阅读说明：本技术 一种快速检测呼吸道b亚属人腺病毒的检测方法 (Detection method for rapidly detecting respiratory tract subgenus B human adenovirus ) 是由 马广源 肖勇 管红霞 鲍静 周琪 於淳安 于 2021-09-16 设计创作，主要内容包括：本发明提出了一种快速检测呼吸道B亚属人腺病毒的检测方法,包括如下步骤：步骤1、采集患者呼吸道标本,并提取人腺病毒核酸作为待检模板；步骤2、针对人腺病毒B亚属中5种呼吸道腺病毒进行引物设计和筛选；步骤3、将步骤2中经引物设计和筛选后的5种呼吸道腺病毒的上下游引物分别按照1:1的体积比例进行混合,并将混合后的产物作为引物混合物；步骤4、配制反应体系,反应体系的总体积为25μL；步骤5、将步骤4中的反应体系进行PCR反应,得到PCR产物；步骤6、将得到的PCR产物进行高分辨率熔解曲线分析,并根据结果判断其亚属,借此,本发明具有不仅可以快速确定是否为腺病毒,并且还可以快速进一步确定亚属的优点。(The invention provides a detection method for rapidly detecting respiratory tract subgenera B human adenovirus, which comprises the following steps: step 1, collecting a respiratory tract specimen of a patient, and extracting human adenovirus nucleic acid as a template to be detected; step 2, designing and screening primers aiming at 5 respiratory tract adenoviruses in human adenovirus subgenus B; step 3, mixing the upstream primers and the downstream primers of the 5 respiratory adenoviruses which are designed and screened in the step 2 according to the volume ratio of 1:1 respectively, and taking the mixed products as a primer mixture; step 4, preparing a reaction system, wherein the total volume of the reaction system is 25 mu L; step 5, carrying out PCR reaction on the reaction system in the step 4 to obtain a PCR product; and 6, carrying out high-resolution melting curve analysis on the obtained PCR product, and judging the subgenera according to the result, so that the method has the advantages of quickly determining whether the PCR product is adenovirus and quickly further determining the subgenera.)

1. A detection method for rapidly detecting human adenovirus of respiratory tract subgroup B is characterized by comprising the following steps:

step 1, collecting a respiratory tract specimen of a patient, and extracting human adenovirus nucleic acid as a template to be detected;

step 2, designing and screening primers aiming at 5 respiratory tract adenoviruses in human adenovirus subgenus B;

step 3, mixing the upstream primers and the downstream primers of the 5 respiratory adenoviruses which are designed and screened in the step 2 according to the volume ratio of 1:1 respectively, and taking the mixed products as a primer mixture;

step 4, preparing a reaction system, wherein the reaction system comprises the following steps: 10 μ L MeltDoctor^TMHRM Master Mix, 8 muL RNase Free Water, 2 muL of the primer mixture in the step 3 and 5 muL of the template to be detected in the step 1, wherein the total volume of the reaction system is 25 muL;

step 5, carrying out PCR reaction on the reaction system in the step 4 to obtain a PCR product;

and 6, carrying out high-resolution melting curve analysis on the obtained PCR product, and judging the subgenera according to the result.

2. The method as claimed in claim 1, wherein the method for collecting the respiratory tract specimen of the patient in step 1 comprises one of nasopharyngeal swab, sputum and lavage.

3. The method for rapidly detecting human adenovirus of subgenera B of respiratory tract according to claim 1, wherein the method for extracting human adenovirus nucleic acid in step 1 comprises one of extraction by a manual extraction kit or extraction by a fully automatic nucleic acid extractor.

4. The method according to claim 1, wherein the primer types for primer design and screening of human adenovirus nucleic acids in step 2 comprise: HAdV-3, HAdV-7, HAdV-11, HAdV-14 and HAdV-55, wherein the upstream and downstream primers are respectively as follows:

HAdV-3 includes the amino acid sequence as shown in SEQ ID NO: 1 and the primer shown as SEQ ID NO: 2, or a reverse primer;

HAdV-7 includes the amino acid sequence as shown in SEQ ID NO: 3 and the primer shown as SEQ ID NO: 4, a downstream primer;

HAdV-11 includes the amino acid sequence as shown in SEQ ID NO: 5 and the primer shown as SEQ ID NO: 6 as shown in the figure;

HAdV-14 includes the amino acid sequence as shown in SEQ ID NO: 7 and the primer shown as SEQ ID NO: 8, a downstream primer;

HAdV-55: comprises the following components as shown in SEQ ID NO: 9 and the primer shown as SEQ ID NO: 10, or a reverse primer.

5. The method for rapidly detecting the human adenovirus of subgenera B of the respiratory tract according to claim 1, wherein the PCR reaction in the reaction system in the step 5 is performed by the following steps: pre-denaturation at 95 ℃ for 1min, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 45s, and 40 cycles.

6. The method for rapidly detecting human adenovirus of subgenera B of respiratory tract according to claim 1, wherein the melting procedure of the high resolution melting curve analysis of the PCR product obtained in step 5 is as follows: melting at 95 deg.C for 2min, melting at 60 deg.C for 2min, heating from 60 deg.C to 95 deg.C at melting rate of 0.2 deg.C/s, and cooling at 37 deg.C.

7. The method according to claim 1, wherein the method for rapidly detecting human adenovirus of subgenera B of respiratory tract in step 6 comprises:

when the Tm value is 86.2 ℃ +/-0.14, the adenovirus is judged to be the human adenovirus type 3;

when the Tm value is 79.5 ℃ +/-0.15, the adenovirus is judged to be the human adenovirus type 7;

when the Tm value is 73.8 ℃ +/-0.10, the adenovirus is judged to be the human adenovirus type 11;

when the Tm value is 76.3 ℃ +/-0.12, the adenovirus is judged to be human adenovirus type 14;

when the Tm value is 82.5 ℃. + -. 0.15, the adenovirus is judged to be the human adenovirus type 55.

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a detection method for rapidly detecting respiratory tract Bsubgenera human adenovirus.

Background

Human Adenovirus (HAdV) is a non-enveloped double-stranded DNA virus, mainly causes respiratory diseases, and also infects digestive tract, urinary tract, and eyes to cause diseases, and its pathogenicity and clinical symptoms are closely related to its type. The International Committee for virology Classification (ICTV) has been divided into 7 subgenera A-G according to their biological characteristics. A. F and G mainly cause infection of digestive tract and diarrhea, C, E and part of subfamily B mainly cause respiratory diseases, other subfamily B mainly cause urinary tract infection, and subfamily D mainly causes keratitis and conjunctivitis. Human adenoviruses can cause a variety of human diseases, among which those associated with respiratory diseases are mainly subgenus B (HAdV-3, 7, 11, 14, 16, 21, 50, 55), subgenus C (HAdV-1, 2, 5, 6, 57) and subgenus E (HAdV-4). While subgenus B, which causes common respiratory diseases, is mainly HAdV-3, 7, 11, 14 and 55.

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. Human adenovirus is highly contagious and can cause outbreak in closed, crowded and humid environments, such as military camps, schools, nursery institutions, medical institutions and the like. The HAdV-3 and HAdV-7 types are the most important human adenovirus types of children lower respiratory tract infection, and the types 11 and 14 are the second; since the HAdV-55 is discovered in China for the first time, the HAdV-55 has become widely popular in China and becomes one of the important pathogens of adult community acquired pneumonia in China.

High Resolution Melting curve analysis (High Resolution Melting) is a new gene analysis technology which forms different form Melting curves based on different Melting temperatures of mononucleotides, and is usually combined with real-time fluorescence PCR to monitor the Melting process of double-stranded DNA in real time when the temperature rises. The current detection methods for adenovirus can be divided into immunological and molecular biological detection methods.

Immunology mainly comprises an ELISA technology, but the technology has certain limitations; the molecular biological method mainly adopts a PCR detection technology, at present, the technology can only determine whether the virus is adenovirus, and the subgenera is further determined by a sequencing technology, so that the subgenera cannot be effectively and rapidly determined, and rapid detection cannot be realized in the emergent public health event.

Disclosure of Invention

The invention provides a method for rapidly detecting human adenovirus of respiratory tract subgenus B, which has the advantages of rapidly determining whether the human adenovirus is adenovirus and further rapidly determining subgenus.

The technical scheme of the invention is realized as follows: a detection method for rapidly detecting respiratory tract subgenera B human adenovirus comprises the following steps:

step 1, collecting a respiratory tract specimen of a patient, and extracting human adenovirus nucleic acid as a template to be detected;

step 2, designing and screening primers aiming at 5 respiratory tract adenoviruses in human adenovirus subgenus B;

step 3, mixing the upstream primers and the downstream primers of the 5 respiratory adenoviruses which are designed and screened in the step 2 according to the volume ratio of 1:1 respectively, and taking the mixed products as a primer mixture;

step 4, preparing a reaction system, wherein the reaction system comprises the following steps: 10 μ L MeltDoctor^TMHRM Master Mix, 8 muL RNase Free Water, 2 muL of the primer mixture in the step 3 and 5 muL of the template to be detected in the step 1, wherein the total volume of the reaction system is 25 muL;

step 5, carrying out PCR reaction on the reaction system in the step 4 to obtain a PCR product;

and 6, carrying out high-resolution melting curve analysis on the obtained PCR product, and judging the subgenera according to the result.

As a preferred embodiment, the method for collecting a respiratory tract specimen of a patient in step 1 comprises one of a nasopharyngeal swab, sputum, or lavage.

As a preferred embodiment, the method for extracting human adenovirus nucleic acid in step 1 comprises one of extraction by a manual extraction kit or extraction by a full-automatic nucleic acid extractor.

As a preferred embodiment, the primer types for primer design and screening of human adenovirus nucleic acids in step 2 include: HAdV-3, HAdV-7, HAdV-11, HAdV-14 and HAdV-55, wherein the upstream and downstream primers are respectively as follows:

HAdV-3 includes the amino acid sequence as shown in SEQ ID NO: 1 and the primer shown as SEQ ID NO: 2, or a reverse primer;

HAdV-7 includes the amino acid sequence as shown in SEQ ID NO: 3 and the primer shown as SEQ ID NO: 4, a downstream primer;

HAdV-11 includes the amino acid sequence as shown in SEQ ID NO: 5 and the primer shown as SEQ ID NO: 6 as shown in the figure;

HAdV-14 includes the amino acid sequence as shown in SEQ ID NO: 7 and the primer shown as SEQ ID NO: 8, a downstream primer;

HAdV-55: comprises the following components as shown in SEQ ID NO: 9 and the primer shown as SEQ ID NO: 10, or a reverse primer.

As a preferred embodiment, the procedure of PCR reaction carried out in the reaction system in step 5 is as follows: pre-denaturation at 95 ℃ for 1min, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 45s, and 40 cycles.

In a preferred embodiment, the melting procedure for analyzing the PCR product obtained in step 5 by a high resolution melting curve analysis is as follows: melting at 95 deg.C for 2min, melting at 60 deg.C for 2min, heating from 60 deg.C to 95 deg.C at melting rate of 0.2 deg.C/s, and cooling at 37 deg.C.

In a preferred embodiment, the method for determining subgenera according to the result in step 6 is as follows:

when the Tm value is 86.2 ℃ +/-0.14, the adenovirus is judged to be the human adenovirus type 3;

when the Tm value is 79.5 ℃ +/-0.15, the adenovirus is judged to be the human adenovirus type 7;

when the Tm value is 73.8 ℃ +/-0.10, the adenovirus is judged to be the human adenovirus type 11;

when the Tm value is 76.3 ℃ +/-0.12, the adenovirus is judged to be human adenovirus type 14;

when the Tm value is 82.5 ℃. + -. 0.15, the adenovirus is judged to be the human adenovirus type 55.

After the technical scheme is adopted, the invention has the beneficial effects that:

the invention adopts the technical means of High Resolution Melting (HRM) and adopts the steps of nucleic acid amplification, real-time fluorescence PCR, HRM and the like. The combination of high resolution melting curve and real time fluorescence PCR (HRM-real time PCR), HRM has very high sensitivity, and saturated dye in HRM can mark PCR product in whole course and judge result in a unique Tm value mode. Therefore, the method realizes the effects of quickness, intuition and good repeatability, and has the advantages of accuracy, high sensitivity, good specificity, low cost, detection time saving, cost saving and the like.

Compared with the technology of the invention, the prior detection technology comprises the following steps:

1. the real-time fluorescence PCR method is used for detecting 5 subtypes, and at least 2-5 tubes of reaction liquid are used for determining the subtypes, and the characteristics are as follows: the operation is complicated, and the fluorescent probe is very expensive;

2. human adenovirus is detected by a real-time fluorescence PCR method, then sequencing is carried out to carry out BLAST analysis, and subtype is determined, and the characteristics are as follows: the operation is complicated, the time consumption is long, and the cost is high;

3. common PCR is used for amplifying target genes, and then gel analysis is carried out to determine subtypes, and the characteristics are as follows: low sensitivity and easy pollution.

Drawings

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

FIG. 1 is a high resolution melting curve of the present invention.

In the figure, 1-human adenovirus type II; 2-human adenovirus type 14; 3-human adenovirus type 7; 4-human adenovirus type 55; 5-human adenovirus type 3.

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, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

As shown in FIG. 1, a method for rapidly detecting respiratory tract subgenera B human adenovirus comprises the following steps:

step 1, collecting a respiratory tract specimen of a patient, and extracting human adenovirus nucleic acid as a template to be detected;

step 2, designing and screening primers aiming at 5 respiratory tract adenoviruses in human adenovirus subgenus B, wherein the results are shown in the following table;

step 3, mixing the upstream primers and the downstream primers of the 5 respiratory adenoviruses which are designed and screened in the step 2 according to the volume ratio of 1:1 respectively, and taking the mixed products as a primer mixture;

step 4, preparing a reaction system, wherein the reaction system comprises the following steps: 10 μ L MeltDoctor^TMHRM Master Mix, 8 muL RNase Free Water, 2 muL of the primer mixture in the step 3 and 5 muL of the template to be detected in the step 1, wherein the total volume of the reaction system is 25 muL;

step 5, carrying out PCR reaction on the reaction system in the step 4 to obtain a PCR product;

and 6, carrying out high-resolution melting curve analysis on the obtained PCR product, and judging the subgenera according to the result.

The method for collecting the respiratory tract specimen of the patient in the step 1 comprises one of a nasopharyngeal swab, sputum or a lavage fluid.

The method for extracting the human adenovirus nucleic acid in the step 1 comprises one of extracting by adopting a manual extraction kit or extracting by adopting a full-automatic nucleic acid extractor.

The primer types for designing and screening the primers for the human adenovirus nucleic acid in the step 2 comprise: HAdV-3, HAdV-7, HAdV-11, HAdV-14 and HAdV-55, wherein the upstream and downstream primers are respectively as follows:

HAdV-3: the upstream primer is 5 'TTTGCCCACTAACACCA 3' (SEQ ID NO.1)

The downstream primer is 5 'GCAATACACGGAAAGGT 3' (SEQ ID NO. 2);

HAdV-7: the upstream primer is 5 'GGCATTGCTTCCACG 3' (SEQ ID NO.3)

The downstream primer is 5 'GGTTGGCTTCCTCTAC 3' (SEQ ID NO.4)

HAdV-11: the upstream primer is 5 'CGGCTTTCAGCGGCT 3' (SEQ ID NO.5)

The downstream primer is 5 'AGATAAGGGATGGACCTAC 3' (SEQ ID NO.6)

HAdV-14: the upstream primer is 5 'CGGCTTTCAGAGGCT 3' (SEQ ID NO.7)

The downstream primer is 5 'GATAAGACCAAGATAAGGGAT 3' (SEQ ID NO.8)

HAdV-55: the upstream primer is 5 'GCCCAACAGACCCAA 3' (SEQ ID NO.9)

The downstream primer is 5 'ACACCTTAGTCCGACAC 3' (SEQ ID NO. 10).

The PCR reaction procedure of the reaction system in the step 5 is as follows: pre-denaturation at 95 ℃ for 1min, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 45s, and 40 cycles.

The melting procedure for analyzing the high-resolution melting curve of the obtained PCR product in the step 6 is as follows: melting at 95 deg.C for 2min, melting at 60 deg.C for 2min, heating from 60 deg.C to 95 deg.C at melting rate of 0.2 deg.C/s, and cooling at 37 deg.C.

The method for judging the subgenera according to the result in the step 7 comprises the following steps:

when the Tm value is 86.2 ℃ +/-0.14, the adenovirus is judged to be the human adenovirus type 3;

when the Tm value is 79.5 ℃ +/-0.15, the adenovirus is judged to be the human adenovirus type 7;

when the Tm value is 73.8 ℃ +/-0.10, the adenovirus is judged to be the human adenovirus type 11;

when the Tm value is 76.3 ℃ +/-0.12, the adenovirus is judged to be human adenovirus type 14;

when the Tm value is 82.5 ℃. + -. 0.15, the adenovirus is judged to be the human adenovirus type 55.

When a saturated fluorescent dye fluoresces when bound to a double strand of DNA and is released from the double strand of DNA, the fluorescence signal is sharply reduced, and if the DNA is denatured by increasing the temperature, the fluorescence signal is plotted on the abscissa as the temperature, a Melting curve can be obtained in which the temperature at which 50% of DNA molecules are denatured is called the Melting temperature (Tm value), and the Tm value is a very important parameter in PCR primer design, which is the temperature at which the primer is precisely complementary to the template and 50% of the primers are paired with the template in the case of an excess amount of the template, and the other 50% of the primers are in a dissociated state. The Tm is generally greater than 55 ℃. Different DNAs have different Tm values, and the higher the G-C content in the DNA, the higher the Tm value. In designing the multiplex PCR, the mismatching between each pair of primers is also considered, and the difference of Tm value can not be less than 2 ℃, so the invention selects 86.2 ℃ +/-0.14 and judges the type of the adenovirus as the type 3.

The design selects the Tm value of the DNA as the basis for distinguishing 5 subtypes in the subgenera B, so that the operation is simple, only one tube of PCR reaction solution is needed to be prepared, and the subtype of the detected pathogen can be easily judged according to the difference of the Tm value.

The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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