CN102485912A - Research on GeXP multi-PCR technology for detecting and parting human papilloma virus (HPV) - Google Patents

Abstract

The invention which belongs to the biotechnological application field relates to a research on GeXP multi-PCR technology for detecting and parting HPV, and concretely relates to the detection and the typing of markers of cervical secretions and the like infected with various HPV subtypes (comprising HPV6, HPV11, HPV31, HPV33 and HPV52) in disease prevention control mechanisms at all levels, hospitals and the like. The research is concretely carried out by analyzing conserved sequences of HPV subtype genes with computer software, respectively designing specific primers aiming at the subtypes, and carrying out single-tube multi-PCR (six-PCR) (comprising Rnasep-DNA), wherein the whole reaction lasts for less than 2h. The research which overcomes a disadvantage of no parting of routine single-tube multiple fluorescent qualitative PCR detections and also overcomes disadvantages of complex operation, long time, high cost and the like of routine chip detection methods provides a new idea for HPV parting technologies, and characteristics of high specificity, high sensitivity and rapidness of the research provide strong technological supports for the realization of the rapid and accurate screening and discrimination of HPV subtype infection, so the research has important meanings to research the HPV infection type distribution characteristic of a patient and improve the control level of cancer pathologies caused by the HPV subtypes.

Description

The GeXP multiple PCR technique is used for the research of human papillomavirus HPV detection and somatotype

Invention field

The invention belongs to biological technology application; Relate to Disease Prevention and Control Institutions at different levels, the various human papilloma virus HPV hypotype that Sentinel point hospital etc. is used for samples such as cervical secretionses (comprises HPV6, HPV11; HPV31, HPV33 and HPV52) detect and somatotype when infecting.Concrete conserved sequence through each subtype gene of computer software analysis human papillomavirus HPV is designed the type specificity primer to each hypotype respectively, carries out single tube multiple (6 weight) PCR (comprising internal control gene Rnasep-DNA), and entire reaction was less than 2 hours.This patent both overcome conventional single tube multi-fluorescence qualitative PCR detect can not somatotype shortcoming; Also overcome the complex operation of conventional chip detecting method; Time is longer; The more high shortcoming of cost; For the typing method of HPV provides new thinking, its height is special, highly sensitive, characteristics are differentiated for the examination quick and precisely that realizes the HPV hypotype and infect fast provides strong technical support, and it is significant with the level of preventing and treating that improves the cancerous lesion that is caused by each hypotype of HPV that research patient HPV is infected the type characteristic distributions.

Background of invention

Cervical cancer is a kind of malignant tumour of serious harm WomanHealth, and sickness rate occupies second in women's malignant tumour, still occupies first in some developing countries and regions.According to the global range statistics, cervical cancer accounts for 10% in all gynecologic malignant tumors, and 471,000 routine new cases and 215,000 routine deaths are arranged every year.Human papillomavirus (HPV) is closely related with cervical cancer; It is a kind of double-stranded cyclic DNA virus; Form by early gene, late gene (late gene L1 and L2 encode respectively main capsid protein and less important capsid protein) and upstream regulation district three parts of not translating; Discover that the cervical cancer more than 99% has HPV to infect, HPV is the main virulence factor of cervical cancer.The relation of foundation and cervical cancer is divided into high-risk-type and low risk with HPV, and high-risk-type comprises: HPV16,18,31,33,35,39,45,51,52,56,58,66, types such as 69; Low risk comprises: 6, types such as 11,43,44.In the high-risk hypotype of HPV, the infection rate of southern area of China 52, the 58 hypotypes north is high, in Taiwan except that HPV16,18; 31,33,35 infection rate is higher; Explanation 16,18,31,33,52,58 is the most common hypotype in Chinese, but because Chinese region is broad, populous; So the data of this respect comparatively lacks, remain further to be studied.In the low danger hypotype, HPV6,11 is common infection hypotype, can cause diseases such as huge condyloma, pointed condyloma, respiratory tract papilloma, papilloma of conjunctiva.To further study pathogenic, treatment and the prognosis situation of different subtype HPV in different ethnic populations; Understand the variation tendency of HPV clinical infection type and generation and the death that reduces cervical cancer, just need set up a kind of novel method clinical application, that can carry out the detection of examination and somatotype simultaneously to all known high-risk types and common low danger type HPV that is suitable for.So the common HPV31 of Chinese is tentatively chosen in this research, 33,52 three kinds of high-risk-types and HPV6,11 two kinds of low risks are as research object.

At present, the HPV classifying method has a lot, and common have cytology detection method and a Protocols in Molecular Biology method, tests (HC), hybridization in situ technique (ISH) and PCR method etc. like liquid-basedcytology technology and smear Automatic Measurement Technique, hybrid capture.Wherein, round pcr is comparatively ripe, has characteristics such as quick, easy, sensitivity.Since substance PCR flux not high limitation; A kind of novel multiple PCR technique based on GeXP is adopted in this research; It adopts universal primer to cause the target gene amplification, can effectively solve the amplification preferences in the multiplex PCR process, the detection sensitivity and the resolving power that adopt capillary electrophoresis and fluorescent mark amplified production to improve; According to amplified production fragment length judged result, help to improve detection specificity.In our previous work, utilization has successfully detected new influenza A virus and seasonal influenza virus based on the multiple PCR technique of GeXP.This research at first detects HPV6,11,31 with the substance round pcr; The HPV clinical sample that 33 and 52 substances infect with the specificity of checking GeXP primer, has been set up a kind of novel multiple GeXP-PCR technology for detection on this basis; To detect and to identify the common HPV6 of Chinese in the RTI simultaneously; Hypotypes such as 11,31,33 and 52.

Summary of the invention

1. the primer sequence of announcing with GenBank is reference; Downloading known array from NCBI uses CLUSTAL X software to carry out sequence alignment; Choose for being directed against each target gene high conservative for the constant gene segment C of other type high special; By eXpress Profiler Software design sixfold primer, like table 1:

Table 1 GeXP detects the nucleotide primer sequence table of human papillomavirus (HPV)

Annotate: what underscore was represented is upstream and downstream universal primer sequences

2. set up following testing process, seen for details as follows:

(1) synthetic primer: synthetic and purifying by Shanghai Ying Jun company.

(2) sample to be measured is pressed the DNA Mini Kit test kit step process of Qiagen company, obtained the DNA of sample.

(3) 6 heavy PCR reactions.

Embodiment

The reaction system of embodiment 1:Gexp multiplex PCR and condition

Adopt 25 μ l PCR reaction systems, wherein contain: Ex Taq12.5 μ l, upstream and downstream chimeric primers mixture (1 μ mol/L) 1.25 μ l, upstream and downstream universal sequence primer (10 μ mol/L) 1.25 μ l, dna profiling 1 μ l, the sterilization distilled water complements to 25 μ l.The pcr amplification condition is: 94 ℃, and 10min; (95 ℃, 30s; 56 ℃, 30s; 72 ℃, 30s) * 5 circulation; (95 ℃, 30s; 68 ℃, 30s; 72 ℃, 30s) * 15 circulation; (95 ℃, 30s; 48 ℃, 30s; 72 ℃, 30s) * 20 circulation; 72 ℃, 5min.

The specificity of embodiment 2:Gexp multiplex PCR

As template, do the multiplex PCR amplification with the hybrid dna of known five hypotypes with six pairs of primers (containing internal control gene RNasep-DNA) mixed solution.The result shows that the gene of 5 kinds of HPV subtype virus can be detected simultaneously, has verified the specificity of the medium-sized special primer of multiple detection architecture.

The sensitivity of embodiment 3:Gexp multiplex PCR

HPV6,11,31,33,52 five cloned plasmids carry out 10 times of serial dilutions after quantitatively, artificially mix five hypotype DNAs, concentration be respectively 10 ⁶Copies/ μ l, 10 ⁵Copies/ μ l, 10 ⁴Copies/ μ l, 10 ³Copies/ μ l, 10 ²Copies/ μ l, 10 ¹The mixed solution of copies/ μ l is made sample to be checked, carries out the sensitivity analysis of GeXP multiple PCR method, and the system that the Gexp multiplex PCR detects is with embodiment 1.The result shows, 10 ³Copy/μ L level can the simultaneously special simulation mixing sample that detects 5 kinds of HPV subtype virus.Each concentration is all at non-triplicate on the same day, the coming to the same thing of acquisition.

Embodiment 4: clinical sample analysis and checking

The result that the GeXP multiplex PCR detects 30 parts of clinical samples shows: the HPV6 substance infects 5 parts, and the HPV11 substance infects 4 parts, and the HPV31 substance infects 2 parts; The HPV33 substance infects 5 parts; The HPV52 substance infects 6 parts, 2 parts of HPV6/52 double infections, 1 part of HPV6/11/33 multiple infection.The result is by human papillomavirus nucleic acid amplification parting detecting reagent (Guangdong Chaozhou Kaipu Biochemistry Co., Ltd.) checking, and is in full accord.GeXP multiplex PCR detection architecture is with embodiment 1.

Claims (5)

1. one kind is used for detecting simultaneously human papillomavirus HPV6, HPV11, and HPV31, the multiple PCR technique of HPV33 and HPV52, comprising: be used for human papillomavirus HPV6, HPV11, HPV31, type specificity primer and universal primer that HPV33 and HPV52 detect.

2. the primer of claim 1 described multiple PCR technique and universal primer comprise the gene order that table 1 is listed and the complementary sequence or the variant of every kind of sequence thereof.

3. claim 1 described multiplex PCR detection technique comprises human papillomavirus HPV6, HPV11, HPV31, HPV33 and HPV52 and variant thereof.

4. claim 3 described ranges of application of the present invention comprise Disease Prevention and Control Institutions at different levels, and hospital is used for human papillomavirus HPV6, HPV11, and HPV31, HPV33 and HPV52 detect and somatotype simultaneously.

5. the claim 1 described human papillomavirus HPV6 that detects simultaneously, HPV11, HPV31, the response procedures of the multiple PCR technique of HPV33 and HPV52 and trace routine.