阅读说明：本技术 一种消化道腺病毒的pcr荧光检测试剂盒及其应用 (PCR fluorescence detection kit for digestive tract adenovirus and application thereof ) 是由 马东礼 刘孝荣 邢志浩 姜含芳 邓栩文 于 2020-04-29 设计创作，主要内容包括：本发明公开了一种消化道腺病毒的PCR荧光检测试剂盒及其应用,属于生物技术领域。消化道腺病毒主要包括人类腺病毒40型和41型,该试剂盒针对人类腺病毒40型和41型设计特异性引物和探针以及PCR反应体系,其中特异性引物和探针由人类腺病毒40型及41型全基因组的共同序列、内参片段的特异性引物和探针组成。该试剂盒能精准检测到40型及41型的人类腺病毒,且不会受到其它非40型、41型人类腺病毒及其它微生物的序列影响,实现高敏感性和高特异性,解决了目前引物设计中敏感性和特异性不能均优的难点问题,同时本发明采用“鸬鹚”生物大数据挖掘系统,根据拷贝数、引物二聚体和序列空间结构等关键技术参数,寻找最佳靶序列,使检测效率、精准度大大提高。(The invention discloses a PCR fluorescence detection kit for digestive tract adenovirus and application thereof, belonging to the technical field of biology. The digestive tract adenovirus mainly comprises human adenovirus type 40 and type 41, and the kit designs specific primers and probes and a PCR reaction system aiming at the human adenovirus type 40 and type 41, wherein the specific primers and probes consist of common sequences of human adenovirus type 40 and type 41 whole genomes and specific primers and probes of internal reference fragments. The kit can accurately detect the human adenoviruses of type 40 and type 41, is not influenced by the sequences of other human adenoviruses of type 40 and type 41 and other microorganisms, realizes high sensitivity and high specificity, solves the difficult problem that the sensitivity and the specificity cannot be excellent in the current primer design, and simultaneously searches an optimal target sequence by adopting a cormorant biological big data mining system according to key technical parameters such as copy number, primer dimer, sequence space structure and the like, so that the detection efficiency and the accuracy are greatly improved.)

1. A PCR fluorescence detection kit for adenovirus in digestive tract comprises a specific primer, a probe and a PCR reaction reagent, wherein the specific primer and the probe consist of a common sequence of a 40-type and a 41-type whole genome of human adenovirus and a specific primer and a probe of an internal reference fragment, and are characterized in that the specific primer and the probe have the following sequences:

the upstream primer of the target fragment is SEQ ID NO. 1: 5'-TTCACCTCTGTTTGCTGCAC-3', respectively;

downstream primer of the target segment SEQ ID NO. 2: 5'-AGGTAGTCCGCAGTTTGGAA-3', respectively;

probe of the fragment of interest SEQ ID NO. 3:

5’-FAM-TGCCATCTGCAGCATTGCCACT-TAMRA-3’；

the upstream primer of the internal reference fragment is SEQ ID NO. 4: 5'-GGCATGTGGAGGAAGGTGGT-3', respectively;

downstream primer of internal reference fragment SEQ ID NO. 5: 5'-CCATGGACTGGCTCTCCGTT-3', respectively;

probe of internal reference fragment SEQ ID NO. 6:

5’-HEX-ACGCAGCCCTGCTTCGTTCGCCG-TAMRA-3’。

2. the use of the kit for PCR fluorescence detection of an adenovirus in the digestive tract according to claim 1.

3. The use according to claim 2, comprising the steps of:

(1) extracting DNA of a sample to be detected;

(2) performing PCR amplification: adding PCR reaction solution A and PCR reaction solution B in a certain proportion by using sample DNA to be detected as template, wherein reaction solution A comprises 12.5mM Tris-HCl, pH9.0, 50mM KCl, 0.13%X-100、3.13mM MgCl₂0.45mM dATP, 0.45mM dGTP, 0.45mM dCTP, 0.79mM dUTP, two pairs of primers SEQ ID No.1 and 2, 4 and 5, both at a concentration of 0.3-0.7. mu.M, and two probes SEQ ID No.3, 6, both at a concentration of 0.14-0.16. mu.M; reaction solution B contained 20mM Tris-HCl, 100mM KCl, 0.1mM EDTA, 1mM DTT, 50% Glycerin, 0.5%20. 1-3U/mu L hot start enzyme and 0.1-0.3U/mu L UNG enzyme, performing PCR amplification, simultaneously replacing the DNA of a sample to be detected with a negative quality control product and a positive quality control product as templates, adding a PCR reaction solution A and a PCR reaction solution B, and performing PCR amplification of the negative quality control product and the positive quality control product;

(3) and (5) judging the result according to the Ct value.

4. The use according to claim 3, wherein the volume ratio of the sample DNA to be tested to the reaction solution A and the reaction solution B is 3: 46: 1.

5. the use according to claim 3, wherein in the step (2), the reaction solution A contains two pairs of primers SEQ ID NO.1 and 2, 4 and 5 at a concentration of 0.5. mu.M, two probes SEQ ID NO.3 and 6 at a concentration of 0.15. mu.M, and the PCR reaction solution B contains hot start enzyme and UNG enzyme at a concentration of 2U/μ L and 0.2U/μ L, respectively.

6. Use according to claim 3, wherein the parameters of the PCR amplification procedure are as follows:

120s at 50 ℃,1 cycle;

600s at 95 ℃ for 1 cycle;

95 ℃, 15s, 55 ℃, 45s (fluorescence collected), 40 cycles;

37 ℃, 20s, 1 cycle.

7. The use of claim 3, wherein the negative quality control substance is physiological saline, and the positive quality control substance is an engineered bacterial suspension of the target gene sequences of cloned human adenovirus type 40 and 41.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a PCR fluorescence detection kit for a digestive tract adenovirus and application thereof.

Background

Adenovirus (adenoviruses) is a particle without an envelope and with a diameter of 70-90 nm, and consists of 252 capsomeres arranged in an icosahedron shape. Inside the capsid are linear double stranded DNA molecules, and since the discovery and successful isolation of adenoviruses in the last 50 th century, over 100 serotypes have been discovered in succession, of which there are 52 human adenoviruses, classified into A, B, C, D, E and six subgroups (subgroups). The gut adenoviruses belong to subgroup F and are predominantly of two types, 40 and 41.

Human adenoviruses can induce a variety of infections, including respiratory infections, ocular infections, digestive tract infections, and the like. Many human adenoviruses replicate in intestinal cells and are excreted in the feces. Most serotypes are not associated with gastrointestinal diseases, but human adenoviruses type 40 and type 41 can cause diseases, and are the main causes of gastroenteritis, abdominal pain and diarrhea of infants and young children (under 4 years old).

A common identification method for gut adenoviruses is as follows: (1) culturing: the culture specificity of the virus strain is high, which is necessary for obtaining the virus strain and carrying out epidemiological investigation, the technical requirement is high, the operation is complex, and clinical laboratories are not developed generally; (2) enzyme-linked immunoassay detection of cytotoxic antibodies: the infection condition is indirectly reflected, the virus antibody detection is rapid, the method has poor repeatability, low sensitivity and weak specificity; (3) colloidal gold method for detecting antigen of cytotoxin: low sensitivity, poor repeatability and better specificity. The laboratory detection methods have the defects of poor sensitivity, low specificity, long detection period, high equipment requirement, poor repeatability and the like, so that false positives or false negatives often appear in the detection result, and misdiagnosis and missed diagnosis are caused.

Disclosure of Invention

In order to solve the technical problems, the invention provides a PCR fluorescence detection kit for the adenovirus in the digestive tract, which can specifically, sensitively, accurately and quickly detect the adenovirus in the digestive tract in a fecal sample.

In order to achieve the purpose, the invention adopts the following technical scheme: the autonomously developed biological big data mining system of cormorant is utilized to download the reference sequences of human adenovirus type 40 and type 41 (type 40 GenBank: KU162869.1, sequence length 34,210 bp; type 41 GenBank: KY316161.1, sequence full length 34,198bp) in the GenBank nucleic acid database (Nucleotide) of the international authoritative database. The reference sequence is used as a template to establish a candidate short fragment library, fragment specificity and sensitivity evaluation, primer design, primer performance evaluation, clinical sample verification and the like, and on the basis, a PCR fluorescence detection kit for the digestive tract adenovirus with high sensitivity, strong specificity, low cost and larger detection flux is developed. The gene is a name card for identifying biological species, and the key for determining the specificity and the sensitivity of the kit is to search a specific and conservative gene sequence like a great sea fishing needle. The innovation of the invention is that: 1) the kit can accurately detect the digestive tract adenoviruses of different serotypes, is not influenced by sequences of other non-digestive tract adenoviruses (particularly respiratory tract adenoviruses) and other microorganisms, realizes high sensitivity and high specificity, and solves the difficult problem that the sensitivity and the specificity cannot be both excellent in the current primer design; 2) the cormorant biological big data mining system adopted by the invention can comprehensively screen the whole genome sequence of tens of millions of base pairs, find dozens to hundreds of specific conserved sequences, and then perform secondary screening according to key technical parameters such as copy number, primer dimer, sequence space structure and the like, wherein the optimal target sequence is selected from the optimal target sequences, so that the detection efficiency, specificity and sensitivity are improved by 10 times compared with those of a conventional design method. The primer design method in the patent of the invention adopts an autonomously developed cormorant big data mining system to search the optimal primer and probe fragment in the whole genome (the length is about 34000bp) range of the alimentary tract adenovirus, compared with the traditional method which only designs the primer aiming at the Hexon gene (about 3000bp), the primer design method has the advantages of expanding the search range by more than 10 times, along with shorter time and higher efficiency.

The PCR fluorescence detection kit for the alimentary tract adenovirus comprises a reaction solution A and a reaction solution B, wherein the reaction solution A comprises two pairs of primers and two probes: specific primer pairs of human adenovirus type 40 and type 41 of SEQ ID NO.1 and SEQ ID NO.2 and corresponding FAM labeled probes SEQ ID NO. 3; the internal reference fragment primer pair SEQ ID NO.4 and SEQ ID NO.5 and the corresponding HEX-labeled probe SEQ ID NO. 6. Wherein HEX and FAM are fluorescent groups, and TAMRA is a quenching group. The reaction solution B contained a hot start enzyme and UNG enzyme.

The negative quality control substance used in the PCR fluorescent detection kit for the adenovirus in the alimentary canal is physiological saline, and the positive quality control substance is engineering bacterial suspension for cloning target gene sequences of human adenovirus type 40 and type 41.

The DNA sequence of the reference fragment SEQ ID NO.7 is as follows:

5’-TCACAAGCAGGAGTGTGCCAGGAGAAGGCCAAACCATCCAGTGCCGGTGGTTTGACCACGAGGAGTGCATCCTGCACGGAGTCACTGAGCTCGTGACCTCCACGCTGCTCGTCCCCTGCGCTATCGAGAGGGCACTCTCTGTGTCTCAGCTGGTGCCGCTGGCGCAGAGTGTTTTGGGCCCCTTAAAGCTCAGCATGGCTGGTTCTGGAGAGATGGAAAAGAGAAAGGATTTCCCCCATTTGGGTGCCTCGGGCATGTGGAGGAAGGTGGTCCGGCGAACGAAGCAGGGCTGCGTGAAGGGGATCTGATAACCCACGTCAACGGAGAGCCAGTCCATGG-3’；

the combination of primers and probes was as follows:

the upstream primer of the target fragment is SEQ ID NO. 1: 5'-TTCACCTCTGTTTGCTGCAC-3', respectively;

downstream primer of the target segment SEQ ID NO. 2: 5'-AGGTAGTCCGCAGTTTGGAA-3', respectively;

probe of the fragment of interest SEQ ID NO. 3:

5’-FAM-TGCCATCTGCAGCATTGCCACT-TAMRA-3’；

the upstream primer of the internal reference fragment is SEQ ID NO. 4: 5'-GGCATGTGGAGGAAGGTGGT-3', respectively;

downstream primer of internal reference fragment SEQ ID NO. 5: 5'-CCATGGACTGGCTCTCCGTT-3', respectively;

probe of internal reference fragment SEQ ID NO. 6:

5’-HEX-ACGCAGCCCTGCTTCGTTCGCCG-TAMRA-3’。

the above two pairs of primers and probes can be used for performing double fluorescence PCR amplification on the specific target fragment and the internal reference fragment of the 40-type and 41-type human adenoviruses.

The invention also provides an application of the kit, which comprises the following steps:

(1) extracting DNA of a sample to be detected;

(2) performing PCR amplification: adding PCR reaction solution A and PCR reaction solution B in a certain proportion by using sample DNA to be detected as template, wherein reaction solution A comprises 12.5mM Tris-HCl, pH9.0, 50mM KCl, 0.13%X-100、3.13mMMgCl₂0.45mM dATP, 0.45mM dGTP, 0.45mM dCTP, 0.79mM dUTP, two pairs of primers SEQ ID No.1 and 2, 4 and 5, both at a concentration of 0.3-0.7. mu.M, and two probes SEQ ID No.3, 6, both at a concentration of 0.14-0.16. mu.M; reaction solution B comprises 20mM Tris-HCl, 100mM KCl, 0.1mM EDTA, 1mM DTT, 50% Glycerin, 0.5%20. 1-3U/mu L hot start enzyme and 0.1-0.3U/mu L UNG enzyme, performing PCR amplification, simultaneously replacing the DNA of a sample to be detected with a negative quality control product and a positive quality control product as templates, adding a PCR reaction solution A and a PCR reaction solution B, and performing PCR amplification of the negative quality control product and the positive quality control product;

(3) and (5) judging the result according to the Ct value.

Further, in the step (2), the reaction solution A contains two pairs of primers SEQ ID NO.1 and 2, 4 and 5 at a concentration of 0.5. mu.M, two probes SEQ ID NO.3 and 6 at a concentration of 0.15. mu.M, and the PCR reaction solution B contains hot-start enzyme and UNG enzyme at a concentration of 2U/. mu. L and 0.2U/. mu. L, respectively.

Further, the volume ratio of the sample DNA to be detected to the reaction solution A and the reaction solution B is 3: 46: 1.

further, the parameters of the PCR amplification procedure are as follows:

120s at 50 ℃,1 cycle;

600s at 95 ℃ for 1 cycle;

95 ℃, 15s, 55 ℃, 45s (fluorescence collected), 40 cycles;

37 ℃, 20s, 1 cycle.

Furthermore, the negative quality control product is physiological saline, and the positive quality control product is engineering bacterial suspension for cloning target gene sequences of human adenovirus type 40 and type 41.

The construction process of the engineering bacteria for cloning the target gene sequences of human adenovirus type 40 and type 41 is as follows:

1. amplification of a fragment of interest

1.1 use Takara Taq^TMHot Start Version (L ot # AIG1122A) the target fragment was amplified as in Table 1

TABLE 1

1.2 PCR parameters

300s at 95 ℃ for 1 cycle;

95 ℃, 15s, 55 ℃, 30s, 72 ℃, 30s, 40 cycles;

72 ℃, 300s, 1 cycle;

37 ℃, 20s, 1 cycle.

1.3 verified by agarose gel electrophoresis.

2. 3' end of the blunt-ended DNA fragment plus "A" reaction

Construction of engineering bacteria Using Takara DNA A-Tailing Kit

2.1A reaction solution "A" was prepared in a microcentrifuge tube in the amount of 50. mu. L in accordance with Table 2.

TABLE 2

Name of reagent	Amount of the composition used
		10×A-Tailing Buffer	5μL
dNTP Mixture	4μlL
		A-Tailing Enzyme	0.5μL
Terminal smoothing DNA fragment	0.5～5μg
		Sterilized water	upto50μL

The reaction was carried out at 2.272 ℃ for 20 minutes.

And 2.3, standing for 1-2 minutes in ice.

Ligation transformation of A-labeling DNA fragment and T vector

3.1 DNA solutions were prepared in a microcentrifuge tube according to Table 3, with a total volume of 5. mu. L.

TABLE 3

Name of reagent	Amount of the composition used
		pMD18-TVector*	1μL
The A-TailingDNA solution of 1- ③ described above	0.1～0.3pmol
		Sterilized water	upto5μL

pMD18-T Vector was the same preparation as CodeNo.6011, and this T Vector 1. mu. L (50ng) was about 0.03 pmol.

3.2 Add 5. mu. L (equiv.) of Solutioni

Solution I is a component of Code No.6022 DNA L alignment Kit Ver.2.1.

Reaction at 3.316 ℃ for 30 min.

Room temperature (25 ℃) also allowed the ligation reaction to proceed normally, but the reaction efficiency was slightly reduced.

The ligation reaction proceeded normally even in 5 minutes, but the reaction efficiency was slightly decreased.

When cloning is performed on a long fragment DNA (2kb or more), the ligation reaction time is required to be extended to several hours.

3.4 full dose (10. mu. L) was added to 100. mu. L JM109 competent cells and left on ice for 30 minutes.

After heating at 3.542 ℃ for 45 seconds, it was left in ice for 1 minute.

3.6 Add 890 u L SOC medium, 37 degrees C shaking culture for 60 minutes.

3.7 culturing on L-agar plate medium containing X-Gal, IPTG and Amp, forming single colony, counting white and blue colonies.

3.8 white colonies were picked, and the length of the insert in the vector was confirmed by PCR.

3.9 PCR method to detect the bacterial strain with correct insert size, namely the engineering bacteria of cloning human adenovirus type 40 and 41 target gene sequence, and preserving them.

Drawings

FIG. 1 is a temperature profile of PCR amplification conditions in example 1.

FIG. 2 is a PCR amplification graph of FAM channels of primer pair 1 and probe 1 set in example 1.

FIG. 3 is a graph showing the HAX channel PCR amplification curves of primer set 1 and probe set 1 in example 1.

FIG. 4 is a PCR amplification graph of FAM channels of primer pair 2 and probe 2 set in example 1.

FIG. 5 is a graph showing the HAX channel PCR amplification of the primer set 2 and the probe set 2 in example 1.

FIG. 6 is a PCR amplification graph of FAM channels of primer pair 3 and probe 3 set in example 1.

FIG. 7 is a graph showing the HAX channel PCR amplification of the primer set 3 and the probe set 3 in example 1.

FIG. 8 is a temperature profile of PCR amplification conditions in example 2.

FIG. 9 is a PCR amplification plot of the specific experimental FAM channel in example 2.

FIG. 10 is a graph of HEX channel PCR amplification curve for the specificity experiment in example 2.

FIG. 11 is a graph of the PCR amplification of the FAM channel for the sensitivity experiment in example 2.

FIG. 12 is a graph showing the HEX channel PCR amplification curve of the sensitivity test in example 2.

Detailed Description