CN113528709B

CN113528709B - Fluorescent quantitative PCR detection method capable of covering 13-type adenovirus and kit

Info

Publication number: CN113528709B
Application number: CN202111065592.1A
Authority: CN
Inventors: 景寅; 张梦; 赵立明
Original assignee: Beijing Huarui Kangyuan Biotechnology Development Co ltd
Current assignee: Beijing Huanuo Aomei Gene Medical Laboratory Co ltd
Priority date: 2021-09-13
Filing date: 2021-09-13
Publication date: 2021-12-10
Anticipated expiration: 2041-09-13
Also published as: CN113528709A

Abstract

The invention discloses a fluorescent quantitative PCR detection method capable of covering 13 types of adenoviruses and a kit, wherein the kit comprises a specific primer, a specific probe, an internal reference primer, an internal reference probe, a PCR amplification reagent, a kit negative reference substance, a kit positive reference substance and ribozyme-free water, wherein the binding site of the specific primer and the specific probe is in a high-conservative section of the whole genome of the 13 types of adenoviruses infecting human respiratory tracts. The fluorescent quantitative PCR detection method and the kit which can cover 13 types of adenoviruses can realize single-tube single-PCR (polymerase chain reaction) and simultaneous detection of 13 adenoviruses infecting human respiratory tracts, realize high coverage, and solve the problem that the coverage, sensitivity, specificity, economy and timeliness cannot be all excellent due to multiple PCR adopted in the current detection link for covering multiple types.

Description

Fluorescent quantitative PCR detection method capable of covering 13-type adenovirus and kit

Technical Field

The invention relates to the technical field of human respiratory pathogen detection, in particular to a fluorescent quantitative PCR detection method capable of covering 13 types of adenoviruses and a kit.

Background

Human adenovirus (HAdV) infection can cause various diseases, including pneumonia, bronchitis, cystitis, conjunctivitis, gastrointestinal diseases, encephalitis and the like, and is one of common pathogens of human respiratory tract infection.

The population is generally susceptible, mainly manifested by recessive infection, acute upper and lower respiratory tract infection, and a few of them can develop severe pneumonia, even die. However, patients with weak immune system or chronic respiratory disease, heart disease, etc. have a high risk of serious diseases after infection, which can cause severe and critical illness and have a high death risk. At present, no specific antiviral drug aiming at human adenovirus infection exists in China, and the clinical application of the specific antiviral drug is symptomatic support, immunoregulation treatment and treatment aiming at complications.

Human adenovirus is popular in the world, and the epidemic pattern is variable and is often related to factors such as the type of human adenovirus, epidemic regions, the age of susceptible people and the like. Human adenovirus infection can occur all the year round, and is common in winter and spring in the north and in spring and summer in the south of China.

In recent years, respiratory tract infection outbreaks caused by human adenoviruses in China often occur, and serious cases of aggregation appear in individual regions. Pneumonia caused by adenovirus is mostly seen in children of 6 months to 5 years old, especially children under 2 years old, and since 2019, the incidence of adenovirus pneumonia in children in partial areas of China is increased to different degrees compared with the incidence of pneumonia in the past years, so that attention to the pneumonia is paid again.

The human adenovirus belongs to the genus of mammalian adenovirus, is a double-stranded DNA virus without envelope, has a virus particle with an icosahedral symmetrical structure and a diameter of 90-100 nm, and can be divided into 7 subgenera of A-G. Due to the different tissue tropism of different human adenoviruses to human body, the human adenoviruses related to respiratory disease mainly belong to subfamily B (HAdV-3, 7, 11, 14, 16, 21, 55), subfamily C (HAdV-1, 2, 5, 6) and subfamily E (HAdV-4). Human adenovirus is often co-infected with other respiratory viruses, and co-infection with different types of human adenovirus has also been reported.

In recent years, rapid pathogenic detection technology is popularized and applied, so that the detection rate of adenovirus is improved, early diagnosis becomes possible, and a plurality of problems in treatment are still needed to be solved. Early recognition and early diagnosis of respiratory diseases caused by adenovirus are crucial to reduce the severe rate and mortality rate of adenovirus infection.

The traditional method for typing adenovirus is mainly serotyping. The culture time is long due to virus isolation, the requirements on the proficiency and the discrimination experience of operators are high, and the culture is difficult to develop in a common laboratory. With the development of molecular biology techniques, methods for identification by molecular means such as PCR amplification and gene sequencing have emerged. However, since there are many types of adenovirus, detection reagents designed based on the genotype prevalent in region A cannot detect the genotype prevalent in region B. In the global trend, when the virus flows widely with the staff, the reagent generated in the area A is effective but cannot be applied to the area B, so that false negative is caused, and misdiagnosis and missed diagnosis are caused.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides a fluorescent quantitative PCR detection method and a kit which can cover 13 types of adenoviruses, and can overcome the defects in the prior art.

In order to achieve the technical purpose, the technical scheme of the invention is realized as follows:

a fluorescent quantitative PCR detection kit capable of covering 13 types of adenoviruses comprises a specific primer, a specific probe, an internal reference primer, an internal reference probe, a PCR amplification reagent, a kit negative control product, a kit positive control product and ribozyme-free water, wherein the binding site of the specific primer and the specific probe is in a high-conservative section of the whole genome of the 13 types of adenoviruses infecting human respiratory tracts, and the types of the 13 types of adenoviruses are 1 type, 2 type, 3 type, 4 type, 5 type, 6 type, 7 type, 11 type, 14 type, 16 type, 21 type, 35 type and 55 type;

the sequence of the specific primer is shown as SEQ ID NO: 1-SEQ ID NO: 6 is shown in the specification;

the sequence of the specific probe is shown as SEQ ID NO: 7-SEQ ID NO: 8 is shown in the specification;

the PCR amplification reagent comprises hot-start DNA polymerase, dNTPs, high-activity UNG enzyme, dUTP and PCR amplification buffer solution.

Preferably, the binding sites of the specific primers and the specific probes are in the internal of the gene sequences corresponding to the human adenovirus pentamer protein.

Preferably, the sequence of the internal reference primer is as shown in SEQ ID NO: 9-SEQ ID NO: 10, the sequence of the reference probe is shown as SEQ ID NO: 11, the regions for detecting and combining the internal reference primer and the internal reference probe are human ribonuclease P gene segments.

Preferably, the kit negative control is normal saline.

Preferably, the positive control substance of the kit is an engineering bacterial suspension for cloning the respiratory adenovirus pentamer protein gene sequence.

Preferably, the sequence of the upstream primer of the specific primer is shown in SEQ ID NO: 1, the sequence of the downstream primer of the specific primer is shown as SEQ ID NO: 4, the sequence of the specific probe is shown as SEQ ID NO: shown in fig. 8.

Preferably, the probe is a Taqman probe, and a fluorescent group is marked on the probe.

Preferably, the fluorescent group is one or more of FAM, VIC, Cy5, TET, HEX and JOE.

According to another aspect of the present invention, there is provided a fluorescent quantitative PCR detection method for non-diagnostic purpose that can cover 13 types of adenovirus, comprising the steps of:

(1) nucleic acid extraction: extracting nucleic acid of a sample to be detected by using a magnetic bead method or a column chromatography;

(2) sample adding: for a sample detection tube to be detected, a negative reference substance detection tube and a positive reference substance detection tube, each tube is a 20 mu l reaction system, and the method specifically comprises the following steps:

10. mu.l of a PCR reaction solution,

mu.l of a primer-probe combination,

5. mu.l of the DNA to be detected,

1 μ l of ribozyme-free water;

the primer probe combination consists of 0.8 mu l of specific forward primer, 0.8 mu l of specific reverse primer, 0.4 mu l of specific probe, 0.8 mu l of internal reference forward primer, 0.8 mu l of internal reference reverse primer and 0.4 mu l of internal reference probe, wherein the sequence of the specific forward primer is shown as SEQ ID NO: 1-SEQ ID NO: 3, the sequence of the specific reverse primer is shown as SEQ ID NO: 4-SEQ ID NO: 6, the sequence of the specific probe is shown as SEQ ID NO: 7-SEQ ID NO: 8, the sequence of the forward primer of the internal reference is shown as SEQ ID NO: 9, the sequence of the reverse primer of the internal reference is shown as SEQ ID NO: 10, the sequence of the reference probe is shown as SEQ ID NO: 11 is shown in the figure;

(3) performing amplification on the machine: the detection instrument can use a Stepone plus model of ABI company or a 7500 fluorescent quantitative PCR instrument, and the detection mode is selected from 'quantification', 'TaqMan Reagents'; program setting: pre-denaturation: 5min at 95 ℃ for 1 cycle; denaturation: 95 ℃ for 20s, annealing + extension: 1min at 60 ℃, signal acquisition, 45 cycles;

(4) data analysis and result judgment:

under the condition that the detection instrument runs normally and the detection results of negative quality control and positive quality control are controlled:

for specific primer detection tubes:

when the ct value is less than or equal to 39, the sample is positive for adenovirus detection;

when the ct value is more than or equal to 40 and less than or equal to 43, repeating the detection of the sample, and if the ct value is still less than or equal to 43, determining that the sample is weakly positive for adenovirus detection; otherwise, the sample is negative to adenovirus detection;

when the ct value is more than or equal to 44, the sample is negative for adenovirus detection;

for internal reference primer detection tubes:

when the ct value is less than or equal to 39, the sample is positive for the detection of the reference gene;

when the ct value is more than or equal to 40 and less than or equal to 43, repeating the detection of the sample, and if the ct value is still less than or equal to 43, determining that the sample is weak and positive for the reference gene detection; otherwise, the sample is negative to the detection of the reference gene;

when the ct value is larger than or equal to 44, the sample is negative to the reference gene detection.

Further, the sample is a clinical sample of a patient with respiratory tract infection, and the sample comprises sputum, nasopharyngeal swab and alveolar lavage fluid.

The invention has the beneficial effects that: the fluorescent quantitative PCR detection method and the kit which can cover 13 types of adenoviruses can accurately cover up to 13 types of adenoviruses, are not influenced by sequences of other non-respiratory adenoviruses (particularly alimentary adenovirus) and other microorganisms, and realize high coverage, high sensitivity and high specificity; by establishing an evolutionary tree model and genetic relationship analysis for the 13 adenoviruses, comprehensively comparing hundreds of thousands of base sequences of the whole genome to find more than ten homologous regions, testing various design software according to various principles (duplex formation hairpin, primer dimer, false primer and cross dimer) of primer design, finally finding out the optimal detection primer and probe combination and detection conditions by depending on multiple rounds of selection in the experiment, realizing that a single-tube single-PCR can cover the 13 adenoviruses infecting human respiratory tracts, judging whether the adenoviruses exist during the amplification period, and solving the difficult problems that the coverage, sensitivity, specificity, economy and timeliness cannot be all excellent due to multiple fluorescence PCR, time-consuming capillary electrophoresis detection or expensive high-throughput sequencing adopted in the conventional detection link for covering multiple types.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is an amplification chart of 13 types of adenovirus that can be effectively detected by using the fluorescent quantitative PCR detection kit according to the embodiment of the present invention;

FIG. 2 is an amplification chart of 13 types of mixed adenovirus effectively detected by the fluorescent quantitative PCR detection kit according to the embodiment of the present invention;

FIG. 3 is a graph showing the amplification curve of a clinical sample known to be positive when the fluorescence quantitative PCR detection kit according to the embodiment of the present invention is used;

FIG. 4 shows the sensitivity of the fluorescence quantitative PCR detection kit according to the embodiment of the present invention at a low concentration of a target to be detected;

FIG. 5 shows the specificity of the fluorescent quantitative PCR detection kit according to the present invention in the detection of various pathogens.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.

The invention adopts the following technical scheme: the full-length adenovirus sequences (GenBank: MH 183293.1; MF 044052.1; KF 006344.1; KX 868466.2; MH 355567.1; MK 886831.1; DQ 105654.4; FJ 597732.1; JX 892927.2; KJ 364592.1; AY 271307.1; FJ 349096.1; AY601636.1) which are mainly popular in the world over the years are downloaded in the NCBI (national authoritative database of International Union, national Committee) NCBI, wherein the full-length adenovirus sequences are of

types

1, 2, 3, 4, 5, 6, 7, 11, 14, 16, 21, 35 and 55, and are used as reference sequence templates, if a pair of primers and a Taqman probe are used for one type according to the conventional scheme, 13-fold PCR of 13 pairs of primers and 13 Taqman probes needs to be realized in a single tube to cover 13 types, so that the sensitivity of single-type detection is reduced and the reagent cost is increased by 13 times.

Therefore, an evolutionary tree model and genetic relationship analysis are established for the 13 adenoviruses, after the specificity and sensitivity of the fragments to be detected are evaluated, the gene sequences corresponding to the pentamer (penton) protein are selected as the combination regions of the primers and the probes, the detection method covers a wide range of types, the gene sequence difference among different types is overlarge, the effective combination regions of the primers and the probes are extremely limited, the combination regions of the primers and the probes are compressed on the basis, and finally the optimal detection combination is selected from nearly fifty pairs of primer combinations through multi-round experimental screening.

The fluorescent quantitative PCR detection kit capable of covering 13 types of adenoviruses comprises a specific primer, a specific probe, an internal reference primer, an internal reference probe, a PCR amplification reagent, a kit negative reference substance, a kit positive reference substance and ribozyme-free water, wherein the binding site of the specific primer and the specific probe is in a high-conservative section of the whole genome of the 13 types of adenoviruses infecting human respiratory tracts, and the types of the 13 types of adenoviruses are 1 type, 2 type, 3 type, 4 type, 5 type, 6 type, 7 type, 11 type, 14 type, 16 type, 21 type, 35 type and 55 type;

the specific primers are formed by a positive primer and a negative primer; the specific probe is a Taqman probe, and the specific primer and the specific probe are combined for detecting whether the respiratory adenovirus exists in the sample. The internal reference primers are formed by a positive primer and a negative primer; the internal reference probe is a Taqman probe, and the internal reference primer and the internal reference probe are combined for detecting the human source DNA in the sample, so that false negative caused by sample acquisition error or detection failure is prevented.

By using the combination of the two primers and the probe, 13 human respiratory adenoviruses and the ribonuclease P of the internal reference sequence can be simultaneously detected in a single tube, and false negative and other missed detections can not be caused while the detection success rate is ensured.

The sequence of the specific primer is shown as SEQ ID NO: 1-SEQ ID NO: 6 is shown in the specification; the binding sites of the specific primers and the specific probes are gene segments corresponding to the quintegrin of the whole genome of 13 types of adenoviruses (1, 2, 3, 4, 5, 6, 7, 11, 14, 16, 21, 35 and 55) infecting human respiratory tracts, and the sequence number of the specific upstream primer of the target segment is SEQ ID NO: 1-SEQ ID NO: 3; the sequence number of the specific downstream primer of the target fragment is SEQ ID NO: 4-SEQ ID NO: 6, wherein, the upstream primer is preferably SEQ ID NO: 1, the downstream primer is preferably SEQ ID NO: 4. the range of the upstream and downstream primer concentrations was 8. mu.M, 10. mu.M, 12. mu.M, 15. mu.M, 18. mu.M, and 20. mu.M for each primer. The concentration of the upstream and downstream primers is preferably 12. mu.M.

The sequence of the specific probe is shown as SEQ ID NO: 7-SEQ ID NO: 8, preferably SEQ ID NO: 8; the specific probe concentrations used were selected from 10. mu.M, 15. mu.M, 25. mu.M, 30. mu.M and 35. mu.M. Among them, the concentration of the specific probe is preferably 25. mu.M.

The sequence of the internal reference primer is shown as SEQ ID NO: 9-SEQ ID NO: 10, wherein the sequence of the internal reference forward F primer is shown as SEQ ID NO: 9, the sequence of the internal reference reverse R primer is shown as SEQ ID NO: 10, the sequence of the reference probe is shown as SEQ ID NO: 11, the detection and combination area of the internal reference primer and the internal reference probe is a human ribonuclease P (RNaseP) gene segment.

The PCR amplification reagent comprises hot-start DNA polymerase, dNTPs, high-activity UNG enzyme, dUTP and PCR amplification buffer solution. The negative control substance of the kit is normal saline. The positive reference substance of the kit is an engineering bacterial suspension for cloning the respiratory adenovirus quintuplet protein gene sequence. The probe is a Taqman probe, a fluorophore is marked on the probe, the fluorophore is one or more of FAM, VIC, Cy5, TET, HEX and JOE, the color of the fluorophore used by the internal reference probe is different from that of the fluorophore used by the specific probe of the adenovirus, for example, when the FAM is used by the adenovirus, the VIC is used by the internal reference.

The invention relates to a fluorescence quantitative PCR detection method capable of covering 13 types of adenoviruses for non-diagnosis purposes, which comprises the following steps:

10 μ l of PCR reaction (containing ROX reference dye),

mu.l of a primer-probe combination (consisting of 0.8. mu.l of a specific forward primer, 0.8. mu.l of a specific reverse primer, 0.4. mu.l of a specific probe, 0.8. mu.l of a forward primer of an internal reference, 0.8. mu.l of a reverse primer of an internal reference, and 0.4. mu.l of an internal reference probe), hereinafter referred to as "primer combination mix",

5. mu.l of the DNA to be detected,

1 μ l of ribozyme-free water;

the sequence of the specific forward primer is shown as SEQ ID NO: 1-SEQ ID NO: 3, the sequence of the specific reverse primer is shown as SEQ ID NO: 4-SEQ ID NO: 6, the sequence of the specific probe is shown as SEQ ID NO: 7-SEQ ID NO: 8, the sequence of the forward primer of the internal reference is shown as SEQ ID NO: 9, the sequence of the forward primer of the internal reference is shown as SEQ ID NO: 10, the sequence of the reference probe is shown as SEQ ID NO: 11 is shown in the figure;

(4) data analysis and result judgment:

for specific primer detection tubes:

for internal reference primer detection tubes:

Example 1

(1) Extraction of nucleic acid:

artificially synthesized sequences (1, 2, 3, 4, 5, 6, 7, 11, 14, 16, 21, 35 and 55) which are purchased from international standards and domestic quality control products and are inactivated by factories and infected with human respiratory adenovirus, pseudovirus and the like are taken one tube each. Extracting adenovirus nucleic acid by using a full-automatic nucleic acid extractor, dissolving in 100 mu l of eluent, measuring the concentration and purity of the nucleic acid by using nanodrop, packaging in a plurality of 0.2ml single tubes according to 10 mu l of each part, marking, and freezing and storing in a refrigerator at-80 ℃.

(2) Dilution of nucleic acid:

and (3) sucking the nucleic acid stock solution in the previous step, extracting 5 microliters of nucleic acid stock solution for each type, adjusting to the same concentration, diluting by 500 times by using ultrapure water without ribozyme, respectively packaging into a plurality of 0.2ml single tubes according to 50 microliters of each portion, marking, and freezing and storing in a refrigerator at the temperature of-20 ℃.

(3) Primer synthesis and dilution:

the flexible Weijie-based synthetic specific primer sequence SEQ ID NO: 1. SEQ ID NO: 4, and specific probes SEQ ID NO: 8, the primer was prepared to a concentration of 12. mu.M with ribozyme-free ultrapure water, the probe was diluted to a concentration of 25. mu.M with ribozyme-free ultrapure water, and then divided into a plurality of aliquots, which were then frozen in a freezer at-20 ℃.

(4) Preparation of an amplification system:

a detection system of a tube of specific primer probe combination is respectively prepared for each type, each tube is a 20 mu l reaction system, and the method specifically comprises the following steps: 10. mu.l of Taqman Universal PCR master mix, 4. mu.l of primer combination mix (consisting of 0.8. mu.l of specific forward primer, 0.8. mu.l of specific reverse primer, 0.4. mu.l of specific probe, 0.8. mu.l of internal reference forward primer, 0.8. mu.l of internal reference reverse primer, 0.4. mu.l of internal reference probe), 5. mu.l of diluted DNA to be tested, and 1. mu.l of ribozyme-free water.

For the negative control detection tube and the positive control detection tube, each tube is a 20 μ l reaction system, which specifically comprises: mu.l of Taqman Universal PCR master mix, 4. mu.l of primer combination mix, 5. mu.l of nucleic acid extracted from the control, and 1. mu.l of ribozyme-free water.

(5) Setting reaction conditions for fluorescence quantitative detection:

the detection mode was selected to be "quantification" or "TaqMan Reagents" using a fluorescent quantitative PCR instrument model 7500 of ABI. Program setting: pre-denaturation: 5min at 95 ℃ for 1 cycle; denaturation: 95 ℃ for 20s, annealing + extension: 1min at 60 ℃, signal acquisition, 45 cycles.

(6) And (4) judging a result:

negative quality control without amplification curve;

positive quality control has an amplification curve;

when 13 types of samples are detected respectively, no amplification exists in the internal reference curve; the ct range detected by the specific primer curve is 22.1-24.5, and the specific primer curve has a typical S-shaped amplification curve as shown in figure 1, so that 13 types of adenoviruses can be effectively detected by using the fluorescent quantitative PCR detection method and the kit which can cover 13 types of adenoviruses.

Example 2

The fluorescent quantitative PCR detection method and the kit which can cover 13 types of adenoviruses and simultaneously detect the mixture of 13 types of adenoviruses

(1) Extraction of nucleic acids

See example 1.

(2) Dilution and mixing of nucleic acids

After the nucleic acid stock solution in the previous step was extracted and the concentration and purity of nucleic acid were measured by nanodrop, 10. mu.l was extracted for each type, 1. mu.l of each of the 13 types of adenovirus DNA was mixed after the same concentration had been adjusted, and then diluted with 87. mu.l of ribozyme-free ultrapure water (hereinafter referred to as "thirteen-in-one nucleic acid diluent") and frozen in a freezer at-20 ℃.

(3) Primer synthesis and dilution

See example 1.

(4) Preparation of amplification System

The detection of the thirteen-in-one nucleic acid diluent is specifically as follows: 10. mu.l of Taqman Universal PCR master mix, 4. mu.l of primer combination mix, 5. mu.l of the diluted DNA to be tested, and 1. mu.l of ribozyme-free water.

See example 1 for negative control and positive control test tubes.

(5) Setting reaction conditions for fluorescence quantitative detection:

see example 1.

(6) And (4) judging a result:

negative quality control without amplification curve;

positive quality control has an amplification curve;

detecting the thirteen-in-one nucleic acid diluent, wherein the internal reference curve is not amplified; the ct value detected by the specific primers was 18.2, and had a typical "S" type amplification curve, as shown in FIG. 2.

Example 3

The invention can cover the condition of detecting clinical positive samples by the fluorescent quantitative PCR detection method and the kit of 13 types of adenoviruses

(1) Extraction of nucleic acids

Taking throat swabs which are clinically detected to be positive to adenovirus, subpackaging the samples for preservation, inactivating, extracting nucleic acid by using a full-automatic nucleic acid extractor, dissolving in 100 mu l of eluent, and freezing and storing in a refrigerator at the temperature of 20 ℃ below zero.

(2) Primer synthesis and dilution:

see example 1.

(3) Preparation of an amplification system:

for a sample to be detected, a 20 mul specific primer detection system is prepared, which specifically comprises the following steps: 10. mu.l of Taqman Universal PCR master mix, 4. mu.l of primer combination mix, 5. mu.l of the diluted DNA to be tested, and 1. mu.l of ribozyme-free water.

See example 1 for negative control and positive control test tubes.

(4) Setting reaction conditions for fluorescence quantitative detection:

see example 1.

(5) And (4) judging a result:

negative quality control without amplification curve;

positive quality control has an amplification curve;

in a detection tube of a known positive clinical sample, a specific primer detects an amplification curve; the internal reference was detected with an amplification curve, as shown in FIG. 3.

Example 4

The fluorescence quantitative PCR detection method and the kit capable of covering 13 types of adenoviruses have the sensitivity under the condition of low concentration of a target to be detected

(1) Extraction and processing of nucleic acids

Taking the inactivated adenovirus type 7 purchased from the outside, extracting adenovirus nucleic acid by using a full-automatic nucleic acid extractor, dissolving the extracted adenovirus nucleic acid in 100 mu l of eluent, performing 5-gradient dilution, subpackaging into a plurality of small parts, and freezing and storing in a refrigerator at the temperature of 20 ℃ below zero.

(2) Primer synthesis and dilution:

see example 1.

(3) Preparation of an amplification system:

for 5 tubes of samples to be detected with different concentrations, a 20 mul specific primer detection system is prepared, which specifically comprises the following steps: 10. mu.l of Taqman Universal PCR master mix, 4. mu.l of primer combination mix, 5. mu.l of the diluted DNA to be tested, and 1. mu.l of ribozyme-free water.

See example 1 for negative control and positive control test tubes.

(4) Setting reaction conditions for fluorescence quantitative detection:

see example 1.

(5) And (4) judging a result:

negative quality control without amplification curve;

positive quality control has an amplification curve;

the detection results of 5 samples with concentration gradients show that no amplification exists in the internal reference curve; the ct range detected by the specific primer curve is 27.7-39.5, the specific primer curve has a typical S-shaped amplification curve, the difference value between the specific primer curve and the S-shaped amplification curve accords with the condition of gradient dilution, and the ct value can be detected to be 39.5 at the lowest concentration, as shown in FIG. 4.

Example 5

The fluorescent quantitative PCR detection method capable of covering 13 types of adenoviruses and the specificity of the kit in the detection of different pathogens

(1) Extraction of nucleic acids

Taking 5 samples (EB virus, syncitial virus A, syncitial virus B, mycoplasma pneumoniae and Coxsackie virus) which are detected as adenovirus negative but positive by other pathogens, inactivating, extracting nucleic acid by using a full-automatic nucleic acid extractor, dissolving in 100 mu l of eluent, and freezing and storing in a refrigerator at the temperature of-20 ℃.

(2) Primer synthesis and dilution:

see example 1.

(3) Preparation of an amplification system:

See example 1 for negative control and positive control test tubes.

(4) Setting reaction conditions for fluorescence quantitative detection:

see example 1.

(5) And (4) judging a result:

negative quality control without amplification curve;

positive quality control has an amplification curve;

the specific primer detection tube has no amplification curve when the sample is detected to be adenovirus negative but other pathogen positive; the internal reference primer detection tube has an amplification curve, as shown in FIG. 5.

In summary, according to the above technical scheme of the present invention, specific primers are designed through the high conserved regions of 13 types of adenoviruses (1, 2, 3, 4, 5, 6, 7, 11, 14, 16, 21, 35, 55) infecting human respiratory tracts, so that single-tube single-plex PCR can be achieved to simultaneously detect 13 types of adenoviruses infecting human respiratory tracts, and high coverage is achieved. The reagent contains high-activity UNG enzyme and dUTP, PCR pollution fragments containing dUTP in residual aerosol in a laboratory can be effectively eliminated when the reagent is prepared, and high sensitivity and high specificity can be ensured while false positive is eliminated. The method solves the difficult problem that the coverage, sensitivity, specificity, economy and timeliness of the existing detection link cannot be all excellent due to the adoption of multiple PCR for covering multiple types.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

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Claims

1. A fluorescent quantitative PCR detection kit capable of covering 13 types of adenoviruses is characterized by comprising specific primers, specific probes, internal reference primers, internal reference probes, PCR amplification reagents, a kit negative reference substance, a kit positive reference substance and ribozyme-free water, wherein the binding sites of the specific primers and the specific probes are in a high-conservative section of the whole genome of the 13 types of adenoviruses infecting human respiratory tracts, and the types of the 13 types of adenoviruses are 1 type, 2 type, 3 type, 4 type, 5 type, 6 type, 7 type, 11 type, 14 type, 16 type, 21 type, 35 type and 55 type;

the PCR amplification reagent comprises hot-start DNA polymerase, dNTPs, UNG enzyme, dUTP and PCR amplification buffer solution.

2. The fluorescence quantitative PCR detection kit capable of covering 13 types of adenoviruses according to claim 1, wherein the binding sites of the specific primers and the specific probes are in the gene sequences corresponding to the human adenovirus quintet protein.

3. The fluorescent quantitative PCR detection kit capable of covering 13 type-specific adenoviruses according to claim 1, wherein the sequence of the internal reference primer is as shown in SEQ ID NO: 9-SEQ ID NO: 10, the sequence of the reference probe is shown as SEQ ID NO: 11, the regions for detecting and combining the internal reference primer and the internal reference probe are human ribonuclease P gene segments.

4. The fluorescent quantitative PCR detection kit capable of covering 13 type-specific adenoviruses according to claim 1, wherein the negative control substance of the kit is normal saline.

5. The fluorescence quantitative PCR detection kit capable of covering 13 types of adenoviruses according to claim 1, wherein the positive control substance of the kit is an engineering bacterial suspension for cloning the gene sequence of the quintuplex protein of the respiratory adenovirus.

6. The kit for detecting fluorescent quantitative PCR of 13-type adenovirus according to claim 1, wherein the upstream primer sequence of the specific primer is as shown in SEQ ID NO: 1, the sequence of the downstream primer of the specific primer is shown as SEQ ID NO: 4, the sequence of the specific probe is shown as SEQ ID NO: shown in fig. 8.

7. The fluorescence quantitative PCR detection kit capable of covering 13 type-specific adenoviruses according to claim 1, wherein the probe is a Taqman probe and a fluorophore is labeled on the probe.

8. The fluorescent quantitative PCR detection kit capable of covering 13 types of adenoviruses according to claim 7, wherein the fluorescent group is one or more of FAM, VIC, Cy5, TET, HEX and JOE.

9. A fluorescence quantitative PCR detection method capable of covering 13 types of adenoviruses for non-diagnosis purposes is characterized by comprising the following steps:

10. mu.l of a PCR reaction solution,

mu.l of a primer-probe combination,

5. mu.l of the DNA to be detected,

1 μ l of ribozyme-free water;

(3) performing amplification on the machine: the program is set as follows: pre-denaturation: 5min at 95 ℃ for 1 cycle; denaturation: 95 ℃ for 20s, annealing and extension: 1min at 60 ℃, signal acquisition, 45 cycles;

(4) data analysis and result judgment:

for specific primer detection tubes:

for internal reference primer detection tubes:

10. The method of claim 9, wherein the sample is a clinical sample from a patient with respiratory tract infection, and the sample comprises sputum, nasopharyngeal swab, and alveolar lavage.