PCR method more is not suitable for the same gene that increases, because in this case except target product and non-single-minded product, also usually can form the amplified production between the non-matching primer of target gene.Yet, great using value is being arranged particularly reducing in the Clinical Laboratory on the false negative that virogene detects with the multiplex PCR same target gene that increases.

PCR has been widely used in the detection that the detection of microorganism in the human body comprises the infectious diseases pathogenic agent, its principle is that the gene order of virus, bacterium or other microorganisms is different with human body, can the single-minded primer of design height according to certain gene order of target microorganism, if the people infects this microorganism, then from some position of human body such as blood, extract DNA or RNA, can increase by PCR method obtains the special genes fragment, otherwise then denys.But Clinical Laboratory practice shows to detect with PCR method and exists serious false negative problem when microorganism particularly detects virus.For example existing tens of pieces of papers are pointed out to have proved hepatitis B male patient according to clinical and immunization method detection, and but often PCR method detects negative.False negative produces except the reason of technological layer such as nucleic acid purification is slipped up and sample contains the material that suppresses the PCR reaction, mainly is that the polytropy of virogene causes.Found and illustrated the HBV of its order, the mutation of virogene such as HCV and HIV is so many, even, also may mate fully with all mutation hardly, just may cause false negative as annealing under very rigorous temperature to such an extent as to any a pair of primer is selected in the most conservative zone.So adopt the standard pcr of a pair of primer can not overcome the false negative that causes owing to mutation order diversity.

We choose each six of positive primer and reactants from 35 couple of reported in literature detects the primer of HBV, analyze each genotypic representative bacterial strain complementary situation in proper order in they and 7 the HBV genotype, result such as table 1 with primer-design software Oligo.

The complementary situation analysis of table 1.12 a HBV primer and 7 representative bacterial strain orders

Most of primers of the presentation of results reported in literature of table 1 only with minority or indivedual genotype fully or matched, some primer then has a large amount of mispairing to exist with all representative strain, and only S8 (-) mates fully with all genotype representative strains in selected 12 primers.But, S8 () is shown that as homology analysis it also has serious mispairing in proper order with quite a few mutation with software BLAST.These facts show: though the conserved regions of virogene also extremely difficulty find a pair of primer, can mate fully with the mutation of all known sequences, though in addition it is not to contain all mutation that gene pool has had the order of a large amount of mutation.Obviously, primer is made multiplex PCR in that the design of the different zones of a virogene is many in a reaction tubes, can reduce or eliminate effectively under the situation that detect step because the false negative that mutation order diversity causes not increasing.Suppose that 3 pairs of primers of design and 20%, 30% or 40% mutation have serious mispairing, 20%, 30% or 40% false negative is then arranged respectively during the application standard PCR method, in various degree reduction must be arranged if make the multiplex PCR false negative with three pairs of primers.Further the supposition variation is that then false negative can be reduced to 2.4% at random.

Multiple PCR method of the present invention each to primer design on, the melting temperature(Tm) of amplified production is low as considering one of standard, each can finish amplification to primer under lower follow-up denaturation temperature, thereby controls each non-single-minded amplification to primer effectively by annealing temperature and the dual sifting machine system of denaturation temperature.Can effectively stop the amplification between the non-matching primer when multiplex PCR of this ultralow denaturation temperature is used to increase same gene, the false negative defective that adopts multiplex PCR to overcome microorganism detection is achieved.Use same principle and can detect virus with a pair or more of universal primer, and with other serotype specific primer virus is carried out somatotype simultaneously, perhaps use other (resistance) sequence specific primers to differentiate mutant strain such as the endurance strain that some has clinical value simultaneously.

Summary of the invention

Technical problem to be solved

Technical problem to be solved by this invention provides a kind of polymerase chain reaction method and reaction solution and application in the preparation detection reagent of multi-primers, to break through the restriction of multi-primers design in the existing multiple PCR method, overcome a plurality of segmental defective that the interior PCR of single tube reacts can not increase simultaneously independently independently same or different dna sequence dnas.

Inventive concept

Multi-primers PCR method of the present invention is also used the primer more than two pairs, but all differs widely with existing multiplex PCR on design of primers principle, product property and PCR reaction process.In the multi-primers of the present invention each all is a order according to To Template to primer, the denaturation temperature characteristic of amplified production as mainly considering standard design, the quantity of primer increases greatly than conventional multiplex PCR, can be adjacent between the To Template, and synthetic non-specific amplification product loses the function of its template in follow-up circulation because of unwinding making in earlier stage after 2 or 3 circulations to make PCR, thereby reduce or eliminate the complicacy of the PCR product that causes owing to the interaction of non-matching primer in the multiplex PCR amplification procedure, increase the specificity and the sensitivity of reaction.

Technical scheme

Polymerase chain reaction method of the present invention is to carry out the PCR reaction at the template DNA chain simultaneously by primer more than 2 pairs, and wherein the template denaturation temperature is 92-97 ℃ in preceding 2 or 3 circulations, is 65-87 ℃ in follow-up circulation, preferred 75-83 ℃.Primer is right to being 2-10 in the reaction, designs in different DNA chains, and perhaps all primers all design in same template DNA chain.

Be used for the reaction solution of above-mentioned multi-primers PCR, the optimal concentration of each bar primer in reaction solution is 0.02-0.2 μ M.

The polymerase chain reaction method of above-mentioned multi-primers is applicable to the various viruses of preparation, bacterium, chlamydozoan, the detection reagent of microorganisms such as mycoplasma, be used to reduce and eliminate the false negative that detects, said virus can be hepatitis B virus, the hepatitis C virus human immunodeficiency virus, papilloma virus etc., said bacterium can be a tubercule bacillus, gonorrhea diplococcus, streptococcus aureus, Pseudomonas aeruginosa, Salmonellas, pneumococcus, intestinal bacteria etc., said chlamydozoan can be a chlamydia trachomatis etc., and said mycoplasma can be a Ureaplasma urealyticum etc.

The polymerase chain reaction method of above-mentioned multi-primers is suitable for preparing serotype, genotype and hypotype thereof or the chemical sproof detection reagent of various microorganisms such as above virus, bacterium, chlamydozoan, mycoplasma, method is that one of multi-primers is designed in serotype, genotype and hypotype thereof or chemical sproof specific sequence, and satisfying the amplified production melting temperature(Tm) simultaneously is 65-87 ℃.

Primer and product method of design are: at first, a type strain of selected target gene, different zones in its order comprises conservative region and hypotype character zone and site, seek with primer software, or draw right zone and the length of multiple PCR primer that satisfies the technique scheme requirement according to the preliminary structure of characteristics such as melting temperature(Tm) of amplified production by primer software; Then, the some or several regions at the variant of this type strain and 2-5 or more tool different subtype feature or other features comprises conservative region and hypotype character zone, and primary election is a pair of or some to primer.At last, consider the complementarity of primer pair and the order of various variants, primer between factors such as 3 ' end complementary characteristic, the primer of selecting multiplex PCR is right.

The Advantage of Clontech company 2 test kits are all adopted in PCR reaction of the present invention.

Beneficial effect (2 and 4 repeat more)

1. when multiplex PCR of the present invention is used to increase different templates, the melting temperature(Tm) of the amplified production that designs in proper order according to each template is all very low, control the specificity of amplification by rigorous annealing temperature and ultralow denaturation temperature double mechanism, the electrophoretic band number of PCR is consistent with the primer logarithm, and the right existing multiplex PCR of logarithmic ratio of multi-primers improves greatly.

2. in these cases, if use annealing temperature than low about 3 to 12 ℃ of the highest permission annealing temperature, even primer and template order have the mispairing about 1 to 4, still can finish amplification smoothly, this has has just reduced or eliminated the false negative that causes because of the variation of template order.Under the situation of unusual low temperature thermal oxidation, by the control denaturation temperature the non-single-minded product that forms in initial 2 to 3 circulations of PCR is miscarried in follow-up circulation, thereby guarantee the specificity of amplified production.And in Standard PC R and existing multiplex PCR, reducing the false negative that causes when annealing temperature causes non-single-minded product and takes rigorous annealing temperature can't make the best of both worlds.

3. when multiplex PCR of the present invention was used to increase same template, if each to primer all and template matches, each target sequence that can increase under rigorous annealing temperature and ultralow denaturation temperature obtained the electrophoretic band from same target gene effectively single-mindedly.The different bands of more same sample can be judged each amplification efficiency to primer; Each band of more different sample rooms can obtain several groups of data simultaneously by an inferior test of pipe, and the viral load or the genetic expression of different samples are carried out sxemiquantitative relatively.

4. in these cases, as using annealing temperature, even then each has the mispairing about 1 to 4 still can finish amplification and non-single-minded band not occur to primer and template order than low about 3 to 12 ℃ of the highest permission annealing temperature.As long as a pair ofly can amplification obtain a target amplified band because have in to primer, just show that target gene exists multiplex PCR some.So ultralow denaturation temperature and low temperature thermal oxidation multiplex PCR provide very wide space for the various variants that detect any virus.Be not difficult to analyze, if choose the primer about 3 pairs in several conservative relatively zones of a virogene, make each amplified production melting temperature(Tm) all quite low, so, no matter how complicated the variation of virus order is, the false negative that causes because of primer order and the mispairing of target gene order can overcome and avoid fully.In other words, the good ultralow denaturation temperature multiplex PCR of design can detect various variants and need not worry false negative.

5. the multiplex PCR at the ultralow denaturation temperature of same template also can be used for detecting simultaneously target gene and differentiates its allelic mutation.Can design in this case and contain two pairs or more ultralow denaturation temperature multiplex PCRs primer, wherein a pair of primer is common to two allelotrope, another is that wild (or sudden change) allelotrope is peculiar to a primer of primer or 3 ' end base or contiguous 3 ' several bases of holding of two primers, under rigorous annealing temperature and ultralow denaturation temperature.Wild (or sudden change) amplified allele obtains two bands, and (or wild) allelotrope that suddenlys change only increases and obtains a band, like this, just simplifies the PCR reaction of having fallen positive control.Conventional PCR method also can be differentiated allelotrope, but must make a positive controlled trial when negative findings occurring to show that negative findings is not that reason owing to the round pcr aspect causes.

6. virus often can be divided into different genotype or serotype with bacterium, big meaning is arranged in clinical diagnosis and treatment, existing multiplex PCR is restriction to some extent in the detection of the less same virus sequence of genome, and the design primer detects virus and differentiates that simultaneously its hypospecificity gene is also difficult.The invention solves this problem.If can find out the distinctive order that is different from other hypotypes to various hypotypes then use the 5th method, can detect virus simultaneously and differentiate the hypotype of virus.

Embodiment

Embodiment 1.

The comparison of ultralow denaturation temperature multi-primers PCR and conventional multiplex PCR.

(gene pool numbering BC016045) goes up three pairs of primers of design at people actomyosin gene, and its order and characteristic are listed in table 2 and 3 respectively.

The primer order of table 2. embodiment 1

The primer characteristic of table 3. embodiment 1

Because thrihydrid is at same target gene and be separated by nearerly, can form the non-matching amplified production under the conventional multiplex PCR condition, its length and melting temperature(Tm) measuring and calculating are shown in table 4.

Table 4. embodiment 1 non-matching primer extension product characteristic

The non-matching primer	Amplified production length (bp)	The amplified production melting temperature(Tm) (℃)
			Positive primer of A01 and A02 anti-primer	360	87.9
Positive primer of A01 and A03 anti-primer	701	85.8
			Positive primer of A02 and A03 anti-primer	444	82.4

The Advantage-2 test kit of Clontech is adopted in the PCR reaction of embodiment 1, every tube reaction volume is 5 microlitres, each primer final concentration is 0.1uM, each reaction tubes contains the cDNA1 microlitre, and cDNA is that primer is synthetic by its regulation with the reverse transcription test kit of Clontech by people's total tissue RNA with Poly (dT) 18.

The PCR reaction conditions divides two groups, and first group is conventional multiple PCR method, i.e. 95 ℃ of sex change first 1 minute are annealed 62 ℃ of 30 second in 95 ℃ of 30 second of circulation sex change, extend 72 ℃ 1 minute, totally 28 circulations, extend at last 72 ℃ 3 minutes.Second group is ultralow denaturation temperature multiple PCR method, sex change first, and cycle annealing, circulation are extended identical with first group with the condition of last extension, and junior three circulation sex change also is 95 ℃ of 30 second, but 25 round-robin sex change thereafter change 80 ℃ of 30 second into.Test-results sees Table 5.

Table 5. embodiment 1 comparative test result

+ expression has the no band of one or several strips, 0 expression, digitized representation band number

Embodiment 2.

The multiplex PCR of 6 pairs of primers.

Design of primers sees Table 6.

The primer characteristic sees Table 7.

Non-matching primer extension product characteristic sees Table 8.

Table 6. embodiment 2 primers order

Table 7. embodiment 2 primer characteristics

People actomyosin base is long less than 2000 bases, and 6 pairs of primers have the possibility of 15 kinds of non-matching combination amplifications, and the amplified production length and the melting temperature(Tm) of measuring and calculating are shown in table 8.

Table 8. embodiment 2 non-matching primer extension product characteristics

The melting temperature(Tm) of 6 pairing amplified productions all is lower than 76.8 ℃, and the melting temperature(Tm) of 15 non-matching amplified productions all is higher than 80.9 ℃.Embodiment 2 reaction reagents are identical with embodiment 1 with condition, but follow-up circulation sex change is 79 ℃ of 30 second, and the result comprises that with the right arbitrary combination of A04-A09 primer the multiple PC of 6 pairs of primers all obtains amplified production, and does not have non-matching product and non-single-minded amplified production.

Embodiment 3.

The ultralow denaturation temperature multiplex PCR design and the implementation condition that contain 10 pairs of primers.

With primer-design software Oligo Cyclin-D gene (gene pool numbering NC_053056) is analyzed, searched 10 pairs and have high preciseness and the melting temperature(Tm) of amplified production all is lower than 81 ℃ primer.The position of 10 pairs of primers, characteristic and 45 non-matchings disturb the characteristic of amplified production to list in table 9 and table 10.

10 pairs of primers that table 10. pair Cyclin D genetic test obtains

Primer is to sequence number	The product position	Length	Amplified production 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

Each test result of table 11. to mutual interference product between the primer

Primer is to sequence number	Disturb primer to sequence number	The 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
					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 result shows that the Tm value of interference product is between 82.1-92.1 ℃, and it is most of between 83-87 ℃, remarkable and normal product is not in same temperature range, that is to say, may obtain 10 pairs of primers in the sequence of 4300 bases of this gene, the melting temperature(Tm) that satisfies amplified production and interference product is not in the design requirements of same scope.Being 81 ℃ as follow-up circulation denaturation temperature can get rid of the interference product sex change and only obtain the target amplification product.

Embodiment 4.

Multi-primers PCR reduces or eliminates hepatitis B virus and detects false positive design and implementation condition.

With the primer (table 1) of detection HBV commonly used, by design philosophy of the present invention its length and order are made amendment, cooperate again and form three pairs of primers, its order and characteristic are shown in table 12 and 13.The virus that the Nucleotide of extrabold letter representative through changing, primer location all are numbered E00010 with gene pool becomes strain and is as the criterion in proper order.

The characteristic of three non-matching interference product of three pairs of primers is listed in table 14.

Table 12. embodiment 4 primer sequences

Table 13. embodiment 4 primer sequence characteristics

Table 14. embodiment 4 non-matchings are disturbed the amplified production characteristic

The non-matching primer	Amplified production length	The amplified production melting temperature(Tm) (℃)
			Positive primer of HBV01 and HBV02 anti-primer	1564	87.5
Positive primer of HBV01 and HBV03 anti-primer	1900	87.2
			Positive primer of HBV02 and HBV03 anti-primer	414	84.6

This shows that pairing product melting temperature(Tm) is all less than 79 ℃, and non-matching disturbs the amplified production melting temperature(Tm) all greater than 84 ℃.According to the result of these data and embodiment 1 and 2, annealing temperature is controlled at 50-63 ℃, follow-up denaturation temperature is controlled at 80-83 ℃, and each all can be to mate with it or the mutation of matched is that template amplifies target product single-mindedly fully to primer.

Embodiment 5.

Detect design and the implementation condition that hepatitis B virus detects the associating sudden change simultaneously with the multi-primers method.

(background note: gene pool store 1000 surplus hepatitis B virogene order and clinical detection analysis revealed both domestic and external, A takes place 1762 and 1764 of C gene promoter and changes the what is called that T and G change A into into and unite sudden change in about 1/4th hepatitis B virus simultaneously, the resultant velocity of transcribing of the mRNA of HBV is decreased, thereby relevant with severity of disease certain clinical relation arranged with treatment.Usually use PCR-RFLP, promptly PCR-restriction enzyme segment polymorphism method differentiates that the associating mutant strain of wild strain and C gene promoter (sees that China tests and the clinical virology magazine 2000,14 (2) 163p; Check magazine 1999,22 (5) is learned by China).This method need be carried out secondary PCR, wherein once introducing T to A at the 1767th artificially suddenlys change, make the PCR product of mutant strain contain the enzyme point of contact order TGATCA of a Bcl-I, and wild strain PCR product is AGTTCA in proper order, do not cut by the Bcl-I enzyme, to amplified production carry out enzymolysis and then electrophoresis mirror can differentiate wild-type and mutant.The complicated operations process has limited its applying in clinical diagnosis.In addition, some mutation is also undergone mutation at the 1763rd, 1765 and 1766, make artificial introduce 1767 sudden change after, 1762 and 1764 associating mutant strains still can not get the TGATCA order, thereby are not cut by enzyme and false negative occurs and judge.)

Present embodiment designs a pair of general primer, in order to check the existence of HBV virus.Design two groups of primers be used to differentiate wild-type and the associating mutant, wherein one group positive primer has two, all with 1764 as its 3 ' end.And the anti-primer of another group has two, all with 1762 as its 3 ' end, total primer and being used to detects the order and the characteristic of the primer of associating sudden change and lists in table 15 and table 16.

The primer order of table 15. embodiment 5

Annotate: the HBV04 primer location expands the mutation that is numbered E00010 with gene and is as the criterion in proper order; The position of HBV05 and HBV06 primer is numbered the AF479684 mutation with gene pool and is as the criterion in proper order.

The characteristic of the primer of table 16. embodiment 5

Detect the general primer HBV04 of HBV and the target amplification product melting temperature(Tm) of detection associating mutant primer HBV05 and HBV06 and all be lower than 82 ℃, when follow-up denaturation temperature is 83 ℃, all can finish denaturing step.The positive primer of " wild-type " anti-primer of HBV05 and " wild-type " of HBV06 all can 65 ℃ with the wild-type template annealing, and under same temperature, all can not finish amplification with associating mutant template annealing.Equally, the positive primer of " mutant " anti-primer of HBV05 and HBV06 " mutant " can 65 ℃ with associating mutant template annealing, and under same temperature can not with the wild-type template annealing.Each sample divides two pipes during detection, and it is right that each pipe all contains the HBV04 primer, positive primer of HBV05 and HBV06 anti-primer.Wherein the first pipe also contains two " wild-type " primers, and the second pipe also contains two " mutant " primers.119,46 and 68 3 band interpret sample occurring as first pipe PCR result is wild-type, and three band interpret sample occurring as the second pipe is 1762 and 1764 associating mutants.All not having the band interpret sample as the first and second two pipes is the HBV feminine gender.

Embodiment 6.

Multi-primers PCR reduces or eliminates hepatitis C virus and detects false-positive design and implementation condition.

The non-coding region of HCV upstream region of gene is quite conservative, a large amount of primers of reported in literature are taken from this zone, choose 4 by design philosophy of the present invention from 4 pairs of primers wherein, and after aspects such as primer length adjust slightly, again be made into 2 pairs of primers, its order and property column and table 17 and table 18.

Table 17. embodiment 6 primers order

^*Be numbered 177039 HCV mutation is as the criterion in proper order with gene pool

Table 18. embodiment 6 primer characteristics

Long 284 bases of amplified production between positive primer of HCV01 and the HCV02 anti-primer, 89.7 ℃ of amplified production melting temperature(Tm)s.So when 55-64 ℃ of control annealing temperature, 84 ℃ of follow-up denaturation temperatures can be finished the synthetic of target amplification product.Disturb amplified production in the initial non-matching that forms of reaction, miscarriage when 84 ℃ of follow-up denaturation temperatures.

Embodiment 7.

Multi-primers PCR reduces and eliminates the human immunodeficiency virus and detect false-negative design and implementation condition.

Conservative region gag and env gene in HIV virus respectively designs 2 pairs of primers respectively, and wherein, it is right that HIV01 is selected from the primer of reported in literature, only primer length made an amendment slightly.The positive primer of other three pairs of primers all is selected from the reported in literature primer, and its length is also made an amendment slightly.The order of 4 pairs of primers and characteristic are listed in table 19 and 20.

Table 19. embodiment 7 primers order

^*Ordinal position is as the criterion with the HIV mutation that gene pool is numbered U23487

Table 20. embodiment 7 primer characteristics

The melting temperature(Tm) of the target amplification product of 4 pairs of primers all is lower than 78 ℃, when annealing temperature is 55-68 ℃, when follow-up denaturation temperature is 80 ℃, can finish amplification single-mindedly.Gag gene and env gene are separated by and are surpassed 5000 bases, so, the primer of HIV01 and HIV02 not can with the interference amplification of the primer generation non-matching of HIV03 and HIV04, and the non-matching of positive primer of HIV01 and HIV02 anti-primer and positive primer of HIV03 and HIV04 anti-primer disturb the length of amplified production and melting temperature(Tm) through measuring and calculating be respectively 280 bases, 82.3 ℃ and 557 bases, 83.3 ℃, when follow-up denaturation temperature is 80 ℃, can not finish amplification because of being obstructed at denaturing step.

Embodiment 8.

Multi-primers PCR detects the design and the implementation condition of different shaped human papillomavirus simultaneously.

What human papillomavirus had been identified has 70 kinds approximately, and the order in each type is more conservative, and the difference between type is bigger.Wherein, 6 types, 11 types are more common or relevant with malignant tumour with 16 types etc.Embodiment 8 a pair of primers of design and 16 types coupling, the primer that it takes from reported in literature only adjusts on length, called after HPV16-01.Other designs two pairs of primers and 11 and 6 types coupling, called after HPV11-01 and HPV11-02.The positive primer of HPV11-01 and the anti-primer of HPV11-02 adjust on length slightly according to the reported in literature primer.The positive primer of the anti-primer of HPV11-01 and HPV11-02 joins, and this position order is used as probe in reported in literature.The order and the characteristic of three pairs of primers are shown in table 21 and table 22.

Table 21. embodiment 8 primers order

^*HPV16 primer order and position are as the criterion with the 16 type HPV that gene pool is numbered NC_001526, and the order of HPV11 primer and position are as the criterion with the 11 type HPV that gene pool is numbered M14119

Table 22. embodiment 8 primer characteristics

The melting temperature(Tm) of above-mentioned 3 pairs of primer target amplification products all is lower than 78.1 ℃, is 50-64 ℃ when controlling annealing temperature, and when follow-up temperature was 80 ℃, target product can increase single-mindedly.The 210bp band occurs behind the PCR reaction solution electrophoresis dying, be illustrated as the HPV16 type, occur 43, if there are not above-mentioned three type HPV in the explanation of no band or/and 64bp band interpret sample is HPV11 or 6 types.If improve follow-up denaturation temperature to 82 ℃, also the 107bp band can appear, and be the amplified production of positive primer of HPV11-01 and HPV11-02 anti-primer, also interpret sample exists HPV11 type or HPV6 C-type virus C.

Embodiment 9.

Multi-primers PCR detects hepatitis b virus hbv, hepatitis C virus HCV and human immunodeficiency virus HIV design and implementation condition simultaneously.

To HBV04, the primer of table 17 is made multiplex PCR to the primer of HCV01 and table 19 to HIV03, is 50-64 ℃ in annealing temperature with the primer of above-mentioned table 16, and follow-up denaturation temperature is under 84 ℃ the condition, three pairs of primers target products that all can increase single-mindedly.119bp behind the electrophoresis dying, 70bp and 146bp band are indicated HBV respectively, the existence of HCV and HIV.

Claims (7)

1. the polymerase chain reaction method of a multi-primers, carry out following reaction at the template DNA chain simultaneously by primer more than 2 pairs:

(1) template sex change;

(2) primer annealing;

(3) synthetic complementary dna chain is extended in archaeal dna polymerase catalysis down;

(4) set by step (1)-(3) circulation carrying out amplified reaction;

It is characterized in that the template denaturation temperature is 92-97 ℃ in preceding 2 or 3 circulations, is 65-87 ℃ in follow-up circulation.

2. polymerase chain reaction method according to claim 1 is characterized in that primer is right to being 2-10 in the reaction.

3. polymerase chain reaction method according to claim 1 is characterized in that all primers all design in different templates or same template DNA chain.

4. according to each described polymerase chain reaction method in the claim 1,2 or 3, it is characterized in that denaturation temperature is 75-83 ℃ in the follow-up circulation.

5. polymerase chain reaction method according to claim 1 is characterized in that the concentration of each bar primer in reaction solution is 0.02-0.2 μ M.

6. the polymerase chain reaction method of the described multi-primers of claim 1 is in preparation virus, bacterium, chlamydozoan, variant, serotype, genotype and the hypotype thereof of mycoplasma or the application in the Resistant strain detection reagent, it is characterized in that multi-primers design in variant, serotype, genotype and hypotype thereof or Resistant strain specific sequence, and each product melting temperature(Tm) is 65-87 ℃.

7. the application of reaction method according to claim 6 is characterized in that said virus, bacterium, chlamydozoan, mycoplasma comprise hepatitis B virus, hepatitis C virus, human immunodeficiency virus, human papillomavirus, tubercule bacillus, gonorrhea diplococcus, streptococcus aureus, Pseudomonas aeruginosa, Salmonellas, pneumococcus, intestinal bacteria, chlamydia trachomatis and Ureaplasma urealyticum.