CN110656203A - Human papilloma virus typing detection method, reagent and application - Google Patents
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Abstract
The invention relates to the technical field of pathogen detection, in particular to a Human Papilloma Virus (HPV) typing detection method, a reagent and application. The method comprises the following steps: detecting at least two types of HPV in the same fluorescent channel of the same PCR reaction tube; determining the type of each HPV of the at least two types of HPV using at least two probes corresponding to the at least two types of HPV, respectively, in the same fluorescent channel; wherein the at least two probes have different Tm values.
Description
Technical Field
The invention relates to the technical field of pathogen detection, in particular to a Human Papilloma Virus (HPV) typing detection method, a reagent and application.
Background
Human Papilloma Virus (HPV) belongs to the papovavirus family, is a double-stranded circular DNA virus without an envelope, and has special tropism for epidermis and mucosal squamous epithelium. Cervical cancer is the second largest malignancy that endangers female health, and recurrent or persistent infections with high-risk HPV are a prerequisite for the development of cervical cancer. The relative risk of cervical cancer increases hundreds of times by infection with HPV, and it takes about 5-10 years from persistent infection with high-risk HPV to the development of common pre-cervical lesions to the final cervical cancer. In fact, more than about 95% of cervical cancers are caused by high risk HPV infections, especially recurrent or persistent infections with HPV16, 18, 31, 33, 39, 45. Therefore, the HPV is pertinently typed and detected, and the kit has important significance for early diagnosis and treatment of cervical cancer.
Currently, the methods for clinically typing and detecting HPV virus mainly include PCR-based target sequence amplification methods such as fluorescence PCR method, lysis curve method, capillary electrophoresis method and signal amplification-based detection methods such as hybridization capture method. The traditional dissolution curve method can only detect one HPV type by a single fluorescence channel (the HPV type is distinguished by the type of the fluorescence channel on the basis that the Tm value of a dissolution curve peak diagram accords with the design expectation), so the number of the fluorescence 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 FIG. 1. FIG. 1 shows the detection results of HPV31 and HPV39 by a conventional TaqMan probe dissolution curve method, wherein a peak represented by a curve 101 is the detection effect of HPV31 on a FAM channel, and a peak represented by a curve 102 is the detection effect of HPV39 on a HEX channel.
At present, both the TaqMan probe fluorescence PCR method and the dissolution curve method for HPV typing detection are limited by technical principles, a signal of one fluorescence channel can only reflect a detection result of one target, the number of targets which can be detected by a single reaction tube is limited by the number of fluorescence channels of a PCR instrument, and if typing detection is carried out on nearly 20 HPV viruses with strong disease occurrence correlation, the HPV viruses need to be divided into multiple tubes for reaction. However, HPV infection is mostly single type infection, and the problems of reagent waste, instrument occupation and complex operation can occur when a multi-tube reaction system is used for detecting a single target.
Disclosure of Invention
The invention provides a human papilloma virus typing detection method, a reagent and application, which can detect targets with more than channel number in the same reaction tube.
In a first aspect, a method for detecting HPV typing of human papillomavirus is provided, comprising:
detecting at least two types of HPV in the same fluorescence channel of the same PCR reaction tube;
determining the type of each HPV of the at least two types of HPV using at least two probes corresponding to the at least two types of HPV, respectively, in the same fluorescent channel; wherein the at least two probes have different Tm values.
In some embodiments, said determining the type of each HPV of said at least two types of HPV in the same fluorescent channel using at least two probes corresponding to said at least two HPV types, respectively, comprises:
detecting the change of fluorescence intensity in the process of renaturation to denaturation of the DNA molecule;
generating a fluorescence curve by a fluorescence quantitative PCR instrument according to the change conditions of the plurality of fluorescence intensities;
and judging the type of each HPV in the at least two types of HPV 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 HPV 39; wherein, the primer pair for amplifying the HPV31 consists of a sequence shown by SEQ ID NO.1 and a sequence shown by SEQ ID NO. 2; the primer pair for amplifying the 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 set forth in SEQ ID No.5 and the nucleotide sequence of the probe corresponding to HPV39 is set forth in SEQ ID No. 6.
In a second aspect, there is provided a set of DNA molecules for HPV typing detection comprising:
a primer pair for amplifying at least two HPVs;
at least two probes corresponding to the at least two HPV types, respectively, the at least two probes differing in Tm value.
In some embodiments, the primer pair for amplifying at least two HPVs comprises: a primer pair for amplifying HPV31 and a primer pair for amplifying HPV 39; wherein the content of the first and second substances,
the primer pair for amplifying the 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 the 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 set forth in SEQ ID No.5 and the nucleotide sequence of the probe corresponding to HPV39 is set forth in SEQ ID No. 6.
In a third aspect, there is provided a PCR reagent for HPV typing detection, comprising the set of DNA molecules of the second aspect.
In a fourth aspect, there is provided a kit for HPV typing detection comprising a set of DNA molecules according to 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 a kit according to the fourth aspect in the manufacture of a product for HPV typing detection.
The method and the reagent for detecting the human papilloma virus can detect multiple HPV types by one tube and perform corresponding grouping, solve the problem of small flux of the conventional quantitative PCR reagent, shorten the detection time of a large batch of samples, and have the advantages of simple and convenient operation and low cost.
Drawings
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 solution of the present invention is further described in detail by the accompanying drawings and embodiments.
The scheme provided by the embodiment of the invention is improved on the basis of the original TaqMan probe method, and the TaqMan probes with different Tm values are designed to identify the types of HPV by the difference of the Tm values besides identifying the types of HPV with different types of fluorescent signals, so that the limit of detecting one type of HPV corresponding to one fluorescent channel is broken through, and the detection effect of single fluorescence on multiple types of HPV is realized. The scheme provided by the embodiment of the invention can realize the typing detection of multiple HPVs by the single fluorescent channel, breaks through the limitation that the number of the fluorescent channels can identify the types of the HPVs in a single-tube reaction system, and effectively reduces the number of reaction tubes.
Next, the scheme provided by the embodiment of the present invention is specifically described in each embodiment.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. 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, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring harbor LABORATORY Press, 1989and third edition, 2001; ausubel et al, Current PROTOCOLS Inmolecular BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATINSTRUCUTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) Methods Inenzymolygy, Vol.304, Chromatin (P.M. Wassarman and A.P.Wolffe, eds.), academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
The scheme provided by the invention aims to overcome the defect that one fluorescence channel can only detect one HPV type in a fluorescence PCR technology and a traditional TaqMan probe dissolution curve technology, realize the typing detection effect of a single fluorescence channel on multiple HPVs, break through the limitation of the number of the fluorescence channels on the number of the HPV types which can be recognized by a single-tube reaction system, and recognize the HPV types from two dimensions of the types and Tm values of fluorescence signals.
The technical principle of the TaqMan probe dissolution curve designed by the scheme provided by the invention is as follows.
This technique involves two parts, asymmetric PCR and a melting curve. The basic principle of asymmetric PCR (asymmetric PCR) is that a large amount of single-stranded DNA is generated by using a pair of primers with different amounts to amplify a template for hybridization with a TaqMan probe, and in order to prevent hydrolysis of the TaqMan probe, the DNA polymerase used in an extension stage is mTaq polymerase lacking 5 '→ 3' exonuclease activity; 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 fluorescence intensity in the reaction system is also maximized as the distance between the fluorescent group and the quenching group of the probe reaches the maximum value, then the temperature is slowly increased, the TaqMan probe and the DNA single-stranded product are denatured and separated, the fluorescence value in the system begins to decrease, the renaturation and the denaturation processes are represented by drawing the fluorescence value by using the temperature, 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.
The scheme provided by the embodiment of the invention can realize the detection of multiple types of HPV in a single-tube reaction system. The multiple 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, HPV68HPV53, HPV66, HPV73, HPV81, HPV82, HPV83, and the like.
Next, in example 1, detection of HPV31 and HPV39 in FAM channel is exemplified, and other types of HPV can be implemented by referring to the scheme described in the examples.
Example 1 detection of HPV31 and HPV39 in the FAM channel
E6 gene sequences of targets HPV31 and HPV39 are downloaded from NCBI databases, primer probes are designed by using Oligo 7.56, probes of HPV31 and HPV39 are designed to have different Tm values, FAM fluorescent groups are marked at 5 'ends, and BHQ1 quenching groups are marked at 3' ends. The E6 genes of HPV31 and HPV39 were then constructed on plasmids as templates and verified by screening with synthetic primer probes. Sequence listing of template and primer probes
TABLE 1
The PCR reaction system is shown in Table 2.
TABLE 2
The reaction was carried out on a real-time fluorescence quantitative PCR instrument with a melting curve function such as Bio-Rad and SLAN 96.
The reaction procedure was as follows:
in band-step, FAM fluorescence was collected to plot the dissolution curve.
Determination of results
As shown in fig. 2, a peak (i.e., a peak indicated by 201) appears at Tm of 64.00, and HPV31 is determined to be positive; a peak (i.e., a peak indicated by 201) appeared at Tm 71.5, and HPV39 was judged to be positive.
To illustrate, example 1 is a protocol for detecting two HPV types, HPV31 and HPV39, in FAM channels using TaqMan probe dissolution curve technology. The embodiment of the invention can be used for detecting more than two HPV types by arranging more probes with different Tm values on a FAM channel, and can also be used for fluorescent channels such as HEX, ROX, CY5 and the like, so that targets with the quantity of 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 is not described in detail.
On the basis of a conventional TaqMan probe dissolution curve, the TaqMan probe for identifying HPV types by the Tm value difference of the same fluorescent group is designed and marked, 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 conventional dissolution curve method, the scheme provided by the embodiment of the invention can realize the detection of multiple HPV types in the same fluorescence channel, and breaks through the limitation of the number of the fluorescence channels to the number of the HPV types which can be detected by a single-tube reaction, so that the reaction number can be effectively reduced when the types of various HPV are detected, and the effects of saving cost, simplifying operation and freeing instruments are achieved.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
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Claims (10)
1. A Human Papillomavirus (HPV) typing detection method, comprising:
detecting at least two types of HPV in the same fluorescent channel of the same PCR reaction tube;
determining the type of each HPV of the at least two types of HPV using at least two probes corresponding to the at least two types of HPV, respectively, in the same fluorescent channel; wherein the at least two probes have different Tm values.
2. The method of claim 1, wherein said determining the type of each HPV of said at least two types of HPV in the same fluorescent channel using at least two probes corresponding to said at least two HPV types, respectively, comprises:
detecting the change condition of fluorescence intensity in the process from renaturation to denaturation of the DNA molecules;
generating a fluorescence curve by a fluorescence quantitative PCR instrument according to the fluorescence intensity change;
and judging the type of each HPV in the at least two types of HPV according to the fluorescence curve and the Tm values of the at least two probes.
3. The method of claim 1, wherein said detecting at least two types of HPV comprises: amplifying at least HPV31 and HPV 39; wherein, the primer pair for amplifying the HPV31 consists of a sequence shown by SEQ ID NO.1 and a sequence shown by SEQ ID NO. 2; the primer pair for amplifying the HPV39 consists of a sequence shown in SEQ ID NO.3 and a sequence shown in SEQ ID NO. 4.
4. The method according to claim 3, wherein 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.
5. A set of DNA molecules for HPV typing detection comprising:
a primer pair for amplifying at least two HPVs;
at least two probes corresponding to the at least two HPV types, respectively, the at least two probes differing in Tm value.
6. The set of DNA molecules of claim 5, wherein the primer pairs for amplifying at least two HPVs comprise: a primer pair for amplifying HPV31 and a primer pair for amplifying HPV 39; wherein the content of the first and second substances,
the primer pair for amplifying the 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 the HPV39 consists of a sequence shown in SEQ ID NO.3 and a sequence shown in SEQ ID NO. 4.
7. The set of DNA molecules of claim 6, wherein 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.
8. A PCR reagent for HPV typing detection comprising the set of DNA molecules according to any one of claims 5 to 7.
9. A kit for HPV typing detection comprising a set of DNA molecules according to any one of claims 5-7.
10. Use of a set of DNA molecules according to any one of claims 5 to 7 or PCR reagents according to claim 8 or a kit according to claim 9 in the manufacture of a product for HPV typing.
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