CN113913556B - Kit for rapidly detecting bat adenovirus and detection method thereof - Google Patents

Abstract

The invention discloses a kit for rapidly detecting bat adenovirus and a detection method thereof. The method comprises the following steps: specific isothermal amplification primers designed aiming at the nucleic acid sequence of a small T-anti gene fragment which is relatively conserved in the hepialus adenovirus Advcxc6 and corresponding rProbe probes which can be digested by RNaseH and contain RNA bases; the fluorescent groups and the quenching groups are respectively designed at two ends of the rProbe, after RNA base is cut by RNaseH to be free, the quick detection of the bat adenovirus can be carried out by utilizing the activity of DNA polymerase and the efficient isothermal amplification primer and probe, and the kit has the advantages of high sensitivity, stability and strong specificity and has very strong practical application value.

Description

Kit for rapidly detecting bat adenovirus and detection method thereof

Technical Field

The invention relates to a kit for rapidly detecting bat adenovirus and a detection method thereof, belonging to the technical field of molecular biology.

Background

In recent years, bat is proved to be a storage host for various important zoonotic viruses, and the bat has the characteristics of complex feeding habit, long flying distance, close relationship with people and livestock and the like, and often brings great threat to human health. The research of Chinese scholars reveals that about 20% of Chinese bats carry adenovirus, and the bat adenovirus presents remarkable genetic diversity due to the difference of bats and regional distribution. Therefore, the method for rapidly detecting the bat carried adenovirus is established, and has important significance for predicting and early warning of adenovirus epidemic situation.

Isothermal amplification of nucleic acids, also known as isothermal amplification of nucleic acids, is a rapidly growing method of nucleic acid amplification since the 21 st century. Isothermal amplification techniques can achieve detection of target nucleic acids within 1 hour, and some can achieve detection within 20 minutes. At present, the judging modes of isothermal amplification results can be basically divided into three categories: running agarose gel electrophoresis to observe a gel diagram; secondly, observing turbidity or color change of the reaction tube before and after amplification by a visual method; thirdly, introducing a fluorescence signal into the reaction liquid, amplifying on simple equipment capable of reading the fluorescence signal, and reading the change of the fluorescence signal. With the development of technology, products of a third class isothermal amplification result judging mode are better popularized and applied in the market.

The enzyme-cutting probe isothermal amplification technology (Enzymatic Probe Isothermal Amplification, EPIA) designs specific isothermal amplification primers and rProbe probes aiming at a conserved region of target nucleic acid to be detected, wherein fluorescent groups and quenching groups are respectively designed at two ends of the rProbe. And amplifying the target nucleic acid sequence to be detected by utilizing the activity of DNA polymerase, combining rProbe to the corresponding target sequence to form a probe-target nucleic acid hybridization double chain, cutting RNA base in the probe-target nucleic acid hybridization double chain by RNaseH, enabling the RNA base and a probe fragment containing a quenching group on the right side of the RNA base to be free, and enabling the fragment containing a fluorescent group on the left side of the RNA base to still form a hybridization chain and be used as a primer to continue to extend, wherein the fluorescent group emits fluorescence. The presence or absence of the target nucleic acid is indicated by the presence or absence of the amplification signal.

At present, preventive medicine and clinic have the requirements of simplicity, convenience, rapidness, high sensitivity and good specificity on the hepialus adenovirus detection technology, and the prior art has limitations, so that development of the rapid, accurate and low-cost hepialus adenovirus detection technology is urgently needed.

Disclosure of Invention

In order to solve the technical problems, the invention discloses a kit for rapidly detecting bat adenovirus and a detection method thereof, wherein a probe (RNHP) containing RNA base and high-temperature resistant RNaseH are introduced on the basis of isothermal amplification, a specific isothermal amplification primer and a specific rProbe probe are designed aiming at a nucleic acid conservation region of the adenovirus, the high-temperature resistant RNaseH is combined, the characteristics of phosphodiester bonds of RNA in a DNA and RNA hybrid chain can be digested by the RNaseH, when a sample to be detected contains target nucleic acid, a large amount of target DNA/cDNA is obtained through isothermal amplification, rProbe can be combined with the target DNA/cDNA to form a probe-target nucleic acid hybrid double chain, then the RNA base in the probe-target nucleic acid hybrid double chain is cut by the RNaseH, so that a probe fragment containing a quenching group on the right side of the RNA base is released, a fragment containing a fluorescent group on the left side of the RNA base still keeps forming a hybrid chain and can be used as a primer to extend, and fluorescent group is emitted, so that the existence of the target nucleic acid can be determined.

The kit is a kit for detecting the enzyme digestion isothermal amplification of the bat adenovirus small T-anti gene, and a primer probe for detecting the bat adenovirus nucleic acid is matched with a primer probe for detecting an internal reference to form a double real-time loop-mediated isothermal amplification process, which comprises a nucleic acid reaction solution, a detection enzyme solution, a positive quality control product and a negative control product,

the nucleic acid reaction solution comprises a specific enzyme-cutting isothermal amplification primer, ribonuclease RNaseH and a fluorescent probe RNHP, wherein the ribonuclease RNaseH is heat-resistant ribonuclease RNaseH, the fluorescent probe RNHP comprises an RNA base, the bases at the two ends of the fluorescent probe RNHP are respectively marked with a fluorescent group and a quenching group,

the detection enzyme solution comprises Bst nucleic acid polymerase and AMV reverse transcriptase,

the positive quality control product is standard plasmid of exogenous internal reference gene fragment and adenovirus pseudovirus cRNA, and the negative control product is RNA/DNA-free water.

Further, the loop-mediated isothermal amplification primer is provided with six primers, and the sequences of the primers are respectively shown in SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO:10 and SEQ ID NO:11, the sequence of the used fluorescent probe RNHP is shown as SEQ ID NO:35.

further, the kit also comprises an internal reference substance, wherein the internal reference substance is a human ACTB gene, six loop-mediated isothermal amplification primers are used, and the sequences of the six loop-mediated isothermal amplification primers are respectively shown in SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39. SEQ ID NO: 40. SEQ ID NO:41, the sequence of the used fluorescent probe RNHP is shown as SEQ ID NO:42.

further, the length of the fluorescent probe RNHP is 19bp, and the sequence is shown in SEQ ID NO:35 and the RNHP probe of SEQ ID NO:42 RNHP probes are respectively marked by different fluorescence, and the specific isothermal amplification primer fragments are 18-39bp; the length of the selected exogenous reference substance is 20-37bp.

Further, the fluorophores are FAM and ROX.

The invention also discloses a method for rapidly detecting the bat adenovirus by using the kit, which comprises the following steps:

step 1: preparation of yang-property control

Construction of a pseudoviral plasmid: adding enzyme cutting sites into a target gene amplification sequence, connecting and inserting a target small fragment into a modified carrier PET42a-MS2 with a pseudovirus induction function through T4 enzyme, and then performing sequencing identification, and preserving bacteria for later use;

preparation of pseudoviruses: adding 50 μl of the bacterial liquid with the target fragment into 5ml of LB liquid culture medium, and shake culturing at 37 ℃ at 200-220rpm for 2.5-3 h to logarithmic phase; adding IPTG to the mixture to make the final concentration of the mixture be 1mM, and then placing the mixture at 37 ℃ and incubating the mixture at 200rpm for 3-4 hours; after incubation, cells were collected, 0.5ml of ultrasound buffer was added and the mixture was sonicated on ice: 350W, stopping for 5s, performing ultrasonic treatment for 5s, performing 30 cycles, centrifuging at 6000rpm for 10min, collecting supernatant to obtain pseudo-virus solution, and extracting RNA to obtain positive quality control product;

finally preparing a nucleic acid reaction solution, a detection enzyme solution, a negative reference substance and an internal reference substance, wherein the nucleic acid reaction solution comprises a primer, a probe, ribonuclease RNaseH, betaine, dNTP, mgSO4 and buffer; the detection enzyme solution comprises Bst polymerase and AMV reverse transcriptase; the positive quality control product is the pseudovirus prepared by the above, and the reference control product is human ACTB gene; the negative reference substance is water without RNA/DNA;

step 2: primer probe screening

The method comprises the steps of selecting small T-anti as a gene target sequence, locating at 1829-2307bp of a bat adenovirus Advcxc6 genome sequence, designing a specific primer and a fluorescent probe RNHP, wherein a group of human ACTB genes are arranged in a system as exogenous internal references to avoid false negative, and the size of the target sequence is 479 bp;

step 3: dual real-time isothermal amplification system

The double real-time isothermal amplification reaction system of adenovirus pseudovirus and primer and probe used as reference is 30 mu L, and the reaction procedure is as follows: amplifying 1min at 63 ℃ for 30 cycles, and collecting the fluorescent group FAM and the quenching group ROX;

step 4: result determination

Target positive: FAM channel Ct is less than or equal to 28, ROX channel is not needed to be seen,

target negative: FAM channel Ct >28, and ROX channel <30,

detection of invalidation: FAM channel Ct >28 or no amplification information, and ROX channel no amplification signal; in this case, the sample needs to be re-extracted and retested.

The beneficial effects are that: the invention realizes the real-time isothermal rapid detection of the bat adenovirus nucleic acid, the detection is completed within 25-30min, the indexes such as the sensitivity of the method and the like are not lower than rRT-PCR, and an effective solution is provided for adenovirus epidemic situation tracing and the detection of the bat adenovirus positive rate. On the basis of the enzyme digestion isothermal amplification technology, primers and probes are designed to correspond to different areas respectively, so that adenovirus nucleic acid is detected, the sensitivity is high, the specificity is high, the practical application value is high, and the method is suitable for basic medical and health units. Under the condition that the target nucleic acid exists, the linear probe labeling group is simple and is easier to combine with the target nucleic acid, so that the sensitivity is improved; compared with a single isothermal amplification mode: the method can be added with the detection of internal references, so that the detection result is more effective and reliable, and the result judgment mode is more objective. The whole process is closed, so that the amplification pollution is avoided; the time is quick, the detection of the pathogen RNA is completed within 1 hour, and a detection report can be sent out within 30min at the highest speed; amplifying in real time: contains a probe capable of being cut by enzyme, and can observe the amplification condition in real time.

Drawings

FIG. 1 is a schematic diagram of the detection principle of the method of the invention,

FIG. 2 is a graph showing the results of a test sample 10Copies/mL repeated 20 times in a kit according to an embodiment of the present invention,

FIG. 3 is a graph showing the result of multi-well verification amplification of other negative human genomic nucleic acids by the kit according to the example of the present invention,

FIG. 4 is a graph showing the results of a blank control for the test kit according to the embodiment of the present invention,

FIG. 5 is a graph showing the results of detection of positive quality control by the kit according to the embodiment of the present invention,

FIG. 6 is a graph showing the results of a test kit for detecting 1 clinical specimen of hepialus in an embodiment of the present invention,

reference numerals: 1-isothermal amplification primer, 2-probe containing RNA base, 3-RNaseH, 4-probe fragment with fluorescent group after enzyme digestion, and free quenching group after 5-enzyme digestion.

Detailed Description

The present invention is further illustrated in the accompanying drawings and detailed description which are to be understood as being merely illustrative of the invention and not limiting of its scope, and various modifications of the invention, which are equivalent to those skilled in the art upon reading the invention, will fall within the scope of the invention as defined in the appended claims.

Unless otherwise indicated, all methods are conventional in the art. In the following examples, reagents and materials used, unless otherwise specified, were purchased from conventional biochemistry agents in the art. Isothermal amplification buffer, mgSO4, bstDNA polymerase, AMV enzyme were purchased from Neugan Biotechnology Co., ltd (New England Biolabs), dNTPs were purchased from Takara Bio-engineering Co., ltd, and primers and probes were synthesized by Shanghai Biotechnology service Co., ltd. Plasmid miniprep kit and viral RNA extraction kit were purchased from beijing tiangen biotechnology limited.

As shown in figure 1, 1 is an isothermal amplification primer, 2 is a probe containing RNA base, 3 is RNaseH,4 is a probe fragment with a fluorescent group after enzyme digestion, 5 is a free quenching group after enzyme digestion, R is an RNA base in the probe, and the invention designs a specific isothermal amplification primer and a probe aiming at bat adenovirus, wherein the fluorescent group and the quenching group are respectively designed at two ends of the probe. Amplifying a target nucleic acid sequence to be detected by utilizing the activity of DNA polymerase, combining a probe to the corresponding target sequence to form a probe-target nucleic acid hybridization double chain, cutting an RNA base in the probe-target nucleic acid hybridization double chain by RNaseH, enabling the RNA base and a probe fragment containing a quenching group on the right side of the RNA base to be free, and enabling the fragment containing a fluorescent group on the left side of the RNA base to still form a hybridization chain and be used as a primer to extend continuously, wherein the fluorescent group emits fluorescence. Whether or not the target nucleic acid is present is indicated by determining whether or not the amplification signal is present. The detection steps are as follows:

step 1: preparation of yang-property control

Construction of a pseudoviral plasmid: adding enzyme cutting sites into a target gene amplification sequence, connecting and inserting a target small fragment into a modified carrier PET42a-MS2 with a pseudovirus induction function through T4 enzyme, and then performing sequencing identification, and preserving bacteria for later use;

preparation of pseudoviruses: adding 50 μl of the bacterial liquid with the target fragment into 5ml of LB liquid culture medium, and shake culturing at 37 ℃ at 200-220rpm for 2.5-3 h to logarithmic phase; adding IPTG to the mixture to make the final concentration of the mixture be 1mM, and then placing the mixture at 37 ℃ and incubating the mixture at 200rpm for 3-4 hours; after incubation, cells were collected, 0.5ml of ultrasound buffer was added and the mixture was sonicated on ice: 350W, stopping for 5s, performing ultrasonic treatment for 5s, performing 30 cycles, centrifuging at 6000rpm for 10min, collecting supernatant to obtain pseudo-virus solution, and extracting RNA to obtain positive quality control product;

finally preparing a nucleic acid reaction solution, a detection enzyme solution, a negative reference substance and an internal reference substance, wherein the nucleic acid reaction solution comprises a primer, a probe, ribonuclease RNaseH, betaine, dNTP, mgSO4 and buffer; the detection enzyme solution comprises Bst polymerase and AMV reverse transcriptase; the positive quality control product is the pseudovirus prepared by the above, and the reference control product is human ACTB gene; the negative reference substance is water without RNA/DNA;

step 2: primer probe screening

The small T-anti is selected as a gene target sequence, is positioned at 1829-2307bp of a bat adenovirus Advcxc6 genome sequence, has the size of 479bp, has the characteristic of high conservation of species in species, designs a specific primer and a fluorescent probe RNHP, wherein a group of human ACTB genes are arranged in a system as exogenous internal references to avoid false negative; target gene synthesis and sequencing was sent to Shanghai Biotechnology Inc. for synthesis and sequencing.

Primer design and screening

A total of 5 sets of primers were designed for the target region as shown in Table 1

TABLE 1 target sequence LAMP prescreening primer sequences

Sensitivity and specificity verification: the second set of primers meets the performance requirements.

Probe design and screening

For the 2 nd set of primers, 2 probes were designed in total, as shown in Table 2

TABLE 2 target primer probe sequences

Primer number	Primer name	Primer sequences
			SEQ ID NO：34	Adv-LF-P	CGGACTCAGCACCTCGT
SEQ ID NO：35	Adv-LB-P	ATTGAGCGGGGTGGCTCCA

Sensitivity and specificity verification: the "Adv-LB-P" meets the performance requirements of sensitivity and specificity.

Screening of exogenous internal reference primers and probes

3 existing exogenous internal references in a laboratory are used as system internal references for optimization screening. Wherein the exogenous internal references 1 and 2 and the target inhibit each other and cannot be used as a system internal reference, the internal reference 3 is selected as the system internal reference, the primer and the probe are shown in the table 3, and finally, the internal reference amplification in a dual detection system is formed by six primers and one probe, wherein the RNA concentration of the extracted internal pseudovirus is 5000 copies/mL.

TABLE 3 exogenous internal reference primer and probe sequences

Primer number	Primer name	Primer sequences
			SEQ ID NO：36	ACTB/F3	GTGGCACCACCATGTACC
SEQ ID NO：37	ACTB/B3	TCCACATCTGCTGGAAGGT
			SEQ ID NO：38	ACTB/FIP	TCTTCATCGTGCTGGGCGCAGGCATCGCCGACAGG
SEQ ID NO：39	ACTB/BIP	TCAAGATCATCGCGCCTCCAGAGCGAGGCCAGGATGG
			SEQ ID NO：40	ACTB/LF	GCCGTGATCTCCTTCTGCAT
SEQ ID NO：41	ACTB/LB	CGCAAGTACTCCGTGTGGAT
			SEQ ID NO：42	ACTB/LF-p	GCCGTGATCTCCTTCTGCAT

Step 3: dual real-time isothermal amplification system

The double detection system of the single target and the internal reference is established, the double real-time isothermal amplification reaction system of the adenovirus pseudovirus and the primer and the probe used by the internal reference is 30 mu L, the components and the final concentration of the nucleic acid reaction liquid are shown in the table 4, and the reaction procedure is as follows: amplifying 1min at 63 ℃ for 30 cycles, and collecting the fluorescent group FAM and the quenching group ROX;

TABLE 4 specific detection of hepialus adenovirus double reaction liquid System Table

The sensitivity of the dual system detection target was 10copies/uL.

Reaction program settings as shown in table 5, the applicable model was a macro SLAN 96 model, and the default threshold line was not used for detection on the macro instrument, but should be set to 0.36 when the reaction program was set.

TABLE 5 reaction procedure for detection systems

Cycle number	Reaction conditions	Whether or not to collect fluorescence
			30cycles	63℃,1min	Is (FAM, ROX)

Step 4: result determination

Target positive: FAM channel Ct is less than or equal to 28, ROX channel is not needed to be seen,

target negative: FAM channel Ct >28, and ROX channel <30,

detection of invalidation: FAM channel Ct >28 or no amplification information, and ROX channel no amplification signal; in this case, the sample needs to be re-extracted and retested.

Performance verification data for detection systems

Sensitivity verification: the multiplex amplification at 10copies/uL, which is shown in FIG. 2, was stable at the lowest limit of detection.

And (3) specificity verification:

the system detects the multi-hole verification of the negative human genome DNA, and the amplification is shown in figure 3: the target channel is free of non-specific amplification.

The system detects the multi-hole verification of the blank control, and the amplification is shown in fig. 4: the target channel is free of non-specific amplification.

And (3) verifying positive quality control:

amplification of 20 multiplex wells of positive quality control extracted RNA, the amplification of which is shown in FIG. 5.

Clinical sample verification:

1 clinical sample of hepialus (B2) was validated, and the results showed normal expansion. The results of the verification are shown in the following table, and the amplification curve is shown in FIG. 6:

it will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.

Sequence listing

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Claims (5)

1. A kit for rapidly detecting bat adenovirus is characterized in that the kit is a kit for detecting enzyme-cutting isothermal amplification of bat adenovirus small T-anti gene, a primer probe for detecting bat adenovirus nucleic acid is matched with a primer probe for detecting internal reference to form a double real-time loop-mediated isothermal amplification process, the kit comprises nucleic acid reaction liquid, detection enzyme liquid, positive quality control product and negative control product,

the amplification primer is provided with six amplification primers, and the sequences of the amplification primers are respectively shown in SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO:10 and SEQ ID NO:11, the sequence of the used fluorescent probe RNHP is shown as SEQ ID NO:35,

the nucleic acid reaction solution comprises a specific enzyme-cutting isothermal amplification primer, ribonuclease RNaseH and a fluorescent probe RNHP, wherein the ribonuclease RNaseH is heat-resistant ribonuclease RNaseH, the fluorescent probe RNHP comprises an RNA base, the bases at the two ends of the fluorescent probe RNHP are respectively marked with a fluorescent group and a quenching group,

the detection enzyme solution comprises Bst nucleic acid polymerase and AMV reverse transcriptase,

the positive quality control product is standard plasmid of exogenous internal reference gene fragment and adenovirus pseudovirus cRNA, and the negative control product is RNA/DNA-free water.

2. The kit for rapidly detecting the bat adenovirus according to claim 1, further comprising an internal reference substance, wherein the internal reference substance is a human ACTB gene, six loop-mediated isothermal amplification primers are used, and the sequences of the six loop-mediated isothermal amplification primers are respectively as shown in SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39. SEQ ID NO: 40. SEQ ID NO:41, the sequence of the used fluorescent probe RNHP is shown as SEQ ID NO:42.

3. the kit for rapid detection of hepialus adenovirus according to claim 1 or 2, wherein the sequence is as set forth in SEQ ID NO:35 and the RNHP probe of SEQ ID NO:42 are each labeled with a different fluorescent label.

4. The kit for rapid detection of hepialus adenovirus according to claim 1, wherein the fluorescent groups are FAM and ROX.

5. A method for rapid detection of hepialus adenovirus for non-diagnostic purposes using the kit according to any one of claims 1-4, comprising the steps of:

step 1: preparation of yang-property control

Construction of a pseudoviral plasmid: adding enzyme cutting sites into a target gene amplification sequence, connecting and inserting a target small fragment into a modified carrier PET42a-MS2 with a pseudovirus induction function through T4 enzyme, and then performing sequencing identification, and preserving bacteria for later use;

preparation of pseudoviruses: adding 50 mu l of bacterial liquid with a target fragment into 5ml of LB liquid culture medium, and shake culturing at a rotating speed of 200-220rpm at 37 ℃ for 2.5-3 h to a logarithmic growth phase; adding IPTG to the mixture to make the final concentration of the mixture be 1mM, and then placing the mixture at 37 ℃ and incubating the mixture at 200rpm for 3-4 hours; after incubation, cells were collected, 0.5ml of ultrasound buffer was added and the mixture was sonicated on ice: 350W, stopping for 5s, performing ultrasonic treatment for 5s, performing 30 cycles, centrifuging at 6000rpm for 10min, collecting supernatant to obtain pseudo-virus solution, and extracting RNA to obtain positive quality control product;

finally preparing a nucleic acid reaction solution, a detection enzyme solution, a negative reference substance and an internal reference substance, wherein the nucleic acid reaction solution comprises a primer, a probe, ribonuclease RNaseH, betaine, dNTP, mgSO4 and buffer; the detection enzyme solution comprises Bst polymerase and AMV reverse transcriptase; the positive quality control product is the pseudovirus prepared by the above, and the reference control product is human ACTB gene; the negative reference substance is water without RNA/DNA;

step 2: primer probe screening

The method comprises the steps of selecting small T-anti as a gene target sequence, locating at 1829-2307bp of a bat adenovirus Advcxc6 genome sequence, designing a specific primer and a fluorescent probe RNHP, wherein a group of human ACTB genes are arranged in a system as exogenous internal references to avoid false negative, and the size of the target sequence is 479 bp;

step 3: dual real-time isothermal amplification system

The double real-time isothermal amplification reaction system of adenovirus pseudovirus and primer and probe used as reference is 30 mu L, and the reaction procedure is as follows: amplifying at 63 ℃ for 1min for 30 cycles, and collecting the fluorescent group FAM and the quenching group ROX;

step 4: result determination

Target positive: FAM channel Ct is less than or equal to 28, ROX channel is not needed to be seen,

target negative: FAM channel Ct >28, and ROX channel <30,

detection of invalidation: FAM channel Ct >28 or no amplification information, and ROX channel no amplification signal; in this case, the sample needs to be re-extracted and retested.

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