CN111172328A - Complete set of primers, complete set of probes, kit and method for HPV nucleic acid typing detection - Google Patents
Complete set of primers, complete set of probes, kit and method for HPV nucleic acid typing detection Download PDFInfo
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Abstract
The invention provides a complete set of primers, a complete set of probes and a kit for HPV nucleic acid typing detection and an HPV nucleic acid typing detection method, and relates to the technical field of in-vitro diagnosis. The sequence of the primer set is shown as SEQ ID NO. 1-SEQ ID NO. 40; the sequence of the probe set is shown in SEQ ID NO. 43-SEQ ID NO. 62. The set of primers, the set of probes and the kit can detect HPV high-risk subtypes HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66 and HPV 68; low risk subtypes HPV6 and HPV 11; and medium risk subtypes HPV26, HPV73 and HPV 82.
Description
Technical Field
The invention relates to the technical field of in-vitro diagnosis, in particular to a complete set of primers, a complete set of probes and a kit for HPV nucleic acid typing detection and an HPV nucleic acid typing detection method.
Background
Human Papillomavirus (HPV), belonging to the genus papillomavirus A of the papovaviridae family. The virus is spherical and has no envelope. The genome is double-stranded DNA, and the total length is about 8000 bp. The viral genome can be divided into an early region (region E), a late region (region L) and a regulatory region (LCR). The E region mainly encodes various proteins required for virus replication initiation, the L region encodes capsid proteins of the virus, and the LCR region is mainly responsible for regulating and controlling virus replication. HPV is an epitheliotropic virus, which only infects humans, the site of infection being most localized in the epidermis and squamous epithelium. The research of international cancer research institution proves that the persistent HPV infection is a key factor for causing the cervical cancer. Persistent HPV infection can cause squamous intraepithelial lesions that may take years or even decades to evolve into cervical cancer. Excessive time to progression of symptoms can lead to patient neglect, and early detection and intervention is one of the effective ways to reduce the rate of cervical cancer.
According to the current HPV subtype distinguishing requirement, the HPV virus genome is considered to be the same subtype when the DNA sequence homology of the L1 region is between 90% and 100%. Over 200 HPV subtypes have been discovered. The subtypes of HPV are classified according to the correlation between the subtypes of HPV and genital tract tumors, and can be divided into three types, namely high-risk type, medium-risk type and low-risk type. Therefore, it is necessary to distinguish the HPV types simultaneously in the process of HPV detection.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a set of primers for HPV genotyping detection and a set of probes for HPV genotyping detection, which can simultaneously distinguish high-risk subtype, medium-risk subtype and low-risk subtype of HPV during HPV detection.
The second purpose of the invention is to provide a kit containing the complete set of primers and/or the complete set of probes for HPV nucleic acid typing detection.
The third purpose of the invention is to provide a method for HPV nucleic acid typing detection, which comprises the step of detecting a sample to be detected by using at least one of the above-mentioned primer set, probe set and kit for HPV nucleic acid typing detection.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to one aspect of the invention, the invention provides a set of primers for HPV nucleic acid typing detection, comprising a primer pair for detecting HPV6, the sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2; a primer pair for detecting HPV11, the sequences of which are shown as SEQ ID NO.3 and SEQ ID NO. 4; a primer pair for detecting HPV16, the sequences are shown as SEQ ID NO.5 and SEQ ID NO. 6; a primer pair for detecting HPV18, the sequences are shown as SEQ ID NO.7 and SEQ ID NO. 8; a primer pair for detecting HPV26, the sequences are shown as SEQ ID NO.9 and SEQ ID NO. 10; a primer pair for detecting HPV31, the sequences are shown as SEQ ID NO.11 and SEQ ID NO. 12; a primer pair for detecting HPV33, the sequences are shown as SEQ ID NO.13 and SEQ ID NO. 14; a primer pair for detecting HPV35, the sequences are shown as SEQ ID NO.15 and SEQ ID NO. 16; a primer pair for detecting HPV39, the sequences are shown as SEQ ID NO.17 and SEQ ID NO. 18; a primer pair for detecting HPV45, the sequences are shown as SEQ ID NO.19 and SEQ ID NO. 20; a primer pair for detecting HPV51, the sequences are shown as SEQ ID NO.21 and SEQ ID NO. 22; a primer pair for detecting HPV52, the sequences are shown as SEQ ID NO.23 and SEQ ID NO. 24; a primer pair for detecting HPV53, the sequences are shown as SEQ ID NO.25 and SEQ ID NO. 26; a primer pair for detecting HPV56, the sequence is shown as SEQ ID NO.27 and SEQ ID NO. 28; a primer pair for detecting HPV58, the sequences are shown as SEQ ID NO.29 and SEQ ID NO. 30; a primer pair for detecting HPV59, the sequences are shown as SEQ ID NO.31 and SEQ ID NO. 32; a primer pair for detecting HPV66, the sequence is shown as SEQ ID NO.33 and SEQ ID NO. 34; a primer pair for detecting HPV68, the sequence is shown as SEQ ID NO.35 and SEQ ID NO. 36; a primer pair for detecting HPV73, the sequences are shown as SEQ ID NO.37 and SEQ ID NO. 38; and a primer pair for detecting HPV82, the sequences are shown as SEQ ID NO.39 and SEQ ID NO. 40.
According to another aspect of the present invention, there is also provided a set of probes for HPV nucleic acid typing detection, comprising a probe for detecting HPV6, the sequence shown in SEQ ID NO. 43; a probe for detecting HPV11, the sequence is shown as SEQ ID NO. 44; a probe for detecting HPV16, the sequence is shown as SEQ ID NO. 45; a probe for detecting HPV18, the sequence is shown as SEQ ID NO. 46; a probe for detecting HPV26, the sequence is shown in SEQ ID NO. 47; a probe for detecting HPV31, the sequence is shown as SEQ ID NO. 48; a probe for detecting HPV33, the sequence is shown as SEQ ID NO. 49; a probe for detecting HPV35, the sequence is shown as SEQ ID NO. 50; a probe for detecting HPV39, the sequence is shown as SEQ ID NO. 51; a probe for detecting HPV45, the sequence is shown as SEQ ID NO. 52; a probe for detecting HPV51, the sequence is shown as SEQ ID NO. 53; a probe for detecting HPV52, the sequence is shown as SEQ ID NO. 54; a probe for detecting HPV53, the sequence is shown as SEQ ID NO. 55; a probe for detecting HPV56, the sequence is shown as SEQ ID NO. 56; a probe for detecting HPV58, the sequence is shown as SEQ ID NO. 57; a probe for detecting HPV59, the sequence is shown as SEQ ID NO. 58; a probe for detecting HPV66, the sequence is shown as SEQ ID NO. 59; a probe for detecting HPV68, the sequence is shown as SEQ ID NO. 60; a probe for detecting HPV73, the sequence is shown as SEQ ID NO. 61; and a probe for detecting HPV82, wherein the sequence is shown as SEQ ID NO. 62.
According to another aspect of the present invention, there is also provided a kit for HPV nucleic acid typing detection, comprising the above-described set of primers for HPV detection, and/or the above-described set of probes for HPV detection.
According to another aspect of the present invention, the present invention also provides a method for HPV nucleic acid typing detection, which comprises detecting a sample to be detected using the above-described set of primers for HPV nucleic acid typing detection, or the above-described kit for HPV nucleic acid typing detection.
Compared with the prior art, the invention has the following beneficial effects:
the complete set of primers for HPV genotyping detection provided by the invention contains 20 primer pairs, and the 20 primer pairs can respectively amplify HPV nucleic acids of 20 subtypes in total, including high-risk subtypes HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66 and HPV 68; low risk subtypes HPV6 and HPV 11; and medium risk subtypes HPV26, HPV73 and HPV 82. The invention also provides a set of probes for HPV nucleic acid typing detection, which can be hybridized with the 20 subtypes of HPV to distinguish the subtypes. The primer set and the probe set provided by the invention can cover multiple subtypes of HPV, improve the detection effect of HPV in a sample, and classify the detected HPV. And the primers and the probes have good sequence amplification and nucleic acid hybridization effects, and are good in detection accuracy, detection limit reference product detection rate and precision.
The kit provided by the invention comprises the complete set of primers and/or the complete set of probes for detecting HPV, when the complete set of primers and the complete set of probes are used in a combined manner, the HPV in a sample to be detected can be detected by adopting a fluorescent quantitative PCR (polymerase chain reaction) method, and the kit has the advantages of short experimental time, low pollution risk, high detection flux, simple experimental operation, low experimental cost and the like, the accuracy of a detection result is high, and the coincidence rate of each subtype of a positive reference product and a negative reference product can reach 100% after experiments are found; the detection rate is high, and the detection rate of the detection limit reference substances of all subtypes can reach 100 percent; high precision, and low variation coefficient of Ct values of the strong positive reference substance and the weak positive reference substance of each subtype.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph of the results of the accuracy (positive reference sample compliance/negative reference sample compliance) test of the kit of example 1;
FIG. 2 is a graph showing the results of the detection rate of the reference sample in the detection limit of the kit according to example 1 of the present invention;
FIG. 3 is a graph showing the results of the precision evaluation experiment of the kit of example 1;
FIG. 4 is a graph showing the results of the clinical specimen testing experiment in example 2 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to one aspect of the present invention, there is provided a set of primers for HPV genotyping detection, the set of primers comprising a primer pair for detecting high risk subtypes HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66 and HPV 68; primer pairs for detecting low-risk subtypes HPV6 and HPV 11; and primer pairs for detecting medium-risk subtypes HPV26, HPV73 and HPV82, wherein specific sequence information of the primer pairs is detailed in Table 1. The primer pair set preferably also comprises a primer pair for amplifying the internal reference gene, and the sequences are shown as SEQ ID NO.41 and SEQ ID NO. 42. The complete set of primers for HPV nucleic acid typing detection provided by the invention can detect and distinguish 20 subtypes of HPV, and the detected subtypes cover high-risk subtypes, medium-risk subtypes and low-risk subtypes and fully cover all subtypes of HPV.
TABLE 1
In some preferred embodiments, the primers are purified by PAGE or HPLC. The PAGE purification method is a method of separating DNA fragments by using denaturing polyacrylamide gel electrophoresis and then recovering the target DNA from the gel, and the purity of the PAGE purification method can reach 95%. The HPLC purification method adopts high performance liquid chromatography for purification, and the purity of the product is high and can reach 99%.
According to another aspect of the present invention, the present invention also provides a set of probes for HPV genotyping detection, specifically comprising probes for detecting HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV6, HPV11, HPV26, HPV73 and HPV82, and the specific sequence information of the probes is detailed in table 2. The probe set can be hybridized with 20 subtypes of HPV for detecting each subtype. The probe set preferably also comprises a probe for detecting an internal reference gene, and the sequence is shown as SEQ ID NO. 63.
TABLE 2
In some preferred embodiments, each probe in the above-mentioned probe set contains a fluorescent label and a quencher, and can be used for fluorescence quantitative PCR in combination with a primer pair for amplifying each subtype. The probe set is preferably combined with the primer set for HPV genotyping detection. The fluorescent label is preferably labeled at the 5' end of the probe sequence, and the fluorescent label can be selected from optional fluorescent labels acceptable in the art, such as, but not limited to, FAM, HEX, ROX, Cy5 or Cy5.5; the quenching group is preferably labeled at the 3' end of the probe sequence, and the quenching group can be selected from optional groups acceptable in the art and capable of quenching the fluorescent label of the probe on which the quenching group is positioned, and can be, for example, but not limited to, BHQ1, BHQ2 or BHQ 3. The probe is preferably purified by HPLC purification with high product purity.
According to another aspect of the present invention, there is also provided a kit for HPV nucleic acid typing detection, comprising the above-described set of primers for HPV detection, and/or the above-described set of probes for HPV detection. The kit contains the primer set and/or the probe set, and can detect 20 subtypes of HPV in a typing mode.
In some alternative embodiments, the kit for HPV genotyping detection comprises the set of primers or set of probes described above for detecting HPV.
In some preferred embodiments, the kit for HPV genotyping detection comprises the above set of primers for detecting HPV and the above set of probes for HPV genotyping detection, the probes preferably comprise a fluorescent label and a quenching group, and the primers and the probes are used together to perform fluorescent quantitative PCR amplification on a sample, which has the advantages of simple experimental operation and low experimental cost; the accuracy of the detection result is high, and the test finds that the coincidence rate of each subtype of the positive reference substance and the negative reference substance can reach 100 percent; the detection rate is high, and the detection rate of the detection limit reference substances of all subtypes can reach 100 percent; high precision, and low variation coefficient of Ct values of the strong positive reference substance and the weak positive reference substance of each subtype.
In some preferred embodiments, in order to increase the detection throughput, multiple pairs of primer pairs in the above primer sets can be used in combination to achieve simultaneous detection of multiple HPV subtypes in the same PCR system in one PCR process. The method specifically comprises the following steps: a first detection unit comprising: a primer pair for detecting HPV6, a primer pair for detecting HPV11, a primer pair for detecting HPV31, a primer pair for detecting HPV59 and a primer pair for amplifying an internal reference gene; a second detection unit comprising: a primer pair for detecting HPV16, a primer pair for detecting HPV18, a primer pair for detecting HPV35, a primer pair for detecting HPV45 and a primer pair for detecting HPV 51; a third detection unit comprising: a primer pair for detecting HPV33, a primer pair for detecting HPV58, a primer pair for detecting HPV52, a primer pair for detecting HPV68 and a primer pair for detecting HPV 39; and, a fourth detection unit comprising: a primer pair for detecting HPV53, a primer pair for detecting HPV56, a primer pair for detecting HPV26, a primer pair for detecting HPV73, a primer pair for detecting HPV82 and a primer pair for detecting HPV 66. It should be noted that the above-mentioned "first", "second", "third" and "fourth" are only for distinguishing different detecting units, and cannot be understood as sequencing the sequence and importance degree of each detecting unit.
In some preferred embodiments, in order to improve the detection efficiency, the first to fourth detection units remove a probe containing a primer and a fluorescent label and a quenching group label to realize fluorescent quantitative PCR detection of multiple HPV subtypes in a single system, specifically: a first detection unit comprising: a primer pair for detecting HPV6, a primer pair for detecting HPV11, a primer pair for detecting HPV31, a primer pair for detecting HPV59 and a primer pair for amplifying an internal reference gene, as well as a probe for detecting HPV6, a probe for detecting HPV11, a probe for detecting HPV31, a probe for detecting HPV31 and a probe for detecting an internal reference gene, wherein fluorescent labels of the probe for detecting HPV6, the probe for detecting HPV11, the probe for detecting HPV31, the probe for detecting HPV31 and the probe for detecting the internal reference gene are different from each other in order to distinguish amplification curves corresponding to respective HPV subtypes during amplification; a second detection unit comprising: a primer pair for detecting HPV16, a primer pair for detecting HPV18, a primer pair for detecting HPV35, a primer pair for detecting HPV45 and a primer pair for detecting HPV51, as well as a probe for detecting HPV16, a probe for detecting HPV18, a probe for detecting HPV35, a probe for detecting HPV45 and a probe for detecting HPV51, wherein the fluorescent labels of the probe for detecting HPV16, the probe for detecting HPV18, the probe for detecting HPV35, the probe for detecting HPV45 and the probe for detecting HPV51 are different from each other; a third detection unit comprising: a primer pair for detecting HPV33, a primer pair for detecting HPV58, a primer pair for detecting HPV52, a primer pair for detecting HPV68 and a primer pair for detecting HPV39, as well as a probe for detecting HPV33, a probe for detecting HPV58, a probe for detecting HPV52, a probe for detecting HPV68 and a probe for detecting HPV39, wherein the fluorescent labels of the probe for detecting HPV33, the probe for detecting HPV58, the probe for detecting HPV52, the probe for detecting HPV68 and the probe for detecting HPV39 are different from each other; and, a fourth detection unit comprising: a primer pair for detecting HPV53, a primer pair for detecting HPV56, a primer pair for detecting HPV26, a primer pair for detecting HPV73, a primer pair for detecting HPV82 and a primer pair for detecting HPV66, a probe for detecting HPV53, a probe for detecting HPV56, a probe for detecting HPV26, a probe for detecting HPV73, a probe for detecting HPV82 and a probe for detecting HPV66, wherein the fluorescent labels of the probe for detecting HPV53, the probe for detecting HPV56 and the probe for detecting HPV66 are different from each other, and the three subtypes HPV26, HPV73 and HPV82 are medium-risk subtypes, and are not classified into three subtypes because of the low infection rate of HPV26, HPV26 and HPV26 types and the generally weaker threat degree of human health than that of the probes other types, i.e., the probe for detecting HPV26 and the fluorescent label of the probe for detecting HPV26 are different from each other subtypes, i.e., the probe for detecting HPV26, the fluorescent label of the probe for detecting HPV26 and the fluorescent label of the HPV26 is different from the fluorescent label of the probe for detecting HPV26, when the experiment shows that the fluorescent-labeled amplification curves corresponding to the HPV26, HPV73 and HPV82 which meet the quality control standard are generated, namely the sample is considered to contain at least one of the medium-risk subtypes HPV26, HPV73 and HPV 82. The complete set of primers and the complete set of probes for HPV nucleic acid typing detection are grouped according to the embodiment, and the probes are subjected to fluorescence labeling and quenching group labeling, so that the kit has the characteristics of short experimental time, low pollution risk, high detection flux, simple experimental operation, low experimental cost and the like when the HPV subtypes are detected and typed.
In some alternative embodiments, the kit may further comprise conventional experimental reagents, such as PCR reaction reagents or fluorescent quantitative PCR reaction reagents, examples of the specific reaction reagents include enzymes, salts, buffer substances, buffers, dntps, stabilizers, etc. used in PCR reaction, and the present invention is not limited to these reagents.
In some alternative embodiments, the kit further comprises a control group to avoid false positives and false negatives in the experimental results. When the kit is designed by adopting the first to fourth detection units, the positive control comprises at least one of an HPV6 gene fragment, an HPV11 gene fragment, an HPV31 gene fragment and an HPV59 gene fragment for comparison with the first detection unit; at least one of an HPV16 gene fragment, an HPV18 gene fragment, an HPV35 gene fragment, an HPV45 gene fragment and an HPV51 gene fragment which are subjected to control by the second detection unit; at least one of an HPV33 gene fragment, an HPV58 gene fragment, an HPV52 gene fragment, an HPV68 gene fragment and an HPV39 gene fragment which are subjected to control by the third detection unit; and at least one of an HPV53 gene fragment, an HPV56 gene fragment, an HPV26 gene fragment, an HPV73 gene fragment, an HPV82 gene fragment, and an HPV66 gene fragment which are controlled with the fourth detection unit. The negative control preferably uses a human genome.
In the first to fourth detecting units, the components, such as the primers, the probes, and the reagents, may be packaged separately and mixed after the reaction; or preparing a premixed system, for example, preparing a premixed detection solution with a concentration suitable for PCR reaction from a probe, a primer, dNTP, a salt, polymerase and a buffer solution, or diluting a concentrated premixed detection solution for use after the reaction; a premixed system containing a part of the reaction system, for example, only the primer and the probe, may be used by mixing with the PCR reaction reagent.
According to another aspect of the present invention, the present invention also provides a method for HPV nucleic acid typing detection, which comprises detecting a sample to be detected using the above-described set of primers for HPV nucleic acid typing detection, or the above-described kit for HPV nucleic acid typing detection. The detection method provided by the invention can carry out nucleic acid typing detection on 20 subtypes of HPV, covers high-risk subtypes, medium-risk subtypes and low-risk subtypes of HPV subtypes and has a comprehensive detection range. It is to be noted that the present invention provides HPV genotyping assays for non-therapeutic purposes.
The technical solution and the advantages of the present invention will be further explained with reference to the preferred embodiments.
Example 1
This example provides a kit for HPV nucleic acid typing detection, comprising the 1 st detection tube detection solution, the 2 nd detection tube detection solution, the 3 rd detection tube detection solution and the 4 th detection tube detection solution, wherein the primers and probes contained in the detection solutions are shown in Table 3:
TABLE 3 detection solution of Human Papillomavirus (HPV) nucleic acid typing detection kit
The detection solution in each detection tube also comprises a PCR reaction solution, wherein the PCR reaction solution is 5U/. mu.L Taq enzyme and 25mM Mg2+500. mu.M dNTP, 10 XPCR reaction buffer;
the kit also contains positive control of HPV31, HPV16, HPV39 and HPV53 type gene fragments and negative control of human genome diluent with the concentration of 2 ng/. mu.L.
Effect example 1
And detecting the product performance of the kit, wherein the product performance comprises three contents of accuracy, detection limit reference product detection rate, precision evaluation and the like. The positive reference substance used by the product can be traced to HPV national standard (HPV L1 genotyping reference substance), and the negative reference substance can be traced to relevant patient samples diagnosed by medical institutions.
1. Accuracy of
5 parts of positive reference products and 3 parts of negative reference products of 20 types are detected by using the finished product kit, the positive reference product coincidence rate and the negative reference product coincidence rate are counted by combining the graph 1, and the results are shown in a table 4.
TABLE 4 Positive reference match/negative reference match
2. Detection limit reference product detection rate
20 detection limit reference products of 20 types are detected by using the finished product kit, the detection rate of the detection limit reference products is counted by combining with the graph 2, and the result is shown in table 5.
TABLE 5 detection limit reference product detection rate
3. Precision degree
Detection of 10 Strong Positive reference products (10) of 20 types by Using finished product kit5copies/mL), 10 parts of weak positive reference substance (10)3copies/mL) and 1 negative reference, and the coefficient of variation (CV value) of Ct values of the 3 references are counted in combination with FIG. 3, and the results are shown in Table 6.
TABLE 6 precision
Example 2
Detection of 78 clinical specimens:
step (1) extraction of sample DNA
200. mu.L of each of the negative control and cervical swab samples in the kit were taken and extracted using the MagaBio plus Virus DNA/RNA purification kit. The extraction procedure is described in "MagaBio plus Virus DNA/RNA purification kit Specification"
Step (2) PCR amplification
And (2) taking the DNA extracted in the step (1) as a template, and simultaneously carrying out fluorescence PCR amplification on the template DNA by using PCR reaction liquid and detection liquid of the 1 st, 2 nd, 3 rd and 4 th detection tubes respectively. Meanwhile, PCR amplification is carried out on the positive control carried by the kit as template DNA.
For each sample (including negative control and positive control), 4 detection tubes are used, and template DNA is added into the 4 detection tubes, wherein each detection tube is composed of different PCR reaction systems. The PCR reaction system is divided into the 1 st, 2 nd, 3 th and 4 th detection tubes. The 1 st detection tube PCR reaction system is 20 mu L and consists of 10 mu L of PCR reaction liquid, 5 mu L of 1 st detection tube detection liquid and 5 mu L of template DNA; the PCR reaction system of the 2 nd detection tube is 20 mu L and consists of 10 mu L of PCR reaction liquid, 5 mu L of the 2 nd detection tube detection liquid and 5 mu L of template DNA; the PCR reaction system of the 3 rd detection tube is 20 mu L and consists of 10 mu L of PCR reaction liquid, 5 mu L of the 3 rd detection tube detection liquid and 5 mu L of template DNA; the 4 th detection tube PCR reaction system is 20 mu L and consists of 10 mu L of PCR reaction solution, 5 mu L of 4 th detection tube detection solution and 5 mu L of template DNA.
The PCR amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 10s, annealing and extension at 60 ℃ for 15s, and reaction is carried out for 40 cycles. FAM/HEX/ROX/Cy5/Cy5.5 channel fluorescence signals were collected at 60 ℃.
Step (3) determining HPV infection type of sample to be detected
The detection result of the kit needs to meet the quality control conditions:
quality control
1. Negative control: there is no typical "S" curve shown.
2. Positive control: channel of test tube ROX and Cy5 No. 1; a 2 nd detection tube HEX channel; the 3 rd detecting tube Cy5.5 channel; the FAM channels of the 4 th detection tube have typical S-shaped curves, and Ct values of corresponding detection channels are all smaller than 31.
3. Internal reference gene: the Cy5 channel of the No.1 detection tube of each real sample has a typical "S" type curve, and the Ct value is less than 35.63.
4. All the conditions need to be met in the same experiment, otherwise, the experiment is judged to be invalid.
And after the experimental result meets the conditions, judging the infection type of the sample according to the condition of result interpretation.
Interpretation of results
1. The detection channel in the detection tube corresponding to each HPV subtype of the actual sample has a typical S-type curve, and if the Ct value is less than or equal to the reference value, the type is judged to be positive; the reference values for each type are shown in table 7.
2. If a typical S-shaped curve exists and the Ct value is larger than the reference value, the sample needs to be repeatedly detected, if the two results are consistent, the type is judged to be positive, and if the two results are inconsistent, the type is judged to be negative;
3. if no typical S-shaped curve exists, the type is judged to be negative.
4. It should be noted that, if the channel "ROX" of the 4 th detection tube shows positive, and Ct is less than or equal to 32.59, it can be determined as "medium-risk type positive", i.e. at least one type of 26, 73, or 82 is positive; if Ct is more than 32.59, repeated detection is needed, if the two detection results are consistent, the medium-risk type is judged to be positive, and if the two detection results are inconsistent, the medium-risk type is judged to be negative.
TABLE 7 HPV results analysis interpretation tables and reference values
The results of 78 clinical specimens were read, and the final results are shown in fig. 4 and table 8. FIG. 4 is a graph showing the amplification curves of 78 samples, and Table 8 shows the results of the above-mentioned samples.
The results in the table were counted to find that the positive coincidence rate of the present kit with the medical institution diagnostic result was 100% (74 (the number of positive samples in the present kit with the medical institution diagnostic result)/74 (the total number of positive samples) was 100%), and the negative coincidence rate was not counted because the number of samples was too small (only 4 samples). Meets the national requirements of clinical test evaluation on in vitro diagnostic reagents.
TABLE 878 sample test results
Note: the mark in the table is "-" is not detected; "intermediate risk" refers to infection with at least one of HPV types 26, 73 and 82.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Hangzhou Bori science and technology Co., Ltd
<120> primer set for HPV nucleic acid typing detection, probe set, kit and method for HPV nucleic acid typing detection
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<213> Artificial sequence
<400>5
tattgtaacc ttttgttgc 19
<210>6
<211>22
<212>DNA
<213> Artificial sequence
<400>6
gcttcggttg tgcgtacaaa gc 22
<210>7
<211>17
<212>DNA
<213> Artificial sequence
<400>7
acgtcacaca atgttgt 17
<210>8
<211>21
<212>DNA
<213> Artificial sequence
<400>8
aagccagaat tgagctagta g 21
<210>9
<211>19
<212>DNA
<213> Artificial sequence
<400>9
ttacagaatt gaagcacaa 19
<210>10
<211>22
<212>DNA
<213> Artificial sequence
<400>10
atgtgtaata gtatagtgca gc 22
<210>11
<211>20
<212>DNA
<213> Artificial sequence
<400>11
<210>12
<211>20
<212>DNA
<213> Artificial sequence
<400>12
<210>13
<211>18
<212>DNA
<213> Artificial sequence
<400>13
<210>14
<211>20
<212>DNA
<213> Artificial sequence
<400>14
<210>15
<211>19
<212>DNA
<213> Artificial sequence
<400>15
ataatattgt aacgtcctg 19
<210>16
<211>21
<212>DNA
<213> Artificial sequence
<400>16
cgacactacg tctgtgtgta c 21
<210>17
<211>20
<212>DNA
<213> Artificial sequence
<400>17
<210>18
<211>20
<212>DNA
<213> Artificial sequence
<400>18
<210>19
<211>19
<212>DNA
<213> Artificial sequence
<400>19
acaaaatttt gtgtgtatg 19
<210>20
<211>23
<212>DNA
<213> Artificial sequence
<400>20
agtgtgacgg cagaattgag ctt 23
<210>21
<211>19
<212>DNA
<213> Artificial sequence
<400>21
ggacaggcta cgtgttaca 19
<210>22
<211>23
<212>DNA
<213> Artificial sequence
<400>22
tgttcaagtg tagtacaact ggc 23
<210>23
<211>20
<212>DNA
<213> Artificial sequence
<400>23
ctacattgtg acatattgtc 20
<210>24
<211>22
<212>DNA
<213> Artificial sequence
<400>24
atagcacatt acggctatgc at 22
<210>25
<211>21
<212>DNA
<213> Artificial sequence
<400>25
tgttacctaa ttgaaacaca g 21
<210>26
<211>23
<212>DNA
<213> Artificial sequence
<400>26
tgtgagtcgt tggtgcagtt ggc 23
<210>27
<211>21
<212>DNA
<213> Artificial sequence
<400>27
gtgttaccta atacacgtac c 21
<210>28
<211>24
<212>DNA
<213> Artificial sequence
<400>28
tcagtgtaag tttgtggtgc agtt 24
<210>29
<211>19
<212>DNA
<213> Artificial sequence
<400>29
ttactacatt gtaacgtgt 19
<210>30
<211>20
<212>DNA
<213> Artificial sequence
<400>30
<210>31
<211>21
<212>DNA
<213> Artificial sequence
<400>31
ttgtgtgtgt gtgttgtaag t 21
<210>32
<211>23
<212>DNA
<213> Artificial sequence
<400>32
atcaacttca gctagtagta gaa 23
<210>33
<211>22
<212>DNA
<213> Artificial sequence
<400>33
tacctaattc acgtaccttg tt 22
<210>34
<211>22
<212>DNA
<213> Artificial sequence
<400>34
gtggtgcagt tggacattca ga 22
<210>35
<211>20
<212>DNA
<213> Artificial sequence
<400>35
<210>36
<211>22
<212>DNA
<213> Artificial sequence
<400>36
acaacccact gcaactagta gt 22
<210>37
<211>20
<212>DNA
<213> Artificial sequence
<400>37
<210>38
<211>20
<212>DNA
<213> Artificial sequence
<400>38
<210>39
<211>20
<212>DNA
<213> Artificial sequence
<400>39
<210>40
<211>19
<212>DNA
<213> Artificial sequence
<400>40
ttggcagtgg aaagcagtg 19
<210>41
<211>24
<212>DNA
<213> Artificial sequence
<400>41
attgtccatg actggtgtgt ggag 24
<210>42
<211>21
<212>DNA
<213> Artificial sequence
<400>42
cagggctttg atagcactat c 21
<210>43
<211>20
<212>DNA
<213> Artificial sequence
<400>43
<210>44
<211>21
<212>DNA
<213> Artificial sequence
<400>44
tgacatcaga caactacaag a 21
<210>45
<211>20
<212>DNA
<213> Artificial sequence
<400>45
<210>46
<211>21
<212>DNA
<213> Artificial sequence
<400>46
agcagctgtt tctgaacacc c 21
<210>47
<211>23
<212>DNA
<213> Artificial sequence
<400>47
ttgtgcagag cagtcgacag aac 23
<210>48
<211>20
<212>DNA
<213> Artificial sequence
<400>48
<210>49
<211>21
<212>DNA
<213> Artificial sequence
<400>49
caacagtaca gcaagtgacc t 21
<210>50
<211>21
<212>DNA
<213> Artificial sequence
<400>50
acatacgtaa attggaagat t 21
<210>51
<211>21
<212>DNA
<213> Artificial sequence
<400>51
gggataccct gcgacaacta c 21
<210>52
<211>21
<212>DNA
<213> Artificial sequence
<400>52
agagctcggc agatgacctt a 21
<210>53
<211>20
<212>DNA
<213> Artificial sequence
<400>53
<210>54
<211>22
<212>DNA
<213> Artificial sequence
<400>54
cggaccttcg tactctacag ca 22
<210>55
<211>24
<212>DNA
<213> Artificial sequence
<400>55
agagttcaac aaaagagctg cgta 24
<210>56
<211>21
<212>DNA
<213> Artificial sequence
<400>56
ttcagagtac caaagaggac c 21
<210>57
<211>20
<212>DNA
<213> Artificial sequence
<400>57
<210>58
<211>20
<212>DNA
<213> Artificial sequence
<400>58
<210>59
<211>20
<212>DNA
<213> Artificial sequence
<400>59
<210>60
<211>22
<212>DNA
<213> Artificial sequence
<400>60
aaggagaacc tgcggaagct ac 22
<210>61
<211>20
<212>DNA
<213> Artificial sequence
<400>61
<210>62
<211>21
<212>DNA
<213> Artificial sequence
<400>62
caggtgttca agtttgctac a 21
<210>63
<211>23
<212>DNA
<213> Artificial sequence
<400>63
tctctctccc actcagcagc tat 23
Claims (10)
1. A set of primers for HPV nucleic acid typing detection, comprising:
a primer pair for detecting HPV6, the sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2;
a primer pair for detecting HPV11, the sequences of which are shown as SEQ ID NO.3 and SEQ ID NO. 4;
a primer pair for detecting HPV16, the sequences are shown as SEQ ID NO.5 and SEQ ID NO. 6;
a primer pair for detecting HPV18, the sequences are shown as SEQ ID NO.7 and SEQ ID NO. 8;
a primer pair for detecting HPV26, the sequences are shown as SEQ ID NO.9 and SEQ ID NO. 10;
a primer pair for detecting HPV31, the sequences are shown as SEQ ID NO.11 and SEQ ID NO. 12;
a primer pair for detecting HPV33, the sequences are shown as SEQ ID NO.13 and SEQ ID NO. 14;
a primer pair for detecting HPV35, the sequences are shown as SEQ ID NO.15 and SEQ ID NO. 16;
a primer pair for detecting HPV39, the sequences are shown as SEQ ID NO.17 and SEQ ID NO. 18;
a primer pair for detecting HPV45, the sequences are shown as SEQ ID NO.19 and SEQ ID NO. 20;
a primer pair for detecting HPV51, the sequences are shown as SEQ ID NO.21 and SEQ ID NO. 22;
a primer pair for detecting HPV52, the sequences are shown as SEQ ID NO.23 and SEQ ID NO. 24;
a primer pair for detecting HPV53, the sequences are shown as SEQ ID NO.25 and SEQ ID NO. 26;
a primer pair for detecting HPV56, the sequence is shown as SEQ ID NO.27 and SEQ ID NO. 28;
a primer pair for detecting HPV58, the sequences are shown as SEQ ID NO.29 and SEQ ID NO. 30;
a primer pair for detecting HPV59, the sequences are shown as SEQ ID NO.31 and SEQ ID NO. 32;
a primer pair for detecting HPV66, the sequence is shown as SEQ ID NO.33 and SEQ ID NO. 34;
a primer pair for detecting HPV68, the sequence is shown as SEQ ID NO.35 and SEQ ID NO. 36;
a primer pair for detecting HPV73, the sequences are shown as SEQ ID NO.37 and SEQ ID NO. 38;
and a primer pair for detecting HPV82, the sequences are shown as SEQ ID NO.39 and SEQ ID NO. 40.
2. The set of primers for detecting HPV according to claim 1, further comprising a primer pair for amplifying an internal reference gene, wherein the sequences are shown as SEQ ID No.41 and SEQ ID No. 42.
3. The set of primers of claim 1 or 2, wherein the primers are purified by PAGE or HPLC.
4. A set of probes for HPV genotyping detection, comprising: a probe for detecting HPV6, the sequence is shown as SEQ ID NO. 43; a probe for detecting HPV11, the sequence is shown as SEQ ID NO. 44; a probe for detecting HPV16, the sequence is shown as SEQ ID NO. 45; a probe for detecting HPV18, the sequence is shown as SEQ ID NO. 46; a probe for detecting HPV26, the sequence is shown in SEQ ID NO. 47; a probe for detecting HPV31, the sequence is shown as SEQ ID NO. 48; a probe for detecting HPV33, the sequence is shown as SEQ ID NO. 49; a probe for detecting HPV35, the sequence is shown as SEQ ID NO. 50; a probe for detecting HPV39, the sequence is shown as SEQ ID NO. 51; a probe for detecting HPV45, the sequence is shown as SEQ ID NO. 52; a probe for detecting HPV51, the sequence is shown as SEQ ID NO. 53; a probe for detecting HPV52, the sequence is shown as SEQ ID NO. 54; a probe for detecting HPV53, the sequence is shown as SEQ ID NO. 55; a probe for detecting HPV56, the sequence is shown as SEQ ID NO. 56; a probe for detecting HPV58, the sequence is shown as SEQ ID NO. 57; a probe for detecting HPV59, the sequence is shown as SEQ ID NO. 58; a probe for detecting HPV66, the sequence is shown as SEQ ID NO. 59; a probe for detecting HPV68, the sequence is shown as SEQ ID NO. 60; a probe for detecting HPV73, the sequence is shown as SEQ ID NO. 61; and a probe for detecting HPV82, the sequence is shown as SEQ ID NO. 62.
5. The kit of claim 4, further comprising a probe for detecting an internal reference gene, the sequence of which is shown in SEQ ID NO. 63.
6. The kit of claim 4 or 5, wherein the probe comprises a fluorescent label and a quencher;
preferably, the 5' of the probe is labeled with a fluorescent label;
preferably, the fluorescent label comprises FAM, HEX, ROX, Cy5, or cy5.5;
preferably, the 3' of the probe is labeled with a quenching group;
preferably, the quenching group comprises BHQ1, BHQ2 or BHQ 3;
preferably, the probe is purified by HPLC.
7. A kit for HPV nucleic acid typing detection comprising the set of primers for HPV detection of any one of claims 1 to 3;
and/or, the set of probes for detecting HPV according to any one of claims 4-6.
8. The kit of claim 7, further comprising a reaction reagent;
preferably, the reaction reagent comprises at least one of an enzyme, a salt, a dNTP and a buffer substance.
9. The kit according to claim 7 or 8, characterized in that the kit comprises a first detection unit, a second detection unit, a third detection unit and a fourth detection unit;
the first detection unit includes: a primer pair for detecting HPV6, a primer pair for detecting HPV11, a primer pair for detecting HPV31, a primer pair for detecting HPV59 and a primer pair for amplifying an internal reference gene;
the second detection unit includes: a primer pair for detecting HPV16, a primer pair for detecting HPV18, a primer pair for detecting HPV35, a primer pair for detecting HPV45 and a primer pair for detecting HPV 51;
the third detection unit includes: a primer pair for detecting HPV33, a primer pair for detecting HPV58, a primer pair for detecting HPV52, a primer pair for detecting HPV68 and a primer pair for detecting HPV 39;
the fourth detection unit includes: a primer pair for detecting HPV53, a primer pair for detecting HPV56, a primer pair for detecting HPV26, a primer pair for detecting HPV73, a primer pair for detecting HPV82 and a primer pair for detecting HPV 66;
preferably, the first detection unit further comprises a probe for detecting HPV6, a probe for detecting HPV11, a probe for detecting HPV31, a probe for detecting HPV31 and a probe for detecting an internal reference gene;
preferably, fluorescent labels of the probe for detecting HPV6, the probe for detecting HPV11, the probe for detecting HPV31, the probe for detecting HPV31 and the probe for detecting an internal reference gene are different from each other;
preferably, the second detection unit further includes: a probe for detecting HPV16, a probe for detecting HPV18, a probe for detecting HPV35, a probe for detecting HPV45 and a probe for detecting HPV 51;
preferably, the fluorescent labels of the probe for detecting HPV16, the probe for detecting HPV18, the probe for detecting HPV35, the probe for detecting HPV45 and the probe for detecting HPV51 are different from each other;
preferably, the third detecting unit includes: a probe for detecting HPV33, a probe for detecting HPV58, a probe for detecting HPV52, a probe for detecting HPV68 and a probe for detecting HPV 39;
preferably, the fluorescent labels of the probe for detecting HPV33, the probe for detecting HPV58, the probe for detecting HPV52, the probe for detecting HPV68 and the probe for detecting HPV39 are different from each other;
preferably, the fourth detection unit further includes: a probe for detecting HPV53, a probe for detecting HPV56, a probe for detecting HPV26, a probe for detecting HPV73, a probe for detecting HPV82 and a probe for detecting HPV 66;
preferably, the fluorescent labels of the probe for detecting HPV53, the probe for detecting HPV56 and the probe for detecting HPV66 are different from each other;
the fluorescent label of the probe for detecting HPV26, the fluorescent label of the probe for detecting HPV73 and the fluorescent label of the probe for detecting HPV82 are the same, and the fluorescent labels of the probe for detecting HPV53, the fluorescent label of the probe for detecting HPV56 and the fluorescent label of the probe for detecting HPV66 are different;
preferably, the first detection unit, the second detection unit, the third detection unit and the fourth detection unit further comprise a reaction reagent;
preferably, the kit further comprises a positive control;
preferably, the positive control comprises at least one of an HPV6 gene fragment, an HPV11 gene fragment, an HPV31 gene fragment, and an HPV59 gene fragment; at least one of an HPV16 gene fragment, an HPV18 gene fragment, an HPV35 gene fragment, an HPV45 gene fragment, and an HPV51 gene fragment; at least one of an HPV33 gene fragment, an HPV58 gene fragment, an HPV52 gene fragment, an HPV68 gene fragment, and an HPV39 gene fragment; and, at least one of an HPV53 gene fragment, an HPV56 gene fragment, an HPV26 gene fragment, an HPV73 gene fragment, an HPV82 gene fragment, and an HPV66 gene fragment;
preferably, the kit further comprises a negative control;
preferably, the negative control comprises a human genome.
10. A method for HPV nucleic acid typing detection, which comprises detecting a sample to be tested using the set of primers for HPV nucleic acid typing detection according to any one of claims 1 to 3, the set of primers for HPV nucleic acid typing detection according to any one of claims 4 to 6, or the kit for HPV nucleic acid typing detection according to any one of claims 7 to 9.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010153061.7A CN111172328A (en) | 2020-03-06 | 2020-03-06 | Complete set of primers, complete set of probes, kit and method for HPV nucleic acid typing detection |
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Application publication date: 20200519 |