CN1824803A - Process for detecting and typing all known genital tract human papillomavirus - Google Patents

Abstract

This invention discloses a method of quickly distinguish and classify existing genital meatus human papilloma virus. The method can distinguish existing 48 type HPV that can infect genital meatus mucous membrane and its normal mixed infection. And it can select Rsa I,Mse I,Pst I and HaeIII four normal using inscribing enzyme combinations according to optimal selection, optimize assort and at the same time the sensitivity and specificity can be ensured. Then HPV gene type is judged according to the established genital HPV gene type MY09/11 amplification product RMPH enzyme inscribing data. The operation is simple and time can be saved, the cost is low, its equipment needed exists in county hospital, and its cost is very low. It can be high pass quantity experienced, the technical operability of developing HPV inflection epidemiology investigation.

Description

A kind of method that detects with typing all known genital tract human papillomavirus

Technical field

The present invention relates to a kind of method that detects genital tract human papillomavirus, relate in particular to a kind of PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism analysis) method that detects with the nearly all known genital tract human papillomavirus of somatotype.

Background technology

Cervical cancer is the modal reproductive tract malignant tumour of women, now confirmer's papilloma virus (Humanpapillomavirus HPV) is the crucial paathogenic factor of cervical cancer and cercinoma prophase pathologic change thereof.The HPV genotype has found that at present 200 is many types of, and wherein about 48 types are relevant with the reproductive tract mucosa infection.Because different reproductive tract mucosal pattern HPV genotype has different biological characteristicses, different risks, so carry out somatotype when need detect to HPV; Carrying out simultaneously along with vaccine research, the classifying method of setting up good HPV uses extremely important with the specific aim that is used for vaccine, but up to now, still not having a kind of suitable method can detect and somatotype all present known HPV that infects the reproductive tract mucous membrane.Many to the research of the somatotype of papilloma virus in recent years, but the method susceptibility that has is very low, as Southern blotting and in situ hybridization method; The method that has can only simply be determined high-risk-type and low risk and can not somatotype such as hybrid capture method; Can't judge during mixing sample though the detection to the term single gene type that has is the most accurate, as order-checking; What have can't avoiding method institute inherent problem, can't definitely avoid the cross hybridization problem as hybridization; The present technical costs that has is very expensive, as the DNA chip.Present existing pvuii restriction fragment (RFLP) method can only detect limited a few type HPV, as HPV16,18 and 6,11.

In a word, up to the present, also do not have a kind of method can reach the specific requirement of susceptibility and can detect present all known genital tract mucosal pattern HPV.

Summary of the invention

The objective of the invention is at the deficiencies in the prior art, provide a kind of and reaching the PCR-RFLP method that can detect under the prerequisite that susceptibility and specificity require with all known reproductive tract mucosal pattern human papillomaviruss of somatotype.Use method of the present invention can detect all known HPV genotype that can infect the reproductive tract mucous membrane, and the PCR that adopts in the method guarantee the susceptibility of detection; The specificity and the accuracy that are used in combination the assurance detection of multiple restriction enzyme.

The objective of the invention is to be achieved through the following technical solutions: a kind of method that detects with typing all known genital tract human papillomavirus, it is characterized in that, comprise the steps:

(1) extracts DNA in cervical secretions or the tissue sample, as the template of pcr amplification;

(2) carry out pcr amplification with universal primer MY09/11 or its improvement primer;

(3) agarose gel electrophoresis is judged PCR result, and the electrophoretic band person who 442～458bp occurs is positive;

(4) with RsaI, MseI, PstI and HaeIII positive PCR product being carried out enzyme respectively cuts;

(5) each enzyme is cut product and carry out agarose gel electrophoresis, measure each endonuclease bamhi length;

(6) cut the fragment length of product according to the enzyme of each enzyme, contrast reproductive tract HPV genotype MY09/11 amplified production RMPH enzyme is cut database and is carried out the genotypic judgement of HPV.

The invention has the beneficial effects as follows: the present invention has redesigned the PCR-RFLP method that is used to detect reproductive tract mucosal pattern HPV fully, particularly selecting for use of restriction enzyme can accomplish to distinguish nearly all known HPV genotype that can infect the reproductive tract mucous membrane, make the clinicist assess patient infect the issuable risk of this HPV genotype, and take therapy measure in time to treat, carry out large-scale examination and can obtain detailed epidemic data and help cervical cancer developing vaccines development and specific aim in the future to use thereby use this method simultaneously.This method is simple to operation, save time, and cost is low, and required equipment (PCR instrument) is popularized in County Hospital, and expense is very cheap, can carry out the high-throughput experiment, and this provides technical operability for the epidemiology survey of carrying out HPV in China and infecting.

Description of drawings

Fig. 1 is a method flow diagram of the present invention.

Embodiment

Describe the present invention below in detail.

The present invention selects reproductive tract mucosal pattern HPV universal primer MY09/11 (or its improve primer, as PGMY09/11) for use, the nucleotide sequence of the high conservative region 450bp in nearly all reproductive tract mucosal pattern HPV L1 district that is used to increase.Finding 48 type HPV of present known PI reproductive tract mucous membrane earlier according to documents and materials, is respectively HPV 6,11,13,16,18,26,30～35,39,40,42～45,51～59,61,62,64,66～74,81～87,89～90.From NCBI, find the genotypic sequence of these HPV, cut on the software at enzyme and with 206 kinds of restriction endonucleases commonly used the pcr amplified fragment of this 48 type HPV is carried out theoretic enzyme and cut, select restriction endonuclease according to the otherness of endonuclease bamhi.Because the HPV genotype is numerous, finding does not have single a kind of restriction endonuclease can fully distinguish these genotype.Under the prerequisite that can distinguish above-mentioned 48 type HPV and common polyinfection thereof, principle according to optimal selection, optimization collocation, select four restriction endonucleases combinations commonly used of RsaI, MseI, PstI and HaeIII, and the enzyme of setting up each HPV genotype MY09/11 amplified fragments is cut database.Consider the practical situation of agarose gel electrophoresis, the following fragment of 100bp is not included interpretation and somatotype in according to scope.

The reproductive tract HPV genotype MY09/11 amplified production RMPH enzyme of setting up is cut database and is seen the following form 1.

Table 1: reproductive tract HPV genotype MY09/11 amplified production RMPH enzyme is cut database

Type	RsaI			MseI		PstI		HaeIII
											hpv11	216	134		399		241	208	218	123	108
hpv13	175	134		201	199	241	214	204	128	123
											hpv16	309			175	135	216	211	443
hpv18	134	125		183		241	214	455
											hpv26	364			123	113	352	103	455
hpv30	449			449		241	208	231	218
											hpv31	379			232	120	216	211	329	123
hpv32	216	160		317	132	449		317	123
											hpv33	236	101		135	132	241	208	449
hpv34	186	160	97	205	94	253	180	335	123
											hpv35	177	158		231	135	427		261	180
hpv39	259	123		204	138	331	124	446
											hpv40	366			140	126	455		446
hpv42	242	134		249		449		449
											hpv43	337			317		278	177	332	123
hpv44	222	160		206	183	455		215	123	108
											hpv45	337			183		241	214	446
hpv51	379			135	80	452		380
											hpv52	449			174	130	424		258	182
hpv53	447			315	132	447		229	218

hpv54	137	125	117	270			452		218	126	108
													hpv55	165	160		206	183		455		215	123	108
hpv56	309			267	107		241	208	275	165
													hpv57	449			449			297	152	449
hpv58	305	112		137	132	105	216	208	449
													hpv59	455			160	91		430		400	55
hpv6	160	149		399			449		218	123	108
													hpv61	185	179		252	123		455		212	210
hpv62	358			205	167		340	109	231	218
													hpv64	186	160		205	141		433		335	123
hpv66	449			115	93		208	150	440
													hpv67	310			243	132		424		267	182
hpv68	259			251	138		455		455
													hpv69	455			198	123		455		224	182
hpv70	231	123		183	138		455		231	117	107
													hpv71	379			207	193		359	91	216	126	108
hpv72	364			207	170		455		221	210
													hpv73	201	160	97	205	94		433		458
hpv74	165	160		204	183		458		218	182
													hpv81	452			208	99		340	112	126	113	108	96
hpv82	309			136			455		446
													hpv83	377			275	99		318	132	384
hpv84	311	141		275	99		452		347	105
													hpv85	382			251	138		455		455
hpv86	377			276	99		318	132	343	107
													hpv87	377			264	99		352	98	232	209
hpv89	379			265	99		452		317	126
													hpv90	310	140		250	132		450		232	209

The step that is used to detect the PCR-RFLP method of reproductive tract mucosal pattern provided by the present invention is:

1. from cervical secretions or tissue sample, extract DNA, as the template of pcr amplification;

2. carry out pcr amplification with universal primer MY09/11 (or its improve primer, as PGMY09/11);

3. agarose gel electrophoresis is judged PCR result, and (± 8bp) electrophoretic band person is positive 450bp to occur;

4. with RsaI, MseI, PstI and HaeIII positive PCR product being carried out enzyme respectively cuts;

5. each enzyme is cut product and carry out agarose gel electrophoresis, measure each endonuclease bamhi length;

6. cut the fragment length of product according to the enzyme of each enzyme, the genotypic judgement of HPV is carried out in contrast " reproductive tract HPV genotype MY09/11 amplified production RMPH enzyme is cut database ".

Embodiment:

1. get the cervical secretions that 200 routine cervical cancers and cervical intraepithelial neoplasia become the patient, extract DNA

1) cotton swab is got cervical secretions, and vibration was abandoned cotton swab after several minutes in 1ml physiological saline, preserves liquid and is sample;

2) get sample 500 μ l, add 200 μ l lysates (containing the 40ug Proteinase K), 55 ℃ of 30-60min;

3) 1: 100 phenol chloroform of adding equivalent is used forced oscillation 15s, leaves standstill 5min on ice;

4) 4 ℃ of centrifugal 10min of 12000rpm get supernatant;

5) add 2 times dehydrated alcohol, leave standstill 30-60min on ice;

6) 4 ℃ of centrifugal 30min of 12000rpm abandon supernatant;

7) 70% ethanol of adding 1000ml, 4 ℃ of centrifugal 10min of 12000rpm abandon supernatant;

8) repeating step 6;

9) dry, add 100 μ l TE (PH=8.0), be the DNA that obtains after the dissolving, as the template of pcr amplification.

2. carry out pcr amplification with universal primer PGMY09/11

PCR reaction system cumulative volume is 50 μ l, is made up of Taq archaeal dna polymerase (5U/ μ l), the DNA of 3 μ l and the distilled water of 34.5 μ l of PGMY11 (10pmol), the 0.5 μ l of PGMY09 (10pmol), the 1 μ l of 10mM dNTP, the 1 μ l of 25mMMgcl, the 1 μ l of 10 * PCR buffer, the 4 μ l of 5 μ l.

Set up positive control and negative control with strictness control experiment quality simultaneously, wherein containing the positive contrast of Siha cell DNA of HPV16, with the negative contrast of DNA of the SKOV3 ovarian cancer cell that do not contain HPV.

The PCR loop parameter is as follows: 96 ℃ of pre-sex change are after 5 minutes, 95 ℃ of 45s, and 57 ℃ of 45s, 72 ℃ of 45s, totally 40 circulations, 72 ℃ were extended 10 minutes then.

3.2% agarose gel electrophoresis, the electrophoretic band person who occurs about 450bp is the PCR positive.

Restriction fragment length polymorphism (RFLP) analyze with RsaI, MseI, PstI and four restriction enzymes of HaeIII to the PCR product respectively enzyme cut.

Endonuclease reaction system cumulative volume is 20 μ l, by 10 * buffer of 2 μ l, and the enzyme of 1 μ l (10U/ μ l), the distilled water of the PCR product of 8 μ l and 9 μ l is formed.

Endonuclease reaction condition: 37 ℃ of incubation 2-4h.

5. each enzyme is cut product and carry out 2% agarose gel electrophoresis, measure each endonuclease bamhi length.

6. cut the fragment length of product according to the enzyme of each enzyme, the genotypic judgement of HPV is carried out in contrast " reproductive tract HPV genotype MY09/11 amplified production RMPH enzyme is cut database ".

7. sequence verification: select several samples to check order respectively at random to detected each genotype of RFLP, detect the accuracy that RFLP detects.

The result

Detecting HPV in 200 routine cervical cancers and intraepithelial neoplasia (cin) patient altogether infects 172 examples (86.0%) and detects HPV genotype 22 types altogether, dual polyinfection 15 examples (7.5%).Each genotype and infection rate thereof are followed successively by HPV16 (45.0%), 58 (12.0%), 52 (6.5%), 33 (4.0%), 45 (3.0%), 18 (2.5%), 31 (2.5%), 51 (2.5%), 59 (2.5%), 6 (2.0%), 39 (2.0%), 11 (1.5%), 66 (1.5%), 68 (1.5%), 53 (1.0%), 61 (0.5%), 67 (0.5%), 69 (0.5%), 82 (0.5%), 26 (0.5%), 40 (0.5%) and 56 (0.5%).Select the result who checks order consistent at random with the judgement of RFLP.

The foregoing description is used for the present invention that explains, rather than limits the invention, and in the protection domain of spirit of the present invention and claim, any modification and change to the present invention makes all fall into protection scope of the present invention.

Claims (2)

1. the method that can detect with typing all known genital tract human papillomavirus is characterized in that, comprises the steps:

(1) extracts DNA in cervical secretions or the tissue sample, as the template of pcr amplification.

(2) carry out pcr amplification with universal primer MY09/11 or its improvement primer.

(3) agarose gel electrophoresis is judged PCR result, and the electrophoretic band person who 442～458bp occurs is positive.

(4) with Rsa I, Mse I, Pst I and HaeIII positive PCR product being carried out enzyme respectively cuts.

(5) each enzyme is cut product and carry out agarose gel electrophoresis, measure each endonuclease bamhi length.

(6) cut the fragment length of product according to the enzyme of each enzyme, contrast reproductive tract HPV genotype MY09/11 amplified production RMPH enzyme is cut database and is carried out the genotypic judgement of HPV.

2. the method that detects with typing all known genital tract human papillomavirus according to claim 1 is characterized in that described improvement primer is PGMY09/11.

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