CN115852057A - High-risk human papilloma virus nucleic acid typing detection kit - Google Patents
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Abstract
The invention relates to the field of molecular biology, in particular to a high-risk human papilloma virus nucleic acid typing detection kit. The kit adopts internal standard positive quality control, judges whether a clinical sample is suitable for amplification detection by detecting beta-Globin in a human genome, and can be used for monitoring whether a PCR inhibitor exists in a reaction system so as to ensure that a result of human papilloma virus false negative does not occur. The kit is used for carrying out high-efficiency typing detection on high-risk types of human papilloma viruses, and ensures the detection sensitivity and specificity by designing a specific primer probe and carrying out multiple optimization on a system.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a high-risk human papilloma virus nucleic acid typing detection kit.
Background
Cervical cancer is one of common gynecological malignant tumors, and the incidence rate of the cervical cancer is the second place in female malignant tumors in China and is behind breast cancer. In 2018, more than 56.9 ten thousand cases of new cervical cancer are discovered worldwide, and more than 31.1 ten thousand cases of death are discovered worldwide. In 2015, about 11.1 ten thousand new cases and 3.4 ten thousand dead cases exist in China. At present, it is clear that persistent infection of high-risk Human Papilloma Virus (HPV) is an essential factor for cervical cancer and precancerous lesion, namely, HPV infection is the most critical link in the process of cervical canceration.
Over 200 subtypes of HPV have been discovered and identified, and about 54 can infect the genital tract mucosa. According to the national relevant guidelines, HPV is classified into two major categories, namely high-risk HPV and low-risk HPV, according to the possibility of causing cervical cancer by HPV virus and the carcinogenicity of HPV infected human genital tract mucosa, and the 14 high-risk HPV causing cervical cancer which are confirmed by WHO and IRAC at present comprise 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68.
The HPV is subjected to typing detection, so that the morbidity risk degree of a detected person can be predicted, and the screening interval of the detected person is determined; can further distinguish persistent infection and transient infection so as to treat as early as possible to reduce incidence and mortality, is used for tracking after operation, determining whether to clear focus, is convenient for follow-up after treatment and guides research and use of HPV vaccine.
Over the years, there have been many detection methods for HPV, such as cytological examination, immunohistochemical detection of HPV antigens, detection of HPV antibodies, etc., but these detection methods have been gradually eliminated because they cannot detect HPV viruses by typing and have the disadvantages of low sensitivity, poor specificity, long time consumption, etc. At present, molecular biological methods such as nucleic acid hybridization, gene chips, polymerase chain reaction and the like are mainly adopted for HPV detection, wherein the most widely applied method is a fluorescent quantitative PCR method.
At present, various kits for detecting HPV by adopting a fluorescent quantitative PCR method exist in the field, wherein the mainstream kits are divided into two types, one type is a kit which covers a high-risk type HPV type in a detection range and can perform typing detection on all HPV types in the detection range; the other type is a kit which covers the high-risk HPV virus type in the detection range and can perform typing detection on part of HPV types in the detection range.
The first type is a kit for typing 15 types of HPV viruses, which needs to perform typing detection in 4 tubes, that is, each sample needs to be typed by using 4 wells, so that in one experiment, a 96-well PCR plate is used to remove a negative and positive control, and only 22 samples can be made at most, thus although typing detection can be achieved, the detection efficiency is low, for some hospitals with a single-day sample amount of more than 100 cases, at least 5 cycles of sample treatment and PCR amplification detection are required, the time of an examining doctor is seriously wasted, and especially in application scenarios such as cervical cancer large screening, the disadvantage of low detection efficiency of the kit is more prominent and obvious.
The second type of kit can detect 14 high-risk types of common HPV viruses and perform typing detection on 16 types and 18 types, has the advantages that single-hole detection is used, a maximum of 94 samples can be made in one experiment except yin-yang quality control on a common 96-micropore plate, the detection efficiency is high, but the disadvantage is that only 16 types and 18 types can be distinguished, and other types cannot be typed. The treatment schemes for different types of HPV infection are different, so that the treatment schemes often bring troubles for the judgment of clinicians, and when the result of repeated examination of patients in many years is positive, the kits cannot distinguish whether the patients are continuously infected with the same type in 12 types, but as is well known, if the patients are continuously infected with the same type HPV for many years, the probability of cervical cancer is high, so that the kits which have high efficiency and can detect types are urgently needed clinically.
In conclusion, the further development of the kit which has high detection efficiency and can carry out typing detection on all high-risk types of HPV has important practical significance.
Disclosure of Invention
In view of the above, the invention provides a high-risk human papilloma virus nucleic acid typing detection kit and a detection method.
In order to achieve the above object, the present invention provides the following technical solutions:
the present invention provides a primer probe combination having:
I. a nucleotide sequence shown as SEQ ID No. 1-60; or
II. A nucleotide sequence which is obtained by modifying, substituting, deleting or adding one or more bases in the nucleotide sequence shown in SEQ ID No. 1-60 and has the same or similar function with the nucleotide sequence shown in SEQ ID No. 1-60; or
III, a nucleotide sequence with at least 80 percent of homology with the nucleotide sequence shown as SEQ ID No. 1-60, or a nucleotide sequence with the same or similar functions with the nucleotide sequence shown as SEQ ID No. 1-60.
In some embodiments of the invention, the primer probe combination comprises one or more of the following combinations:
combination X:
I. the upstream primer has a nucleotide sequence shown as SEQ ID No. (3X-2); and
II. The downstream primer has a nucleotide sequence shown as SEQ ID No. (3X-1); and
the III probe has a nucleotide sequence shown as SEQ ID No. 3X; or
IV, a nucleotide sequence which is obtained by modifying, substituting, deleting or adding one or more bases in the nucleotide sequence shown in I, II or III and has the same or similar functions with the nucleotide sequence shown in I, II or III; or
V, a nucleotide sequence with at least 80% homology with the nucleotide sequence shown as I, II or III;
x is selected from any integer from 1 to 20;
when X =1, detecting human papillomavirus types 16, 31 and 35;
when X =2, detecting human papillomavirus type 33, 52 and 58;
when X =3, detecting human papillomavirus types 51, 56 and 66;
when the X =4, detecting the human papillomavirus types 18, 45 and 68;
when X =5, detecting human papillomavirus type 39, 59;
detecting an internal reference when the X = 6; the internal reference is human genome beta-Globin;
when the X =7, detecting the human papillomavirus type 16;
when the X =8, detecting the human papillomavirus type 31;
when the X =9, detecting the human papillomavirus type 35;
when X =10, detecting human papillomavirus type 33;
detecting human papillomavirus type 52 when the X = 11;
detecting human papillomavirus type 58 when the X = 12;
when X =13, detecting human papillomavirus type 51;
detecting human papillomavirus type 56 when the X = 14;
detecting human papillomavirus type 66 when the X = 15;
when the X =16, detecting the human papillomavirus type 18;
when the X =17, detecting the human papillomavirus type 45;
detecting human papillomavirus type 68 when the X = 18;
when the X =19, detecting the human papillomavirus type 39;
and when the X =20, detecting the human papillomavirus 59 type.
The invention also provides application of the primer probe combination in preparation of a reagent or a kit for detecting human papilloma virus.
In some embodiments of the invention, the human papillomavirus comprises high risk human papillomavirus types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68.
The invention also provides a detection reagent which comprises the primer probe combination and an acceptable auxiliary agent.
In some embodiments of the present invention, the detection reagent comprises human papillomavirus primer probe mixed liquor 1, human papillomavirus primer probe mixed liquor 2, human papillomavirus primer probe mixed liquor 3 and human papillomavirus primer probe mixed liquor 4, wherein:
the human papilloma virus primer probe mixed solution 1 comprises a combination 1, a combination 2, a combination 3, a combination 4, a combination 5 and a combination 6 in the primer probe combination, wherein two ends of a probe in the combination 1 are respectively marked with a fluorescence reporter group FAM and a fluorescence quenching group BHQ1, two ends of a probe in the combination 2 are respectively marked with a fluorescence reporter group HEX and a quenching group MGB, two ends of a probe in the combination 3 are respectively marked with a fluorescence reporter group ROX and a quenching group MGB, two ends of a probe in the combination 4 are respectively marked with a fluorescence reporter group CY5 and a fluorescence quenching group BHQ2, two ends of a probe in the combination 5 are respectively marked with a fluorescence reporter group Quasar and a fluorescence quenching group BHQ3, and two ends of a probe in the combination 6 are respectively marked with a fluorescence reporter group Atto425 and a fluorescence quenching group BHQ1;
the human papilloma virus primer probe mixed solution 2 comprises a combination 7, a combination 8, a combination 9, a combination 10, a combination 11 and a combination 6 in the primer probe combination, wherein two ends of a probe in the combination 7 are respectively marked with a fluorescence reporter FAM and a fluorescence quenching group BHQ1, two ends of a probe in the combination 8 are respectively marked with a fluorescence reporter HEX and a fluorescence quenching group BHQ1, two ends of a probe in the combination 9 are respectively marked with a fluorescence reporter ROX and a fluorescence quenching group BHQ2, two ends of a probe in the combination 10 are respectively marked with a fluorescence reporter CY5 and a fluorescence quenching group BHQ2, two ends of a probe in the combination 11 are respectively marked with a fluorescence reporter Quasar705 and a fluorescence quenching group BHQ3, and two ends of a probe in the combination 6 are respectively marked with a fluorescence reporter Atto425 and a fluorescence quenching group BHQ1;
the human papilloma virus primer probe mixed solution 3 comprises a combination 13, a combination 14, a combination 15, a combination 16, a combination 17 and a combination 6 in the primer probe combination, wherein two ends of a probe in the combination 13 are respectively marked with a fluorescence reporter group FAM and a fluorescence quenching group BHQ1, two ends of a probe in the combination 14 are respectively marked with a fluorescence reporter group HEX and a fluorescence quenching group BHQ1, two ends of a probe in the combination 15 are respectively marked with a fluorescence reporter group ROX and a fluorescence quenching group BHQ2, two ends of a probe in the combination 16 are respectively marked with a fluorescence reporter group CY5 and a fluorescence quenching group BHQ2, two ends of a probe in the combination 17 are respectively marked with a fluorescence reporter group Quasar705 and a fluorescence quenching group BHQ3, and two ends of a probe in the combination 6 are respectively marked with a fluorescence reporter group Atto425 and a fluorescence quenching group BHQ1;
the human papilloma virus primer probe mixed solution 4 comprises a combination 12, a combination 18, a combination 19, a combination 20 and a combination 6 in the primer probe combination, wherein two ends of a probe in the combination 12 are respectively marked with a fluorescence reporter group FAM and a fluorescence quenching group BHQ1, two ends of a probe in the combination 18 are respectively marked with a fluorescence reporter group HEX and a fluorescence quenching group BHQ1, two ends of a probe in the combination 19 are respectively marked with a fluorescence reporter group ROX and a fluorescence quenching group BHQ2, two ends of a probe in the combination 20 are respectively marked with a fluorescence reporter group CY5 and a fluorescence quenching group BHQ2, and two ends of a probe in the combination 6 are respectively marked with a fluorescence reporter group Quasar705 and a fluorescence quenching group BHQ3;
the probe comprises a Taqman probe and an MGB probe, wherein a fluorescence reporter group and a fluorescence quenching group are respectively marked at two ends of the Taqman probe, and a fluorescence reporter group and a quenching group MGB are respectively marked at two ends of the MGB probe.
The invention also provides application of the detection reagent in preparation of a kit for detecting human papilloma virus.
In some embodiments of the invention, the human papillomavirus comprises high risk human papillomavirus types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68.
The invention also provides a kit comprising the primer probe combination or the detection reagent.
The invention provides a high-risk human papilloma virus nucleic acid typing detection kit, which at least comprises 7 components, wherein the components are as follows: the kit comprises human papillomavirus PCR reaction liquid A, human papillomavirus primer probe mixed liquid 1, human papillomavirus primer probe mixed liquid 2, human papillomavirus primer probe mixed liquid 3, human papillomavirus primer probe mixed liquid 4, human papillomavirus positive control and human papillomavirus negative control. Wherein:
the human papillomavirus PCR reaction solution A is commercialized multiplex PCR amplification MIX;
the human papillomavirus positive control comprises human papillomavirus 16 type clone plasmid, human papillomavirus 31 type clone plasmid, human papillomavirus 35 type clone plasmid, human papillomavirus 33 type clone plasmid, human papillomavirus 52 type clone plasmid, human papillomavirus 58 type clone plasmid, human papillomavirus 51 type clone plasmid, human papillomavirus 56 type clone plasmid, human papillomavirus 66 type clone plasmid, human papillomavirus 18 type clone plasmid, human papillomavirus 45 type clone plasmid, human papillomavirus 68 type clone plasmid, human papillomavirus 39 type clone plasmid, human papillomavirus 59 type clone plasmid and human genome beta-Globin clone plasmid, wherein the human papillomavirus positive control is formed by mixing 15 plasmids and can be specifically amplified by using the primer probe combination.
In some embodiments of the invention, the concentration of each plasmid is 1.00-5.00E +05copies/mL.
The invention also provides a detection method of the human papilloma virus, which comprises the following steps of taking a sample to be detected to detect:
(I) The primer probe combination; or
(II), the detection reagent; or
(III) the kit.
In some embodiments of the invention, the detection method comprises:
in the face of detection mechanisms with large clinical sample size or cervical cancer large screening, the following detection methods can be used:
taking out a detection reagent in the kit, mixing human papillomavirus PCR reaction liquid A and human papillomavirus primer probe mixed liquid 1 according to the following mode, wherein the addition amount of the human papillomavirus PCR reaction liquid A is 12.5 mu L per person, the addition amount of the human papillomavirus primer probe mixed liquid 1 is 7.5 mu L per person, the final concentration of each primer is 0.1-0.5 mu mol/L, the final concentration of each probe is 0.05-0.25 mu mol/L, oscillating and uniformly mixing, and subpackaging the mixture into PCR reaction tubes one by one according to 20 mu L of each reaction;
secondly, extracting nucleic acid of a clinical sample by using a paramagnetic particle method;
thirdly, sucking 5 mu L of each of the negative quality control product, the positive quality control product and the extracted clinical sample nucleic acid, respectively adding the negative quality control product, the positive quality control product and the extracted clinical sample nucleic acid into a PCR reaction tube filled with reaction liquid, uniformly mixing and centrifuging;
fourthly, carrying out amplification detection on a 6-channel fluorescent quantitative PCR instrument, wherein the reaction procedure is as follows: (1) at 25 ℃ for 5-10min; (2) 95 ℃ for 1-5min; (3) 95 ℃,5-15sec; performing 40-45 cycles at 60 deg.C for 20-30 sec;
fifthly, after PCR amplification detection is finished, judging whether the detection target is negative or positive according to an amplification curve and a Ct value, judging that the detection target is positive when the amplification curve is in a standard S type and the Ct value of the detection target is less than or equal to 39 and the beta-Globin amplification curve is in a standard S type and the Ct value is less than or equal to 40, determining that the detection target is positive, determining next operation according to the detection result, and directly reporting the negative without performing the next operation if the detection result of the sample is negative; and if the detection result is positive in a certain channel, performing secondary detection. Specifically, if the detection result is positive in the FAM channel, which indicates that the sample is positive in one or more of human papillomavirus types 16, 31 and 35, the human papillomavirus PCR reaction solution a and the human papillomavirus primer probe mixed solution 2 are repeatedly operated according to the first step to the fourth step to confirm the positive type of the sample; if the detection result is positive, the sample is positive in one or more of human papillomavirus types 33, 52 and 58, and the human papillomavirus PCR reaction liquid A is respectively mixed with the human papillomavirus primer probe mixed liquid 2 and the human papillomavirus primer probe mixed liquid 4 for secondary detection; if the detection result is positive in ROX channel, the sample is positive in one or more of human papillomavirus types 51, 56 and 66, and the human papillomavirus PCR reaction liquid A and the human papillomavirus primer probe mixed liquid 3 are mixed for secondary detection; if the detection result is positive in CY5 channel, the sample is positive in one or more of human papillomavirus 18, 45 and 68 types, and the human papillomavirus PCR reaction liquid A, the human papillomavirus primer probe mixed liquid 3 and the human papillomavirus primer probe mixed liquid 4 are respectively mixed for secondary detection; if the detection result is that the Quasar705 channel is positive, the sample is positive to one or more of human papillomavirus 39 and human papillomavirus 59 types, and the human papillomavirus PCR reaction liquid A and the human papillomavirus primer probe mixed liquid 4 are mixed for secondary detection;
in the case of a detection facility or hospital with a small clinical sample size, when the daily sample size does not exceed 31 cases, the following detection method can be used:
taking out detection reagents in the kit, mixing human papillomavirus PCR reaction liquid A with human papillomavirus primer probe mixed liquid 2, human papillomavirus primer probe mixed liquid 3 and human papillomavirus primer probe mixed liquid 4 respectively to prepare 3 reagents (1), (2) and (3) in the following way, wherein the addition amount of each reaction human papillomavirus PCR reaction liquid A in the reagent (1) is 12.5 mu L per person, the addition amount of the human papillomavirus primer probe mixed liquid 2 is 7.5 mu L per person, the addition amount of each reaction human papillomavirus PCR reaction liquid A in the reagent (2) is 12.5 mu L per person, the addition amount of the human papillomavirus primer probe mixed liquid 3 is 7.5 mu L per person, the addition amount of each reaction human papillomavirus PCR reaction liquid A in the reagent (3) is 12.5 mu L per person, the addition amount of the human papillomavirus primer probe mixed liquid 4 is 7.5 mu L per person, wherein the final concentration of each primer is 0.1-0.5 mu L, the final concentration of each reaction probe in the reagent (3) is 0.5 mu L, and each reaction probe mixed liquid A is prepared according to 20 mu L by oscillation one by one, and the reaction is uniformly mixed in a reaction tube;
secondly, extracting nucleic acid of a clinical sample by using a magnetic bead method;
thirdly, sucking 5 mu L of each of the negative quality control product, the positive quality control product and the extracted clinical sample nucleic acid, respectively adding the negative quality control product, the positive quality control product and the extracted clinical sample nucleic acid into a PCR reaction tube filled with reaction liquid, uniformly mixing and centrifuging;
fourthly, carrying out amplification detection on a 6-channel fluorescence quantitative PCR instrument, wherein the reaction procedure is as follows: (1) 25 ℃,5-10min; (2) 95 ℃ for 1-5min; (3) 95 ℃,5-15sec; performing 40-45 cycles at 60 deg.C for 20-30 sec;
and fifthly, after the PCR amplification detection is finished, judging the negative and positive of the detection target according to the amplification curve and the Ct value, judging that the target is positive when the amplification curve is of a standard S type and the Ct value of the detection target is less than or equal to 39 and the beta-Globin amplification curve is of a standard S type and the Ct value is less than or equal to 40, and judging the human papillomavirus according to the used detection reagent and the positive channel.
The kit adopts internal standard positive quality control, judges whether a clinical sample is suitable for amplification detection by detecting beta-Globin in a human genome, and can be used for monitoring whether a PCR inhibitor exists in a reaction system and ensuring that a result of human papillomavirus false negative does not appear.
The kit is used for carrying out high-efficiency typing detection on high-risk types of human papilloma viruses, and ensures the detection sensitivity and specificity by designing a specific primer probe and carrying out multiple optimization on a system.
The kit is used for typing and efficiently detecting high-risk types of human papilloma viruses, the application scenes are mainly 2, the first method is to carry out primary screening by carrying out primary amplification detection when a detection mechanism with larger clinical sample amount or a cervical cancer large screening is faced, the single-hole detection is adopted, on a conventional PCR instrument, 94 clinical samples can be detected at most by one-time experiment, and the other two holes are used for detecting positive quality control and negative quality control. Because most of the human papilloma viruses are negative, when the detection results of the samples are negative, secondary detection is not needed, negative is directly reported, and for the samples with positive, secondary detection is needed to determine a specific type;
the second application scenario is that when the daily sample size of a hospital does not exceed 31 cases, in order to save time of medical staff, a secondary detection strategy can be directly used, the human papillomavirus PCR reaction liquid A is respectively mixed with the human papillomavirus primer probe mixed liquid 2, the human papillomavirus primer probe mixed liquid 3 and the human papillomavirus primer probe mixed liquid 4 in proportion, then the sample is added and amplified, the high-risk typing detection of the human papillomavirus is directly carried out, and the high-risk typing detection can be completed through one-time experiment.
The invention has the following beneficial effects:
firstly, when a detection mechanism with a large sample size or a cervical cancer large screening is carried out, a clinical sample can be primarily screened through a single hole of the kit, most negative samples in normal people are negative HPV, the negative samples with a negative detection result are directly reported, and when the positive samples appear, typing detection is carried out through a secondary experiment. The kit not only meets the requirement of clinically carrying out typing detection on high-risk HPV virus types, but also greatly improves the detection efficiency and saves the time of medical personnel;
secondly, the human papillomavirus primer probe mixed solution 1 in the kit comprises 6 pairs of primer probes, the 6 probes are respectively marked with different fluorescence emission groups and correspond to different channels of a fluorescence quantitative PCR instrument, so that the different channels correspond to different detection types, the primer probes of the human papillomavirus primer probe mixed solution 1 in the application patent cover 2 or 3 types, in the human papillomavirus primer probe mixed solution 2, the human papillomavirus primer probe mixed solution 3 and the human papillomavirus primer probe mixed solution 4, the primer probes of each type only specifically detect the type, all the primer probes are carefully compared and designed, and are screened from the pairs of primer probes through the verification of a large number of clinical samples, so that the detection sensitivity is ensured, and meanwhile, the kit has higher specificity;
thirdly, most negative samples can be screened out during primary screening, and a secondary experiment is carried out on a small part of positive samples, but the secondary experiment is not simply screened from all 14 high-level types, but a channel with a positive signal is recorded after a primary experiment, and at most 3 types are contained in the channel, namely the positive type is one of the 3 types, so that during the secondary experiment, a targeted primer and probe mixed solution is adopted for detection, and the infection type can be determined by using one hole or 2 holes, so that 1 hole can complete the detection of the sample if one clinical sample is negative, and positive typing detection can be completed by using 2 or 3 holes if the clinical sample is positive, and other manufacturers generally need 4 holes or more for typing, therefore, the kit not only achieves the purpose of HPV typing, but also saves the use of reagents and has higher economic value;
fourthly, the kit can be used for detection mechanisms with large sample size or cervical cancer screening periods, is also suitable for detection mechanisms with small sample size, if the single sample size does not exceed 31 samples, the kit can adopt a scheme of 3-hole detection, directly carry out typing detection on all samples in one experiment, can detect 31 samples at most in one experiment, and add positive control and negative control into the rest 2 holes. Therefore, the kit meets the requirements of various application scenes, when the sample size is large or the cervical cancer is screened greatly, a rapid and efficient typing detection scheme is provided, when the sample size is small, a 3-hole rapid detection scheme is adopted, the goal of both typing and high efficiency is really achieved, and the kit can exert the characteristics of the kit no matter large hospitals or large-scale screening.
Drawings
In order to more clearly illustrate the embodiments or prior art solutions of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below.
FIG. 1 shows the original curve obtained by preparing amplification reagent from the reaction solution A and the mixed solution 1 in proportion and amplifying and detecting the FAM channel of LCBD002 sample in example 1;
FIG. 2 is a graph showing an original curve obtained by preparing an amplification reagent from the reaction solution A and the mixed solution 1 in proportion and amplifying and detecting the CY5 channel of a sample LCBD023 in example 1;
FIG. 3 shows the original curve obtained by preparing amplification reagent from the reaction solution A and the mixed solution 1 in proportion and detecting HEX channel of the LC007-LC012 sample by amplification in example 1;
FIG. 4 shows the results of linear regression analysis of Ct values of positive samples in the test results of the kit of the present application and the kit of the same family as Jiangsu in example 1;
FIG. 5 shows the results of linear regression analysis of Ct values of positive samples in the test results of the kit of the present application and the kit of the same family as Jiangsu in example 3.
Detailed Description
The invention discloses a high-risk human papilloma virus nucleic acid typing detection kit, and a person skilled in the art can use the contents for reference and appropriately improve process parameters to realize the detection. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The nucleotide sequences of the primers and probes for specifically amplifying multiple HPV subtypes and internal reference beta-globins are shown in the following table:
TABLE 1
The high-risk human papilloma virus nucleic acid typing detection kit provided by the invention comprises the following components: (1) PCR reaction solution a, commercial multiplex PCR amplification MIX, purchased from saint next biotechnology (shanghai) gmbh and having a product name:
universal TaqMan mu. Ltiplex qPCR master mix (UDG plus) with product number 11212ES03; (2) the human papillomavirus primer probe mixed solution 1 comprises primer probe combinations shown as a combination 1, a combination 2, a combination 3, a combination 4, a combination 5 and a combination 6; (3) human papillomavirus primer probe mixed solution 2 comprises primer probe combinations shown as combination 7, combination 8, combination 9, combination 10, combination 11 and combination 6; (4) human papillomavirus primer probe mixed liquor 3 comprises primer probe combinations shown as combination 13, combination 14, combination 15, combination 16, combination 17 and combination 6; (5) the human papillomavirus primer probe mixed solution 4 comprises primer probe combinations shown as a combination 12, a combination 18, a combination 19, a combination 20 and a combination 6; (6) human papillomavirus positive controls including human papillomavirus 16, 31, 35, 33, 52, 58, 51, 56, 66, 18, 45, 68, 39, 59 type cloning plasmids and human genome beta-Globin cloning plasmids, wherein each plasmid is synthesized by bio-engineering (Shanghai) GmbH, and the concentration is 1.00-5.00E +05copies/mL; (7) human papillomavirus negative control, ultrapure water.
Each primer and probe in the human papillomavirus primer probe mixed solution 1-4 are synthesized by biological engineering (Shanghai) corporation, the final concentration of each primer in the reaction system is 0.1-0.5 mu mol/L, and the final concentration of each probe in the reaction system is 0.05-0.25 mu mol/L.
The invention provides a use method of a high-risk human papilloma virus nucleic acid typing detection kit, which comprises the following steps:
firstly, mixing a human papillomavirus PCR reaction solution A and a human papillomavirus primer probe mixed solution 1 according to the following mode, wherein according to the use instruction of PCR amplification MIX of the Hippon company, the addition amount of the human papillomavirus PCR reaction solution A is 12.5 mu L per person, the addition amount of the human papillomavirus primer probe mixed solution 1 is 7.5 mu L per person, the final concentration of each primer is 0.1-0.5 mu mol/L, the final concentration of each probe is 0.05-0.25 mu mol/L, oscillating and uniformly mixing, and subpackaging the mixture into PCR reaction tubes one by one according to 20 mu L of each reaction; secondly, extracting nucleic acid of a clinical sample by using a magnetic bead method;
thirdly, sucking 5 mu L of each of the negative quality control product, the positive quality control product and the extracted clinical sample nucleic acid, respectively adding the negative quality control product, the positive quality control product and the extracted clinical sample nucleic acid into a PCR reaction tube filled with reaction liquid, uniformly mixing and centrifuging;
fourthly, amplification detection is carried out on a 6-channel fluorescence quantitative PCR instrument purchased from Shanghai Hongshi medical science and technology Limited company, and the reaction procedure is as follows: (1) at 25 ℃ for 5-10min; (2) 95 ℃ for 1-5min; (3) 95 ℃,5-15sec; performing 40-45 cycles at 60 deg.C for 20-30 sec;
fifthly, after PCR amplification detection is finished, judging whether the detection target is negative or positive according to an amplification curve and a Ct value, judging that the detection target is positive when the amplification curve is in a standard S type and the Ct value of the detection target is less than or equal to 39 and the beta-Globin amplification curve is in a standard S type and the Ct value is less than or equal to 40, determining that the detection target is positive, determining next operation according to the detection result, and directly reporting the negative without performing the next operation if the detection result of the sample is negative; and if the detection result is positive in a certain channel, performing secondary detection. Specifically, if the detection result is positive in the FAM channel, which indicates that the sample is positive in one or more of human papillomavirus types 16, 31 and 35, the human papillomavirus PCR reaction solution a and the human papillomavirus primer probe mixed solution 2 are repeated according to the first to fourth steps, so as to confirm the positive type of the sample; if the detection result is positive, the sample is positive in one or more of human papillomavirus 33, 52 and 58 types, and the human papillomavirus PCR reaction liquid A is respectively mixed with the human papillomavirus primer probe mixed liquid 2 and the human papillomavirus primer probe mixed liquid 4 for secondary detection; if the detection result is positive in ROX channel, the sample is positive in one or more of human papillomavirus types 51, 56 and 66, and the human papillomavirus PCR reaction liquid A and the human papillomavirus primer probe mixed liquid 3 are mixed for secondary detection; if the detection result is positive in CY5 channel, the sample is positive in one or more of human papillomavirus 18, 45 and 68 types, and the human papillomavirus PCR reaction liquid A, the human papillomavirus primer probe mixed liquid 3 and the human papillomavirus primer probe mixed liquid 4 are respectively mixed for secondary detection; if the detection result is that the Quasar705 channel is positive, the sample is positive to one or more of human papillomavirus 39 and human papillomavirus 59 types, and the human papillomavirus PCR reaction liquid A and the human papillomavirus primer probe mixed liquid are mixed for secondary detection.
In the examples provided by the present invention, the instruments and sources of reagents used are shown in the following table:
TABLE 2
In the high-risk human papilloma virus nucleic acid typing detection kit provided by the invention, used raw materials and reagents can be purchased from the market.
The invention is further illustrated by the following examples:
example 1
In order to embody the detection effect of the primer probe combination and the kit, the kit is used for detecting clinical samples and is compared with a nucleic acid detection kit for human papilloma virus of the same family as Jiangsu.
1.1 sample information
Sample source: 35 clinical specimens of cervical exfoliated cells from Zhengzhou Sonote medical laboratory;
sample number: LC001-LC035.
1.2 Instrument and reagent sources in the experiment
The instrument and reagent sources used in this example are shown in Table 2, and the sequences of the specific primers and probes used for typing detection of human papillomavirus are shown in Table 1.
1.3 Experimental methods
1.3.1 reagent preparation
Mixing the human papillomavirus PCR reaction liquid A and the human papillomavirus primer probe mixed liquid 1 according to the following mode, wherein the addition amount of the human papillomavirus PCR reaction liquid A in each reaction is 12.5 mu L per person, the addition amount of the human papillomavirus primer probe mixed liquid 1 is 7.5 mu L per person, preparing 37 reactions in total, oscillating and uniformly mixing after preparation, and subpackaging the prepared 20 mu L reactions one by one into a PCR reaction tube; the reaction system is as follows:
TABLE 3
| Components | Dosage of |
| Human papilloma virus PCR reaction liquid A | 12.5μL |
| Human papillomavirus primer probe |
7.5μL |
| Form panel | 5μL |
1.3.2 sample handling
The 35 cases of clinical samples were shaken and mixed well, and nucleic acid extraction was performed on the clinical samples using nucleic acid extraction reagents of Henan Sainuo biologies in combination with a fully automatic nucleic acid extractor of Jifan Biotech Co., ltd.
1.3.3 sample adding machine
Sucking extracted clinical sample nucleic acid, negative quality control product and positive quality control product, adding into PCR reaction tube filled with reaction liquid, mixing, centrifuging, and performing amplification detection on SLAN-96S/48P full-automatic medical PCR analysis system instrument, wherein the reaction procedure is as follows: (1) at 25 ℃ for 5-10min; (2) 95 ℃ for 1-5min; (3) 40-45 cycles of 95 ℃ 5-15sec,60 ℃ 20-30 sec.
1.4 analysis of results
The samples LC001-LC035 were tested according to the above test procedure, and the first test results are as follows:
TABLE 4
The FAM channel of the sample LC001-LC006 is positive in the detection result, which indicates that the LC001-LC006 contains one or more types of human papillomavirus 16, 31 and 35 positive, the human papillomavirus PCR reaction liquid A and the human papillomavirus primer probe mixed liquid 2 are repeatedly operated according to the experimental method of 1.3, and the positive type of the sample is confirmed;
in the detection result, HEX channels of the samples LC007-LC012 are positive, which indicates that the LC007-LC012 contains one or more types of human papillomaviruses 33, 52 and 58 which are positive, the PCR reaction liquid A of the human papillomaviruses is respectively repeatedly operated with the primer probe mixed liquid 2 and the primer probe mixed liquid 4 of the human papillomaviruses according to the experimental method of 1.3, and the positive type of the sample is confirmed;
in the detection result, ROX channels of the samples LC013-LC018 are positive, which indicates that the LC013-LC018 contain one or more types of human papillomavirus 51, 56 and 66, and the positive types of the samples are confirmed by repeatedly performing the operation on the human papillomavirus PCR reaction liquid A and the human papillomavirus primer probe mixed liquid 3 according to the experiment method 1.3;
in the detection result, the CY5 channel of the sample LC019-LC024 is positive, which indicates that the LC019-LC024 contains one or more types of human papillomaviruses of 18, 45 and 68, and the positive type of the sample is confirmed by repeatedly performing the operation on the human papillomavirus PCR reaction solution A, the human papillomavirus primer probe mixed solution 3 and the human papillomavirus primer probe mixed solution 4 according to the experiment method 1.3;
in the detection result, the Quasar705 channel of the sample LC025-LC028 is positive, which indicates that the LC025-LC028 contains one or more positive types of human papillomavirus 39 or 59, and the human papillomavirus PCR reaction liquid A and the human papillomavirus primer probe mixed liquid 3 are repeatedly operated according to the experiment method of 1.3 to confirm the positive type of the sample;
and in the detection result, the detection result of the sample LC029-LC035 is negative, and secondary detection is not needed.
In the first detection results shown in table 4, the original curve of the sample LC002 fluorescence quantitative PCR detection result is shown in fig. 1, the sample detection channel is FAM, the Ct value is 22.91, and the channel detection result is positive; the original curve of the sample LC023 fluorescence quantitative PCR detection result is shown in figure 2, the detection channel of the sample is CY5, the Ct value is 30.77, and the detection result of the channel is positive; the original curve of the fluorescent quantitative PCR detection result of the sample LC007-LC012 is shown in FIG. 3, the detection channel of the sample is HEX, the Ct value of the sample LC007 is 25.38, and the detection result of the channel is positive; the Ct value of the sample LC008 is 26.05, and the detection result of the channel is positive; the Ct value of the sample LC009 is 31.34, and the detection result of the channel is positive; the Ct value of the sample LC010 is 24.93, and the detection result of the channel is positive; the Ct value of the sample LC011 is 22.73, and the detection result of the channel is positive; the Ct value of the sample LC012 is 26.53, and the channel detection result is positive.
The samples LC001 to LC028 were subjected to secondary detection according to the above experimental procedure, and the detection results are shown in table 5.
Detecting samples LC001-LC035 according to the kit instruction of the same department of Jiangsu, and comparing the detection result with the detection result of the kit of the invention, the result is as follows:
TABLE 5
Through comparative analysis, the negative and positive coincidence rates of the detection results of the kit and the comparative kit are both 100%, the Ct value of the positive sample detection is made into a linear regression equation for linear regression analysis, the linear function is obtained and is y =1.0756x-1.5622, and the absolute value of the correlation R of the positive sample detection and the linear regression equation is 0.99.
1.5 conclusion
According to the analysis of the comparison detection experiment result, the consistency of the detection results of the kit and the comparison kit is better, which indicates that the kit can achieve the same detection effect as the kit of the Jiangsu same family.
Example 2
In order to embody the present invention, the final concentration of each primer probe of each type in the kit provided by the present invention is verified by using the optimization of clinical samples through specific experiments, and this example takes the optimal final concentration screening and optimization process of the 16, 31, 35 type primers as a representative, and explains the optimal final concentration screening and optimization process of all primers and probes.
2.1 sample information
Sample source: type 16 positive 3 clinical samples of cervical exfoliated cells from Zhengzhou cenote medical laboratory;
sample number: LCBD1033-LCBD1035.
2.2 Instrument and reagent sources in the experiment
The instrument and reagent sources used in this example are shown in Table 2, and the sequences of the specific primers and probes used for typing detection of human papillomavirus are shown in SEQ ID Nos. 1-3 in Table 1.
2.3 Experimental methods
2.3.1 reagent preparation
Reagent preparation is carried out according to the purpose of experiment, control variables are adopted, the quantity of other components is kept unchanged, and the optimal final concentration screening and optimization are carried out by only changing primers.
The purpose of this example assay was to screen the optimal concentration range of primers, thus retaining the 16, 31, 35 type probes and ddH 2 Changing the addition amount of the primers without changing the O content to prepare 16, 31 and 35 type primer probe mixed solutions (1) - (5), wherein the preparation method of the primer probe mixed solutions (1) - (5) is as follows:
TABLE 6
TABLE 7
TABLE 8
TABLE 9
The primer probe mixed liquid (1) - (5) is prepared according to the methods shown in tables 6-10, 5 corresponding reagents are respectively prepared according to the next PCR amplification MIX system after the preparation is completed, 5 persons are prepared for each reagent, and the system is as follows:
TABLE 11
| Components | The dosage of each part of the medicine is |
| Human papilloma virus PCR reaction liquid A | 12.5μL |
| Primer Probe mixture (1) - (5) | 7.5μL |
2.3.2 sample handling
The 3 clinical samples were separately shaken and mixed, and nucleic acid extraction was performed on the clinical samples using a nucleic acid extraction reagent of Henan Sainuo biologies in combination with a fully automatic nucleic acid extractor of Jifan Biotech Co., ltd.
2.3.3 sample adding machine
Sucking extracted clinical sample nucleic acid, negative quality control product and positive quality control product, adding into PCR reaction tube filled with reaction liquid, mixing, centrifuging, and performing amplification detection on SLAN-96S/48P full-automatic medical PCR analysis system instrument, wherein the reaction procedure is as follows: (1) at 25 ℃ for 5-10min; (2) 95 ℃ for 1-5min; (3) 40-45 cycles of 95 ℃ 5-15sec,60 ℃ 20-30 sec.
2.4 analysis of results
2.4.1 the test results are as follows:
TABLE 12
2.4.2 conclusion
Analysis on the detection result shows that the detection sensitivity is better when the final concentration of the primer is 0.1-0.5 mu mol/L.
Example 3
The second application scene of this application patent is when the hospital sample size of the day is no longer than 31 cases, in order to save medical staff's time, can directly use the strategy of secondary detection, mix human papillomavirus PCR reaction liquid A respectively with human papillomavirus primer probe mixed liquid 2, human papillomavirus primer probe mixed liquid 3, human papillomavirus primer probe mixed liquid 4 mixes in proportion, then the application of sample amplifys, directly carry out the high-risk type typing detection of human papillomavirus, high-risk type typing detection can be accomplished in the experiment once, reach simple high efficiency's purpose. This embodiment will detail the implementation process of the detection method.
3.1 sample information
Sample source: 17 clinical specimens of cervical exfoliated cells from Zhengzhou cenot medical laboratory;
sample number: LC036-LC052.
3.2 Instrument and reagent sources in the experiment
The instrument and reagent sources used in this example are shown in Table 2, and the sequences of the specific primers and probes used for typing detection of human papillomavirus are shown in SEQ ID Nos. 16-60 in Table 1.
3.3 Experimental methods
3.3.1 reagent preparation
Mixing the human papillomavirus PCR reaction liquid A with a human papillomavirus primer probe mixed liquid 2, a human papillomavirus primer probe mixed liquid 3 and a human papillomavirus primer probe mixed liquid 4 respectively to prepare 3 reagents (1), (2) and (3), wherein 19 reactions are prepared for each reagent, the addition amount of each reaction human papillomavirus PCR reaction liquid A in the reagent (1) is 12.5 mu L per person, the addition amount of the human papillomavirus primer probe mixed liquid 2 is 7.5 mu L per person, the addition amount of each reaction human papillomavirus PCR reaction liquid A in the reagent (2) is 12.5 mu L per person, the addition amount of the human papillomavirus primer probe mixed liquid 3 is 7.5 mu L per person, the addition amount of each reaction human papillomavirus PCR reaction liquid A in the reagent (3) is 12.5 mu L per person, the addition amount of the human papillomavirus primer probe mixed liquid 4 is 7.5 mu L per person, the 3 reagents are prepared and then are uniformly mixed, and the PCR reaction liquid is subpackaged into 20 mu L reaction tubes one by one; the reaction system is as follows:
watch 13
| Reagent (1) Components | Dosage of |
| Human papilloma virus PCR reaction liquid A | 12.5μL |
| Human papillomavirus primer probe mixed |
7.5μL |
TABLE 14
| Reagent (2) Components | Amount of the composition |
| Human papilloma virus PCR reaction liquid A | 12.5μL |
| Human papillomavirus primer probe mixed |
7.5μL |
| Reagent (3) Components | Dosage of |
| Human papilloma virus PCR reaction liquid A | 12.5μL |
| Human papillomavirus primer probe mixed liquor 4 | 7.5μL |
3.3.2 sample handling
The 17 cases of clinical samples were shaken and mixed well, and nucleic acid extraction was performed on the clinical samples using nucleic acid extraction reagents of Henan Sainuo organisms in combination with a fully automatic nucleic acid extractor of Jifan Biotech Co.
3.3.3 sample adding machine
Sucking extracted clinical sample nucleic acid, negative quality control product and positive quality control product, adding into PCR reaction tube filled with reaction liquid, mixing, centrifuging, and performing amplification detection on SLAN-96S/48P full-automatic medical PCR analysis system instrument, wherein the reaction procedure is as follows: (1) at 25 ℃ for 5-10min; (2) 95 ℃ for 1-5min; (3) 95 ℃,5-15sec; 40-45 cycles at 60 ℃ for 20-30 sec.
3.4 detection result:
the detection results of the kit of the present invention are shown in Table 16. Detecting a sample LC036-LC052 by using a Jiangsu Cochinchinensis kit according to the instruction, and carrying out comparative analysis on the detection results of the two reagents, wherein the results are as follows:
TABLE 16
Through comparison analysis, the negative and positive coincidence rates of the detection results of the kit and the comparison kit are both 100%, the Ct value of the positive sample detection is made into a linear regression equation for linear regression analysis, the linear function is obtained and is y =1.0649x-1.5071, and the absolute value of the correlation R of the positive sample detection and the linear regression equation is 0.99 (the result is shown in figure 5).
3.5 conclusion
According to the analysis of the comparison detection experiment result, the consistency of the detection results of the kit and the comparison kit is better. Thus, the second application scenario of the present patent: when the daily sample size of a hospital does not exceed 31 cases, in order to save the time of medical staff, a secondary detection strategy can be directly used and can be smoothly realized, and the strategy has high speed and high efficiency when the sample size is less than 31 cases.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A primer probe set comprising:
I. a nucleotide sequence shown as SEQ ID No. 1-60; or
II. A nucleotide sequence which is obtained by modifying, substituting, deleting or adding one or more bases in the nucleotide sequence shown in SEQ ID No. 1-60 and has the same or similar function with the nucleotide sequence shown in SEQ ID No. 1-60; or
III, a nucleotide sequence with at least 80 percent of homology with the nucleotide sequence shown as SEQ ID No. 1-60, or a nucleotide sequence with the same or similar functions with the nucleotide sequence shown as SEQ ID No. 1-60.
2. The primer probe combination of claim 1, wherein the primer probe combination comprises one or more of the following combinations:
combination X:
I. the upstream primer has a nucleotide sequence shown as SEQ ID No. (3X-2); and
II. The downstream primer has a nucleotide sequence shown as SEQ ID No. (3X-1); and
III, the probe has a nucleotide sequence shown as SEQ ID No. 3X; or
IV, a nucleotide sequence which is obtained by modifying, substituting, deleting or adding one or more bases in the nucleotide sequence shown in I, II or III and has the same or similar function with the nucleotide sequence shown in I, II or III; or
V, a nucleotide sequence with at least 80% homology with the nucleotide sequence shown as I, II or III;
wherein X is selected from any integer of 1-20.
3. The primer probe combination of claim 2,
when X =1, detecting human papillomavirus types 16, 31 and 35;
when X =2, detecting human papillomavirus type 33, 52 and 58;
when the X =3, detecting the human papillomavirus types 51, 56 and 66;
when X =4, detecting human papillomavirus types 18, 45 and 68;
when X =5, detecting human papillomavirus type 39, 59;
detecting an internal reference when the X = 6;
when the X =7, detecting the human papilloma virus type 16;
detecting human papillomavirus type 31 when the X = 8;
when the X =9, detecting the human papilloma virus type 35;
when X =10, detecting human papillomavirus type 33;
detecting human papillomavirus type 52 when the X = 11;
detecting human papillomavirus type 58 when the X = 12;
when X =13, detecting human papillomavirus type 51;
detecting human papillomavirus type 56 when the X = 14;
detecting human papillomavirus type 66 when the X = 15;
detecting human papillomavirus type 18 when the X = 16;
when X =17, detecting human papillomavirus type 45;
detecting human papillomavirus type 68 when the X = 18;
when X =19, detecting human papillomavirus type 39;
and when the X =20, detecting the human papilloma virus 59 type.
4. Use of the primer probe combination according to any one of claims 1 to 3 for preparing a reagent or a kit for detecting human papillomavirus.
5. The use of claim 4, wherein the human papillomavirus comprises high risk human papillomavirus types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68.
6. A detection reagent comprising the primer-probe combination according to any one of claims 1 to 3 and an acceptable auxiliary.
7. Use of the detection reagent according to claim 6 in the preparation of a kit for detecting human papillomavirus.
8. The use of claim 7, wherein the human papillomavirus comprises high risk human papillomavirus types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68.
9. A kit comprising the primer-probe combination according to any one of claims 1 to 3 or the detection reagent according to claim 6.
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