CN110684867B - Complete set of primers, kit and detection method for detecting GIV.1 norovirus by RT-LAMP method - Google Patents

Complete set of primers, kit and detection method for detecting GIV.1 norovirus by RT-LAMP method Download PDF

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CN110684867B
CN110684867B CN201910976389.6A CN201910976389A CN110684867B CN 110684867 B CN110684867 B CN 110684867B CN 201910976389 A CN201910976389 A CN 201910976389A CN 110684867 B CN110684867 B CN 110684867B
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韩一芳
叶福强
张锦海
王太武
张琪
汪春晖
曹猛
王长军
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Abstract

The invention relates to a complete set of primers, a kit and a detection method for detecting GIV.1 norovirus by an RT-LAMP method. The complete set of primers comprises an outer primer pair GIV-F3 and GIV-B3, an inner primer pair GIV-FIP and GIV-BIP, and a loop primer pair GIV-LF and GIV-LB; the sequence is shown in SEQ ID NO 1-6. The kit contains the primer set. The detection method adopts the kit. The invention has reliable result, relatively low cost, simple, quick and sensitive characteristics, is particularly suitable for on-site instant detection, and is suitable for most domestic hospitals and epidemic prevention department laboratories lacking the real-time fluorescent quantitative PCR instrument.

Description

Complete set of primers, kit and detection method for detecting GIV.1 norovirus by RT-LAMP method
Technical Field
The invention relates to a complete set of primers, a kit and a detection method for detecting GIV.1 norovirus by an RT-LAMP method, and belongs to the technical field of biology.
Background
Norovirus (Norovirus, NV), also known as Norovirus, belongs to the family caliciviridae, is a single-stranded positive-strand RNA virus that is the leading pathogen causing outbreaks of acute non-bacterial gastroenteritis worldwide. The virus is widely prevalent worldwide, is mainly transmitted by polluted food and water sources, can also be transmitted by human-human contact, and is often epidemic in places where people gather, such as hospitals, schools, military and the like. It is reported that more than 1 million people worldwide develop disease each year due to norovirus infection. The clinical symptoms are mainly nausea, vomiting, abdominal pain and diarrhea, most patients show self-limitation, but a few infants, the elderly and patients with immunodeficiency can develop severe illness and even death. The rapid diagnosis of norovirus has important significance for clinical diagnosis and treatment and disease prevention and control.
Depending on the coding sequence of the norovirus capsid protein VP1, norovirus can be divided into 7 genomes (GI to GVII), where GI type, GII type and GIV type infect humans. The GIV type is divided into two genotypes, GIV.1 type infects humans, GIV.2 type infects cats and dogs.
Most of the current tests for norovirus mainly focus on the GI type and GII type common to human infection, and are mainly performed by serology, such as ELISA and other technical means. The global prevalence of the GIV.1 norovirus is unknown, and is usually detected in sporadic cases and environments, with relatively low detection rates, which reflect the lack of specific and sensitive detection methods.
Loop-mediated isothermal amplification (LAMP) is a isothermal nucleic acid amplification technique developed by Notomi in 2000; the LAMP method designs 3 pairs of specific primers aiming at 8 gene segments of a target gene, realizes high-efficiency amplification of the target gene within 1h under the isothermal condition of 60-65 ℃ by depending on strand displacement DNA polymerase (Bst DNA polymerase), forms macroscopic pyrophosphoric acid precipitate during amplification, and can judge positive reaction according to the precipitate; the method has the advantages of convenient operation, strong specificity and high sensitivity, does not need expensive equipment such as a PCR instrument and the like, and only needs one water bath kettle or constant-temperature metal bath. And a magnesium ion indicator Hydroxyl Naphthol Blue (HNB) can be added into a reaction system, the result judgment is carried out through the color change after magnesium ions are consumed in the amplification process, the blue color is positive reaction, the purple color is negative reaction, the result is directly observed by eyes, the aerosol pollution caused by uncovering and glue running and the like is avoided, and the method is suitable for field detection and basic hospital laboratories lacking equipment such as a PCR instrument and the like.
The retrieval finds that Chinese patent with application number CN200710030430.8 and grant publication number CN101153341B disclose a food-borne pathogen rapid detection technology based on loop-mediated isothermal amplification technology, wherein a primer for detecting norovirus GII type can amplify a specific base sequence of a target gene, and the target gene is a capsid protein of the norovirus GII type-GenBank landing number: x86557, a primer complementary to a part of the nucleic acid sequence from position 5095 to position 5319 of the target gene or a complementary strand thereof. The technical scheme utilizes a kit containing the primer group to detect whether a norovirus GII type specific gene fragment exists in a specimen, and further determines whether the norovirus GII type exists in the specimen. However, this technical solution is directed only to the GII type norovirus, not to the GIV type norovirus, and is not directly applicable.
Chinese patent inventions with application numbers CN201310690315.9 and grant publication numbers CN103642943B disclose a loop-mediated isothermal amplification detection primer group, a detection method and a kit for GI type norovirus. The detection primer group comprises an upstream outer primer F3, a downstream outer primer B3, an upstream inner primer FIP and a downstream inner primer BIP, and specific sequences are shown in the original text. The technical scheme establishes an LAMP detection method of the GI type norovirus, and the detection method designs and screens a set of specific primers (4 primers) aiming at RNA polymerase genes of the GI type norovirus, wherein the specific primers comprise two specific inner primers and two specific outer primers. However, this technical solution is only directed to GI type norovirus, not to GIV type norovirus, and is not directly applicable.
Chinese patent application No. CN201610079851.9 and No. CN105648111B disclose a reverse transcription loop-mediated isothermal amplification detection primer group, a detection method and a kit for norovirus. The detection primer group comprises an upstream outer primer F3, a downstream outer primer B3, an upstream inner primer FIP and a downstream inner primer BIP, and specific sequences are shown in the original text. The technical scheme establishes a one-step RT-LAMP technology for detecting the NV, and can detect the NV in allantoic fluid and tissue samples. However, analysis based on the primer sequences provided by this method only targets a portion of GII.4 type NV gene sequences, i.e., only a portion of GII.4 type norovirus can be detected.
Because of differences of NV genomes and genotypes, commercial NV universal kits (such as real-time fluorescent quantitative PCR methods and the like) only contain partial GI type and GII type NV detection reagents.
At present, no method for detecting the GIV type norovirus by using RT-LAMP technology exists.
Disclosure of Invention
The main purposes of the invention are: the problem in the prior art is solved, and the complete set of primers for detecting the GIV.1 type norovirus by the RT-LAMP method are provided, so that the GIV.1 type norovirus can be effectively detected. Meanwhile, a corresponding kit and a detection method are also provided, and the kit and the detection method can be used for detecting the GIV.1 norovirus.
The technical scheme for solving the technical problem is as follows:
a complete set of primers for detecting the GIV.1 norovirus by the RT-LAMP method is characterized by comprising an outer primer pair GIV-F3 and GIV-B3, an inner primer pair GIV-FIP and GIV-BIP, and a loop primer pair GIV-LF and GIV-LB; the sequence of GIV-F3 is shown as SEQ ID NO. 1, the sequence of GIV-B3 is shown as SEQ ID NO. 2, the sequence of GIV-FIP is shown as SEQ ID NO. 3, the sequence of GIV-BIP is shown as SEQ ID NO. 4, and the sequence of GIV-LF is shown as SEQ ID NO. 5; the sequence of GIV-LB is shown in SEQ ID NO 6.
After the complete set of primers is adopted, the detection of the GIV.1 type norovirus by an RT-LAMP method can be realized.
The present invention also provides:
a kit for detecting GIV.1 type norovirus by RT-LAMP, characterized in that the kit comprises a primer mixture containing the set of primers described above.
By adopting the kit, the detection of the GIV.1 norovirus can be smoothly realized.
The technical scheme for further perfecting the kit provided by the invention is as follows
Preferably, the molar ratio of the outer primer pair GIV-F3 and GIV-B3, the inner primer pair GIV-FIP and GIV-BIP, and the loop primer pair GIV-LF and GIV-LB is 1 (2-4).
Preferably, the kit further comprises an enzyme solution, a 2 × reaction solution, a color development solution and ultrapure water.
More preferably, the enzyme solution is composed of BstDNA polymerase with the concentration of 6-10U/. Mu.l, AMV reverse transcriptase with the concentration of 8-16U/. Mu.l and water;
the 2 × reaction solution is prepared from 40mmol/L Tris-HCl with pH8.8, 20mmol/L KCl and 20mmol/L (NH) 4 ) 2 SO 4 Triton X-100 with a mass concentration of 0.2% and composed of dATP, dTTP and dCT with a concentration of 2.8mmol/LdNTPs consisting of P and dGTP, 1.6mmol/L betaine C 5 H 11 NO 2 16mmol/L MgSO 4 And water;
the color development liquid is metal ion indicator HNB storage liquid with the concentration of 4 mmol/L.
Preferably, the kit also comprises a positive control of the norovirus type GIV.1 at a concentration of 20-100mg/L.
More preferably, the concentration of the positive control for GIV.1 norovirus is 50mg/L and is obtained by: constructing a plasmid containing the RdRp gene of the GIV.1 type norovirus, carrying out in-vitro transcription reaction to obtain an RNA fragment of the RdRp gene, and purifying to obtain a positive control of the GIV.1 type norovirus; the sequence of the RdRp gene is shown as SEQ ID NO. 7.
By adopting the preferable scheme, the detection of the GIV.1 norovirus by RT-LAMP technology can be better realized.
The present invention also provides:
a non-diagnosis purpose GIV.1 type norovirus RT-LAMP detection method is characterized in that the kit is adopted; the method comprises the following steps:
firstly, adding 2 Xreaction solution of the kit, primer mixed solution, enzyme solution, color development solution and ultrapure water into a transparent reaction tube, and then mixing to obtain reaction solution.
Secondly, adding a nucleic acid sample to be detected into the reaction tube, covering the reaction tube and uniformly mixing;
thirdly, placing the reaction tube under a constant temperature condition for RT-LAMP amplification, and then placing the reaction tube under a reaction ending condition to terminate the reaction;
fourthly, observing the color of the reaction solution, wherein if the color of the reaction solution is sky blue, the nucleic acid sample to be detected contains the GIV.1 type norovirus; if the color of the reaction solution is violet, the nucleic acid sample to be detected does not contain the GIV.1 type norovirus.
By adopting the method, the detection of the GIV.1 norovirus by the RT-LAMP method can be smoothly realized, and the result can be visually judged according to the color of the reaction solution, so that the method is rapid and accurate.
Preferably, in the second step, after the nucleic acid sample to be detected is added, the volume ratio of the kit 2 × the reaction solution, the primer mixture, the enzyme solution, the color development solution, the ultrapure water and the nucleic acid sample to be detected is 12.5:2.5:1:0.7:7.3:1, and the volume of the reaction solution in the reaction tube is 25 mul; in the third step, the RT-LAMP amplification conditions are as follows: standing at constant temperature of 62 deg.C for 60min; the conditions for finishing the reaction are as follows: standing at 85 deg.C for 5min.
By adopting the preferred scheme, the detection of the GIV.1 norovirus by the RT-LAMP method can be better realized.
The present invention also provides:
the detection method of the kit for detecting the GIV.1 norovirus by the RT-LAMP method is characterized by comprising the following steps of:
the first step, preparing two reaction tubes containing reaction liquid: adding 2 multiplied reaction liquid, primer mixed liquid, enzyme liquid and color development liquid in the kit into a transparent reaction tube, and then adding ultrapure water and mixing to obtain reaction liquid with a preset volume;
secondly, adding distilled water into the first reaction tube, covering the reaction tube and uniformly mixing the mixture to serve as a negative control; adding a GIV.1 type norovirus positive control into a second reaction tube, covering the reaction tube, and uniformly mixing to serve as a positive control; the adding volumes of the distilled water and the positive control are the same;
thirdly, placing the reaction tube at a constant temperature of 62 ℃ for 60min for RT-LAMP amplification, and then placing the reaction tube at 85 ℃ for 5min to finish the reaction;
fourthly, observing the colors of the negative control reaction liquid and the positive control reaction liquid, wherein if the color of the negative control reaction liquid is violet and the color of the positive control reaction liquid is sky blue, the kit used in the first step is qualified; if the color of each reaction solution does not completely match or completely does not match the above condition, the reagent kit used in the first step is not qualified.
By adopting the verification method, the use of a normal kit can be ensured to obtain reliable results.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the RdRp gene which is conservative for the GIV.1 type norovirus is taken as a target fragment, the related fragment is cloned from a virus isolate of a patient actually diagnosed of the GIV.1 type norovirus infection, a standard plasmid is constructed, and an RT-LAMP visual rapid detection method for the GIV.1 type norovirus is established, so that if the RT-LAMP visual rapid detection method is applied to clinical specimen detection, missed diagnosis and misdiagnosis of the GIV.1 type norovirus infection can be avoided, the prevalence rate of the GIV.1 type norovirus infection can be known, and the method has important significance for the work of preventing and controlling the GIV.1 type norovirus infection.
The invention aims at the detection of the GIV.1 type norovirus by an RT-LAMP method, wherein a special primer set, a kit containing the primer set and a detection method adopting the kit are adopted. The kit is simple to use, and the experimental operation can be completed by one step; large complex and expensive instruments are not needed; the result identification is convenient and intuitive, the cover opening is not needed after the reaction is finished, the problem of false positive pollution caused by subsequent aerosol due to the cover opening can be avoided, and the subsequent tedious processes such as nucleic acid electrophoresis are not needed; the method is rapid and efficient, and the whole process of amplification and result judgment can be completed within half more hours; the detection sensitivity is high, and through experimental comparison tests, the lowest sensitivity of the invention for detecting the RdRp gene of the GIV.1 norovirus can detect 10 2 And (6) copying.
The invention has reliable result, relatively low cost, simple, quick and sensitive characteristics, is particularly suitable for on-site instant detection, and is suitable for most domestic hospitals and epidemic prevention department laboratories lacking the real-time fluorescent quantitative PCR instrument.
Drawings
FIG. 1 is a comparative diagram of the primer screening of example 1 of the present invention.
FIG. 2 is a comparison graph of the sensitivity test in example 5 of the present invention, in which RT-LAMP was performed to detect a gradient-diluted GIV.1 type norovirus positive control by the nephelometry, and a positive determination was made when the turbidity value was > 0.1.
FIG. 3 is a comparison chart of the sensitivity test in example 5 of the present invention, in which a gradient-diluted GIV.1 type norovirus positive control was detected by RT-LAMP using a color development method, and a sky blue test was positive and an violet test was negative. In the figure, 10 1 、10 0 NC is violet, and the rest is sky blue.
FIG. 4 is a graph showing the results of the turbidimetric assay kit for different types of norovirus in example 6 of the present invention.
FIG. 5 is a specific result chart of the turbidimetric and colorimetric assay kits according to example 6 of the present invention, wherein the numbers of the specific result chart correspond to 1. Negative control norovirus type 2.GIV.1, astrovirus type 3, rotavirus type 4, typhoid bacillus, shigella bacillus, salmonella type 7, staphylococcus aureus, and azure is positive and violet is negative, respectively. In the color development result chart, no. 2 is sky blue, and the rest is violet.
Detailed Description
The present invention will be described in further detail with reference to the drawings in conjunction with embodiments. The invention is not limited to the examples given.
The experimental procedures described below are carried out under conventional conditions unless the specific experimental conditions are specified, and for example, the conditions described in Molecular Cloning, A Laboratory Manual, new York, cold Spring Harbor Laboratory Press,2017, which is published by SAMBROOK.J., or the conditions suggested by the manufacturer may be referred to.
The test materials and reagents described below are commercially available unless otherwise specified.
Example 1: primer set for GIV.1 type norovirus
First, in NCBI database (https://www.ncbi.nlm.nih.gov) Searching to obtain a conserved region RdRp gene sequence of the GIV.1 type norovirus, and synthesizing by Shanghai biological engineering Co., ltd; LAMP Primer design is carried out on the specific RdRp gene sequence through LAMP Primer Explorer software (version 5.0), manual selection and correction are carried out according to professional experience, then a final Primer group is screened out from the synthesized 3 sets of Primer combinations according to multiple experimental tests (Japanese Rongyan LA-500 turbidimeter test), and as shown in figure 1, a set of 1 Primer is determined as a preferred Primer group for subsequent experiments.
The complete set of primers comprises an outer primer pair GIV-F3 and GIV-B3, an inner primer pair GIV-FIP and GIV-BIP, and a loop primer pair GIV-LF and GIV-LB; the sequence of GIV-F3 is shown as SEQ ID NO. 1, the sequence of GIV-B3 is shown as SEQ ID NO. 2, the sequence of GIV-FIP is shown as SEQ ID NO. 3, the sequence of GIV-BIP is shown as SEQ ID NO. 4, the sequence of GIV-LF is shown as SEQ ID NO. 5, and the sequence of GIV-LB is shown as SEQ ID NO. 6.
Example 2: kit for detecting GIV.1 type norovirus by RT-LAMP method
The kit of this example includes the primer set of example 1. The molar ratio of the outer primer pair GIV-F3 and GIV-B3, the inner primer pair GIV-FIP and GIV-BIP, and the loop primer pair GIV-LF and GIV-LB is 1 (2-4), preferably 1.
For example: each 2.5. Mu.l of the primer mixture contained 2pmol of GIV-F3, 2pmol of GIV-B3, 16pmol of GIV-FIP, 16pmol of GIV-BIP, and 8pmol of GIV-LF, 8pmol of GIV-LB.
The kit of the embodiment further comprises:
(1) Positive control: the concentration of the positive control substance of the GIV.1 type norovirus is 20-100mg/L, and preferably 50mg/L.
The positive control is obtained by the following method: sequencing and bioinformatic analysis of viral isolates from patients infected with norovirus type GIV.1, with NCBI database (https://www.ncbi.nlm.nihGov), the target sequence was determined to be the conserved region RdRp gene sequence (shown in SEQ ID NO: 7), the virus strain was delivered to Shanghai Bioengineering Co., ltd., the target gene was cloned into a PUC57 vector (Invitrogen, USA) and sequenced, and named Noro-GIV plasmid. The Noro-GIV plasmid was gel-purified with PstI restriction enzyme (Takara, japan), and used as a template, and In Vitro Transcription was performed using RiboMax T7 In Vitro Transcription System (Promega, usa), and DNA templates were completely degraded with Dnase enzyme reagent, and the obtained RNA products were purified with RNA purification kit (Qiagen), and the concentration was measured using NanoDrop, and the obtained RNA fragments were GIV-type norovirus positive control.
In addition, the method for obtaining the plasmid in the above process may also be: in NCBI database (https:// www.ncbi.nlm.nih.gov) Searching to obtain the conserved region RdRp gene sequence (shown as SEQ ID NO: 7) of the GIV type norovirus, synthesizing, cloning to a vector andsequencing to obtain a plasmid.
(2) 2 × reaction solution: comprises 40mmol/L Tris-HCl with pH8.8, 20mmol/L KCl,20mmol/L (NH) 4 ) 2 SO 4 Triton X-100 with mass concentration of 0.2%, dNTPs composed of dATP, dTTP, dCTP and dGTP with concentration of 2.8mmol/L, and betaine C with concentration of 1.6mmol/L 5 H 11 NO 2 And 16mmol/L MgSO 4 ；
(3) Enzyme solution: including BstDNA polymerase (large fragment, NEB, USA) at a concentration of 6-12U/. Mu.l (preferably 8U/. Mu.l), and AMV reverse transcriptase (NEB, USA) at a concentration of 8-16U/. Mu.l (preferably 10U/. Mu.l).
(4) Color development liquid: adding 0.5-1.0 mu L (preferably 0.7 mu L) of metal ion indicator HNB (hydroxynaphthol blue) storage solution with the concentration of 4mmol/L into a reaction system before reaction, thereby realizing uncapping visual detection. The indicator is available from Sigma-Alorich (Supelco) of U.S. having cat # 63451-35-4, cat # 1045930025; the preferred final concentration of the indicator after addition to the reaction system is 112. Mu. Mol/L.
Example 3: rapid detection method for non-diagnosis-purpose detection of GIV.1 type norovirus by RT-LAMP method
The detection method of the embodiment adopts the kit of the embodiment 2 for detection.
The detection method comprises the following steps:
firstly, adding a 2 Xreaction solution of a kit, a primer mixed solution, an enzyme solution, a color development solution and ultrapure water into a transparent reaction tube, and then mixing; the volume ratio of the primer mixture, the enzyme solution, the color development solution and the ultrapure water is 12.5;
for example, the reaction solution can be obtained as follows:
Figure BDA0002233780070000091
secondly, adding a nucleic acid sample to be detected into the reaction tube, covering the reaction tube, and uniformly mixing;
for example, 1. Mu.l of a nucleic acid sample to be tested is added to a reaction tube so that the final volumes of both reaction solutions are 25. Mu.l, and the mixture is mixed and centrifuged briefly.
The nucleic acid sample to be tested can be obtained by using an existing commercial viral nucleic acid EXtraction Kit (e.g., QIAamp MinElute Virus Spin Kit of QIAGEN, germany, miniBEST viral DNA/RNA EXtraction Kit of Takara, japan, and TIANAmp viral RNA EXtraction Kit of Tiangen, beijing) according to the Kit instructions of the manufacturer for 30 to 45 minutes. The extraction process should be carried out under strict biosafety protection conditions.
Thirdly, placing the reaction tube at the constant temperature of 62 ℃ for 60min to carry out RT-LAMP amplification, and then placing the reaction tube at the temperature of 85 ℃ for 5min to inactivate the enzyme, thus finishing the reaction;
in particular, the thermostated conditions may be provided by means of a water bath, a thermostated metal bath, or a nephelometer, for example.
Fourthly, observing the color of the reaction solution, wherein if the color of the reaction solution is sky blue, the nucleic acid sample to be detected contains the GIV.1 type norovirus; if the color of the reaction solution is violet, the nucleic acid sample to be detected does not contain the GIV.1 type norovirus.
This visualization step can also be replaced by scanning turbidity in real time using a turbidity meter. For example, the turbidity during the reaction was scanned in real time (wavelength: 650mm, measurement 1 time per 6 seconds) using a LA-500 turbidimeter (manufactured by Nippon Hippon Kagaku Co., ltd.), and the turbidity value exceeding 0.1 was judged to be positive. If the turbidity value of the reaction solution is more than 0.1, prompting that the nucleic acid sample to be detected contains the GIV.1 type norovirus; if the turbidity value is less than 0.1, the nucleic acid sample to be tested does not contain the GIV.1 type norovirus.
Example 4: kit detection method
The method of this example can test whether the kit of example 2 is acceptable.
The method comprises the following steps:
the first step, preparing two reaction tubes containing reaction liquid: taking a transparent reaction tube, adding the 2 multiplied reaction solution, the primer mixed solution, the enzyme solution and the color development solution in the kit, then adding ultrapure water and mixing to obtain a reaction solution with a predetermined volume;
specific formulation volumes are referred to in example 3.
Secondly, adding distilled water into the first reaction tube, covering the reaction tube and uniformly mixing the mixture to serve as negative control; adding a GIV.1 type norovirus positive control into a second reaction tube, covering the reaction tube, and uniformly mixing to serve as a positive control; the adding volumes of the distilled water and the positive control are the same;
for example, 1. Mu.l of distilled water or a positive control is added to the reaction tube so that the final volume of the reaction solution is 25. Mu.l.
And thirdly, placing the reaction tube at the constant temperature of 62 ℃ for 60min to carry out RT-LAMP amplification, and then placing the reaction tube at the temperature of 85 ℃ for 5min to inactivate the enzyme, thus finishing the reaction.
Fourthly, observing the colors of the negative control reaction liquid and the positive control reaction liquid, wherein if the color of the negative control reaction liquid is violet and the color of the positive control reaction liquid is sky blue, the kit used in the first step is qualified; if the color of the reaction solution does not completely match or completely does not match the above condition, the reagent kit used in the first step is not qualified, and the reagent kit needs to be replaced.
Example 5: kit detection sensitivity analysis
The concentration of the positive control of the norovirus type giv.1 in the kit of example 2 was determined using NanoDrop.
Then, 10-fold gradient dilution was carried out to obtain a concentration of 1.0X 10 0 copies/μl、1×10 1 copies/μl、1.0×10 2 copies/μl、1.0×10 3 copies/μl、1.0×10 4 copies/μl、1.0×10 5 copies/μl、1.0×10 6 copies/. Mu.l of positive control line dilution.
The serial dilutions of the above concentrations were used as the nucleic acid samples to be tested, and the color of the reaction solution was observed using the kit of example 2 according to the method of example 3.
The test results show that the concentration of the positive control for GIV.1 norovirus is greater than or equal to 1.0 × 10 2 The color of the reaction solution is sky blue, namely the detection limit (detection sensitivity) of the kit in the example 2 on the GIV.1 type norovirus can reach 1.0 multiplied by 10 2 copies/μl。
In order to verify the visual visualization result, an LA-320C turbidity meter is used at the same time, the kit in the embodiment 2 is adopted to carry out detection according to the method in the embodiment 3, the turbidity is scanned in real time, positive judgment is carried out when the turbidity value exceeds 0.1, and the result of the turbidity method is consistent with the visual visualization result.
FIG. 2 shows RT-LAMP which detects a gradient-diluted GIV.1 norovirus positive control by a nephelometry method, and the positive control is judged to be positive when the nephelometry value is more than 0.1.
FIG. 3 shows RT-LAMP color development method for detecting gradient diluted GIV.1 type norovirus positive control, determining sky blue as positive and violet as negative.
Example 6: kit for detecting specificity analysis
To verify the specificity of the kit in example 2, a GI type norovirus positive control, a GII type norovirus positive control, and a giv.1 type norovirus positive control are selected and detected by the kit in example 2 according to the method in example 3. This example uses LA-320C turbidimetry to observe experimental results. The results are shown in FIG. 4, only GIV.1 type norovirus was positive, GI type and GII type were both negative.
Alternatively, astrovirus, rotavirus, typhoid bacillus, shigella, salmonella and staphylococcus aureus which may have potential cross reactions or may cause similar intestinal symptoms are taken as templates, nucleic acids thereof are extracted, and then the detection is carried out by the method of example 3 by using the kit of example 2. This example uses LA-320C turbidimetry and color development to simultaneously observe the results. The results are shown in FIG. 5, which shows that each of viruses and bacteria except for the GIV.1 type norovirus are negative results, indicating that the kit of the present invention has good specificity.
In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the present invention.
Sequence listing
<110> center for preventing and controlling diseases of eastern war zone of people's liberation army of China
<120> RT-LAMP method detection primers, kit and detection method for GIV norovirus
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 1
aggtgatgag ggaccaactt 20
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 2
acgttgtctt gtcaagcga 19
<210> 3
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 3
tcttgactgc ggcctgcaag aacatttcac agctcctcgt 40
<210> 4
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 4
cgagaacgtg ctggaacaga ccctggcaag cctgtgcata t 41
<210> 5
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 5
ggggtttgac tggtctccc 19
<210> 6
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 6
attgatcccc caacaccatg gac 23
<210> 7
<211> 1530
<212> DNA
<213> Artificial Sequence
<400> 7
aacactgata acggcacgta ctgcggcgcc ccaatagtgt caaaaggtaa tgcaccccag 60
ctatcctcaa agacaaaatt ctggaggagc tctgtcgagc ctctcccgcc tggcacgttt 120
gagcccgcct acctcggggg tcgcgacccc agagtcgacg gtgggccctc actttaccag 180
gtgatgaggg accaacttaa acatttcaca gctcctcgtg ggagaccagt caaaccccac 240
ctcttgcagg ccgcagtcaa gacaatcgag aacgtgctgg aacagaccat tgatccccca 300
acaccatgga catatgcaca ggcttgccag tcgcttgaca agacaacgtc gagtggctgg 360
ccccaccatg tgcagaaaaa cacccattgg aatggtgaag catttacagg acccttggct 420
gaccaagcca gcaaggccaa tttaatgtat gaacaaggga agtccatgac cccccagtac 480
actgctgccc ttaaggatga actagtgaaa ccagacaagg tgtacaaaaa ggtcaagaaa 540
aggctgctgt ggggtgcaga tctcggaacc atggtccggt gcgccagagc attcggcccg 600
ttcactgacg cacttaagaa gtgctgcacc caacttccag tcaaagttgg actcaacatt 660
aatgaagagg gacccatcat ctttgagaaa cacgcccaat atgagctcca ctatgatgcg 720
gattattcgc ggtgggattc cacccagcaa cgggaggtcc ttgccgccgc acttggcatc 780
atgacaaaat tcactgctga gccacagctg gcctccgttg tggcagaaga cttgatttcc 840
ccaagtatgc ttgatgttgg cgactacgtc gtccaggtca atgaagggct accctctggt 900
gtcccctgca cctcacagtt aaacagtatt gcccattgga tcatcaccct cacatccatg 960
gctgaggcca ctggcctgga tcctgacatc gtccaggcta acagctactt ctccttctat 1020
ggtgatgatg aaattgtgtc aacagacata aaattcaatc cagaggtcct caccctgaaa 1080
cttaaagcaa ttggccttgt cccaacccgc ccagacaaga cagaaggccc attggtggtg 1140
tcaaacaaat tagaaggtct gacttttctg aggcgcacca taactagaga caaagtgggc 1200
ttctttggtc ggttggataa ggattccatt ctcaggcaaa tgtactggac caagggcccg 1260
aaccatcaag acccatctga aagcatgtta ccccaccaga atcgcgccac acaattgatg 1320
gccctgttgg gcgagtcagc actccatggt caaaacttct acaaaaagat tagtggcatg 1380
gtaatcaaag aagtgaagaa tggtgggcat gagttctatg tgccaaagtt tgagtccatg 1440
tacaagtgga tgcggttctc agacttgagc acttgggagg gggatcgcga tctcgctccc 1500
gattttgtga atgaagatgg cgtcgagtga 1530

Claims (9)

1. A complete set of primers for detecting the GIV.1 norovirus by the RT-LAMP method is characterized by comprising an outer primer pair GIV-F3 and GIV-B3, an inner primer pair GIV-FIP and GIV-BIP, and a loop primer pair GIV-LF and GIV-LB; the sequence of GIV-F3 is shown as SEQ ID NO. 1, the sequence of GIV-B3 is shown as SEQ ID NO. 2, the sequence of GIV-FIP is shown as SEQ ID NO. 3, the sequence of GIV-BIP is shown as SEQ ID NO. 4, and the sequence of GIV-LF is shown as SEQ ID NO. 5; the sequence of GIV-LB is shown in SEQ ID NO 6.
2. A kit for RT-LAMP detection of GIV.1 norovirus, characterized in that it comprises a primer mixture containing the set of primers of claim 1.
3. The kit of claim 2, wherein the molar ratio of the outer primer pair GIV-F3 and GIV-B3, the inner primer pair GIV-FIP and GIV-BIP, and the loop primer pair GIV-LF and GIV-LB is 1.
4. The kit according to claim 2, wherein the kit further comprises an enzyme solution, a 2 x reaction solution, a color developing solution and ultrapure water.
5. The kit according to claim 4, wherein the enzyme solution is composed of BstDNA polymerase at a concentration of 6-10U/. Mu.l, AMV reverse transcriptase at a concentration of 8-16U/. Mu.l, and water;
the 2 × reaction solution is prepared from 40mmol/L Tris-HCl with pH8.8, 20mmol/L KCl and 20mmol/L (NH) 4 ) 2 SO 4 Triton X-100 with mass concentration of 0.2%, dNTPs composed of dATP, dTTP, dCTP and dGTP with concentration of 2.8mmol/L, and betaine C with concentration of 1.6mmol/L 5 H 11 NO 2 16mmol/L MgSO 16 4 And water;
the color development liquid is metal ion indicator HNB storage liquid with the concentration of 4 mmol/L.
6. The kit of claim 2, wherein the kit further comprises a positive control for norovirus type giv.1 at a concentration of 20-100mg/L.
7. The kit of claim 6, wherein the concentration of the positive control for GIV.1 norovirus is 50mg/L and is obtained by: constructing a plasmid containing the RdRp gene of the GIV.1 type norovirus, carrying out in-vitro transcription reaction to obtain an RNA fragment of the RdRp gene, and purifying to obtain a positive control of the GIV.1 type norovirus; the sequence of the RdRp gene is shown as SEQ ID NO. 7.
8. A method for detecting GIV.1 norovirus RT-LAMP for non-diagnostic purposes, characterized in that a kit according to any one of claims 2 to 7 is used; the method comprises the following steps:
firstly, adding 2 Xreaction solution of a kit, primer mixed solution, enzyme solution, color development solution and ultrapure water into a transparent reaction tube, and then mixing to obtain reaction solution;
secondly, adding a nucleic acid sample to be detected into the reaction tube, covering the reaction tube, and uniformly mixing;
thirdly, placing the reaction tube under a constant temperature condition for RT-LAMP amplification, and then placing the reaction tube under a reaction ending condition to terminate the reaction;
fourthly, observing the color of the reaction solution, wherein if the color of the reaction solution is sky blue, the nucleic acid sample to be detected contains the GIV.1 type norovirus; if the color of the reaction solution is violet, the nucleic acid sample to be detected does not contain the GIV.1 type norovirus.
9. The detection method according to claim 8, wherein in the second step, after the nucleic acid sample to be detected is added, the volume ratio of the reagent kit 2 x the reaction solution, the primer mixture solution, the enzyme solution, the color-developing solution, the ultrapure water and the nucleic acid sample to be detected is 12.5:2.5:1:0.7:7.3:1, and the volume of the reaction solution in the reaction tube is 25 mul; in the third step, the RT-LAMP amplification conditions are as follows: standing at constant temperature of 62 deg.C for 60min; the conditions for finishing the reaction are as follows: standing at 85 deg.C for 5min.
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