CN110656203B - Human papilloma virus typing detection method, reagent and application - Google Patents
Human papilloma virus typing detection method, reagent and application Download PDFInfo
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Abstract
The invention relates to the technical field of pathogen detection, in particular to a method, a reagent and application for human papillomavirus (human papilloma virus, HPV) typing detection. The method comprises the following steps: detecting at least two types of HPVs in the same fluorescent channel of the same PCR reaction tube; judging the type of each HPV in at least two types of HPVs by using at least two probes respectively corresponding to the at least two types of HPVs in the same fluorescent channel; wherein the Tm values of the at least two probes are different.
Description
Technical Field
The invention relates to the technical field of pathogen detection, in particular to a method, a reagent and application for human papillomavirus (human papilloma virus, HPV) typing detection.
Background
Human papillomaviruses (Human Papillomavirus, HPV) belong to the family papilloma virus, the non-enveloped double-stranded circular DNA virus, with specific tropism for the epidermis and mucosal squamous epithelium. Cervical cancer is the second largest malignancy that jeopardizes female health, and recurrent or persistent infection with high-risk HPV is a necessary condition for cervical cancer to occur. Infection with HPV increases the relative risk of cervical cancer by a factor of hundred, whereas it takes about 5-10 years for cervical cancer to eventually progress from persistent infection with high-risk HPV to general pre-cervical lesions. In fact, more than about 95% of cervical cancers are caused by high risk HPV infections, especially recurrent or persistent infections of HPV16, 18, 31, 33, 39, 45. Therefore, the HPV is subjected to typing detection in a targeted manner, and the method has important significance for early diagnosis and treatment of cervical cancer.
The current methods for typing HPV virus in clinic mainly include PCR-based target sequence amplification methods such as fluorescent PCR method, dissolution curve method, capillary electrophoresis method and signal amplification-based detection methods such as hybrid capture method. The traditional dissolution curve method can only detect one HPV type by a single fluorescent channel (the types of HPV are distinguished by the fluorescent channel on the basis that the Tm value of the dissolution curve peak diagram accords with the design expectation), so the number of the fluorescent channels limits the number of the HPV types which can be identified by a single-tube reaction system, and the effect diagram is shown in figure 1. FIG. 1 shows the detection results of HPV31 and HPV39 by the conventional TaqMan probe dissolution curve method, wherein the peak represented by curve 101 is the detection effect of HPV31 on the FAM channel and the peak represented by curve 102 is the detection effect of HPV39 on the HEX channel.
At present, the TaqMan probe fluorescent PCR method and the dissolution curve method for HPV typing detection are limited by the technical principle, the signal of one fluorescent channel can only reflect the detection result of one target, the number of targets which can be detected by a single reaction tube is limited by the number of fluorescent channels of a PCR instrument, and if nearly 20 HPV viruses with stronger correlation with disease occurrence are typed to be detected, the HPV viruses need to be divided into multiple tubes to react. The HPV infection is mostly single-type infection, and the problems of reagent waste, instrument occupation and complicated operation can occur when a multitube reaction system is used for detecting a single target.
Disclosure of Invention
The invention provides a human papillomavirus typing detection method, a reagent and application thereof, which can detect targets with more than channels in the same reaction tube.
In a first aspect, there is provided a method for human papillomavirus HPV typing detection comprising:
detecting at least two types of HPVs in the same fluorescent channel of the same PCR reaction tube;
judging the type of each HPV in at least two types of HPVs by using at least two probes respectively corresponding to the at least two types of HPVs in the same fluorescent channel; wherein the Tm values of the at least two probes are different.
In some embodiments, the determining the type of each HPV of the at least two HPV types using at least two probes respectively corresponding to the at least two HPV types in the same fluorescent pathway comprises:
detecting a change in fluorescence intensity during the renaturation to denaturation of the DNA molecule;
generating a fluorescence curve by a fluorescence quantitative PCR instrument according to the change conditions of the fluorescence intensities;
and judging the type of each HPV in the at least two types of HPVs according to the fluorescence curve and the Tm values of the at least two probes.
In some embodiments, the amplifying at least two types of HPV comprises: amplifying at least HPV31 and HPV39; wherein, the primer pair for amplifying HPV31 consists of a sequence shown in SEQ ID NO.1 and a sequence shown in SEQ ID NO. 2; the primer pair for amplifying HPV39 consists of a sequence shown in SEQ ID NO.3 and a sequence shown in SEQ ID NO. 4.
In some embodiments, the nucleotide sequence of the probe corresponding to HPV31 is shown in SEQ ID No.5 and the nucleotide sequence of the probe corresponding to HPV39 is shown in SEQ ID No. 6.
In a second aspect, there is provided a set of DNA molecules for HPV typing detection comprising:
primer pairs for amplifying at least two HPVs;
at least two probes corresponding to the at least two HPV types, respectively, the at least two probes having different Tm values.
In some embodiments, the primer pair for amplifying at least two HPV comprises: a primer pair for amplifying HPV31 and a primer pair for amplifying HPV39; wherein, the liquid crystal display device comprises a liquid crystal display device,
the primer pair for amplifying HPV31 consists of a sequence shown in SEQ ID NO.1 and a sequence shown in SEQ ID NO. 2; the primer pair for amplifying HPV39 consists of a sequence shown in SEQ ID NO.3 and a sequence shown in SEQ ID NO. 4.
In some embodiments, the nucleotide sequence of the probe corresponding to HPV31 is shown in SEQ ID No.5 and the nucleotide sequence of the probe corresponding to HPV39 is shown in SEQ ID No. 6.
In a third aspect, there is provided a PCR reagent for HPV typing detection comprising the DNA kit of the second aspect.
In a fourth aspect, there is provided a kit for HPV typing detection comprising a kit of DNA molecules as described in the second aspect.
In a fifth aspect, there is provided the use of a set of DNA molecules according to the second aspect or PCR reagents according to the third aspect or kits according to the fourth aspect in the preparation of a product for HPV typing detection.
The method and the reagent for detecting human papilloma virus can detect multiple HPV types in one tube and make corresponding groups, solve the problem of small flux of the conventional quantitative PCR reagent, shorten the detection time of a large number of samples, and have simple and convenient operation and low cost.
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FIG. 1 shows the detection results of HPV31 and HPV39 by the conventional TaqMan probe dissolution curve method;
FIG. 2 shows the detection results of HPV31 and HPV39 by the detection method of the embodiment of the invention.
Detailed Description
The technical scheme of the invention is further described in detail through the drawings and the embodiments.
The scheme provided by the embodiment of the invention is improved based on the original TaqMan probe method, besides the types of fluorescent signals are used for identifying the HPVs of different types, the TaqMan probes with different Tm values are designed for identifying the types of the HPVs according to the difference of the Tm values, the limit of one fluorescent channel corresponding to the detection of one HPV type is broken through, and the single-fluorescence multi-type detection effect is realized. The scheme provided by the embodiment of the invention can realize the parting detection of multiple HPVs by a single fluorescent channel, break through the limitation of the number of fluorescent channels on the identification of HPV type types by a single-tube reaction system, and effectively reduce the number of reaction tubes.
Next, in each embodiment, a scheme provided by the embodiment of the present invention will be specifically described.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. These techniques are well described in the prior art literature and see, in particular, sambrook et al MOLECULAR CLONING: a LABORATORY MANUAL, second edition, cold Spring Harbor Laboratory Press,1989and Thirdedition,2001; ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, john Wiley & Sons, new York,1987and periodic updates; the series METHODS IN ENZYMOLOGY, academic Press, san Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, academic Press, san Diego,1998; METHODS IN ENZYMOLOGY, vol.304, chromatin (p.m. wassman and a.p. wolffe, eds.), academic Press, san Diego,1999; and METHODS IN MOLECULAR BIOLOGY, vol.119, chromatin Protocols (p.b. becker, ed.) Humana Press, totowa,1999, etc.
The scheme provided by the invention aims to solve the defect that one fluorescent channel can only detect one HPV type in the fluorescent PCR technology and the traditional TaqMan probe dissolution curve technology, realizes the effect of typing detection of multiple HPVs by a single fluorescent channel, breaks through the limitation of the number of the fluorescent channels on the number of the types of the HPVs which can be identified by a single-tube reaction system, and identifies the types of the HPVs from two dimensions of the types of fluorescent signals and the Tm values.
The scheme provided by the invention designs the technical principle of a TaqMan probe dissolution curve, wherein the technical principle is as follows.
The technique involves two parts, an asymmetric PCR and a dissolution profile. The basic principle of the asymmetric PCR (asymmetric PCR) is that a large amount of single-stranded DNA is generated by amplifying a template by adopting a pair of primers with unequal amounts, so that the template is hybridized with a TaqMan probe, and in order to prevent the hydrolysis of the TaqMan probe, the DNA polymerase used in the extension stage is mTaq polymerase with the 5 '. Fwdarw.3' exonuclease activity deleted; after the asymmetric PCR reaction is completed, the incubation temperature is reduced, so that the TaqMan probe is hybridized with the single-stranded DNA product of the asymmetric PCR, at the moment, the distance between the fluorescent group and the quenching group of the probe reaches the maximum, the fluorescence intensity in the reaction system also reaches the maximum, then the temperature is slowly increased, the TaqMan probe is denatured and separated from the DNA single-stranded product, the fluorescence value in the system begins to decrease, the renaturation and the denaturation process are represented by using a graph of temperature and the fluorescence value, a peak of the change of the fluorescence value can appear, and the type of the target can be judged according to the Tm value corresponding to the peak.
According to the scheme provided by the embodiment of the invention, detection of multiple types of HPV can be realized in a single-tube reaction system. The plurality of types of HPV include, but are not limited to, any or all of the following combinations.
HPV6, HPV11, HPV42, HPV43, HPV44, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV53, HPV66, HPV73, HPV81, HPV82, HPV83 and the like.
Next, in example 1, detection of HPV31 and HPV39 in FAM channels is exemplified, and other types of HPV can be realized by referring to the schemes described in the examples.
Example 1 detection of HPV31 and HPV39 in FAM channel
E6 gene sequences of target HPVs 31 and HPV39 are downloaded from NCBI database, primer probe design is carried out by using Oligo 7.56, probes of HPV31 and HPV39 are designed to be different Tm values, FAM fluorescent groups are marked at the 5 'end, and BHQ1 quenching groups are marked at the 3' end. Then constructing E6 genes of HPV31 and HPV39 on the plasmid as templates, and screening and verifying by using synthesized primer probes. Template and primer probe sequence table 1 shows
TABLE 1
The PCR reaction system is shown in Table 2.
TABLE 2
Reactions were performed on a real-time fluorescent quantitative PCR apparatus with a dissolution profile function, such as Bio-Rad, SLAN 96, etc.
The reaction procedure was as follows:
in the band-x step FAM fluorescence was collected to draw a dissolution curve.
Result determination
As shown in fig. 2, a peak appears at tm=64.00 (i.e., a peak denoted by 201), and HPV31 is determined to be positive; a peak (i.e., a peak denoted by 201) appears at tm=71.5, and HPV39 is judged to be positive.
In addition, example 1 is a protocol for detecting both HPV31 and HPV39 types in FAM channel using TaqMan probe lysis curve technique. The embodiment of the invention can be used for detecting more than two HPV types by arranging more probes with different Tm values on the FAM channel, and can also be used for fluorescent channels such as HEX, ROX, CY5, so that targets with a plurality of channels can be detected in the same reaction tube. The specific implementation manner may refer to the description of embodiment 1, and will not be described in detail.
According to the embodiment of the invention, on the basis of a conventional TaqMan probe dissolution curve, the TaqMan probes for identifying HPV types by designing the Tm value difference of the same fluorescent groups are designed, so that the detection effect of identifying multiple HPV types in the same fluorescent channel is realized, the number of reaction tubes can be effectively reduced, the reagent cost is saved, the operation flow is simplified, and the detection flux is improved.
Compared with the traditional dissolution curve method, the method provided by the embodiment of the invention can realize detection of multiple HPV types in the same fluorescent channel, breaks through the limit of the number of the traditional fluorescent channels on the number of HPV types which can be detected by single-tube reaction, and therefore, when the types of various HPVs are detected, the reaction number can be effectively reduced, and the effects of saving cost, simplifying operation and freeing instruments are achieved.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the scope of the invention, but to limit the invention to the particular embodiments, and any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.
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Claims (4)
1. A set of DNA molecules for HPV typing detection comprising:
primer pairs for amplifying two HPVs;
two probes respectively corresponding to the two HPV types, wherein the Tm values of the two probes are different;
the primer pair for amplifying two HPVs comprises: a primer pair for amplifying HPV31 and a primer pair for amplifying HPV39; wherein, the liquid crystal display device comprises a liquid crystal display device,
the primer pair for amplifying HPV31 consists of a sequence shown in SEQ ID NO.1 and a sequence shown in SEQ ID NO. 2; the primer pair for amplifying HPV39 consists of a sequence shown in SEQ ID NO.3 and a sequence shown in SEQ ID NO. 4; the nucleotide sequence of the probe corresponding to HPV31 is shown in SEQ ID NO.5, and the nucleotide sequence of the probe corresponding to HPV39 is shown in SEQ ID NO. 6.
2. A PCR reagent for HPV typing detection comprising the DNA kit of claim 1.
3. A kit for HPV typing detection comprising the DNA kit of claim 1.
4. Use of a set of DNA molecules according to claim 1 or PCR reagents according to claim 2 or a kit according to claim 3 for the preparation of a product for HPV typing.
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| CN101871007A (en) * | 2010-05-07 | 2010-10-27 | 无锡锐奇基因生物科技有限公司 | Method for detecting by using labeled probe and analyzing fusion curve |
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| CN106957903A (en) * | 2016-11-01 | 2017-07-18 | 上海泽因生物科技有限公司 | One kind detection folic acid metabolism key gene pleomorphism site genotyping kit and its detection method |
| CN107475446A (en) * | 2017-08-24 | 2017-12-15 | 复旦大学附属儿科医院 | Multi-PCR detection method and its probe groups and kit a kind of while that detect various respiratory road virus |
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| CN101871007A (en) * | 2010-05-07 | 2010-10-27 | 无锡锐奇基因生物科技有限公司 | Method for detecting by using labeled probe and analyzing fusion curve |
| CN105087763A (en) * | 2014-05-16 | 2015-11-25 | 北京万泰生物药业股份有限公司 | Multiplex nucleic acid detecting method based on real-time fluorescence PCR (polymerase chain reaction) and probe method melting curve |
| CN105506118A (en) * | 2016-01-08 | 2016-04-20 | 智海生物工程（北京）股份有限公司 | Primer pair used for detecting CYP2C19 genotypes, fluorescent probe, reagent kit and method |
| CN106957903A (en) * | 2016-11-01 | 2017-07-18 | 上海泽因生物科技有限公司 | One kind detection folic acid metabolism key gene pleomorphism site genotyping kit and its detection method |
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