CN107574261B - Detection primer, detection kit and detection method for detecting hantavirus - Google Patents
Detection primer, detection kit and detection method for detecting hantavirus Download PDFInfo
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Abstract
The invention discloses a detection primer for detecting hantavirus, which comprises the following components: the detection primers comprise detection primers of Hantaan virus, detection primers of Hancheng virus and detection primers of Primala virus. The invention also discloses a detection kit for detecting the hantavirus, which comprises detection primers of the hantavirus, detection primers of the hancheng virus and detection primers of the prama virus. The invention also discloses a detection method for detecting the hantavirus, which comprises the step of using the detection kit for detecting the hantavirus.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a detection primer, a detection kit and a detection method for detecting hantavirus.
Background
The hemorrhagic fever with renal syndrome (HFRS) is a serious infectious disease which is caused by Hantavirus (HV) and is transmitted by rodents (mainly rodents) and seriously harms the health of people, and the disease is an important epidemic area in our province. The clinical manifestations of HFRS are fever, bleeding and kidney damage, which are acute in onset, serious in illness and high in death rate. The incidence and the death rate of the HFRS are high, and the HFRS is continuously positioned in the first ten infectious diseases in China for many years. Therefore, early diagnosis and early treatment of HFRS are crucial.
There are four types of HV that cause HFRS: hantavirus (HTNV), hancheng virus (SEOV), pomela virus (PUUV) and dobrav-beergard virus (DOBV). For years, HTNV and SEOV which are main hazards in China are developed, and Chinese scholars have conducted intensive research on diagnosis and treatment of the two subtype viruses. PUUV is only popular in Europe and the far east of Russia in the past, and the popularity of PUUV is not existed in China. PUUV is found in the bodies of rats in the region of the joss area in Jilin province for the first time in 2003, which indicates that PUUV is introduced into China, but no PUUV is found to be infected by people at present. However, in recent years, a phenomenon has become noticeable, and clinical studies have shown that: the clinical symptoms of new HFRS patients in a plurality of areas (areas such as Changchun, Jilin, Tonghua and the like) in Jilin province show an atypical change trend, which is specifically manifested as unclear disease stages (fever stage, hypotension shock stage, oliguria stage, diuresis stage and recovery stage) (direct transition of part of patients from fever stage to oliguria stage and even diuresis stage), atypical clinical symptoms (fever, bleeding and kidney damage) (lack of typical symptoms of three red and three pain), serious harm and poor prognosis of patients, are very similar to the clinical manifestations of PUUV infection, so that the existence of PUUV cannot be ignored. The general susceptibility of the population in China to PUUV is realized, but no specific detection means exists at present, no effective vaccine prevention exists, and once PUUV is transmitted in the population, serious consequences can be caused. Therefore, there is a need to develop a specific PUUV detection method, which enhances the detection strength of PUUV and provides basis and guarantee for prevention and treatment of HFRS.
Laboratory tests for HV are currently mainly serological and nucleic acid detection methods. Serological methods have low sensitivity, poor specificity, easy omission and failure of early diagnosis and treatment. The nucleic acid detection method is mainly a real-time fluorescence quantitative PCR method, needs special instruments, and has high cost, long time consumption and difficult observation of results. The loop-mediated isothermal amplification (RT-LAMP) is a novel nucleic acid isothermal amplification technology, can rapidly, sensitively, efficiently and specifically amplify target genes under a constant temperature condition, and has the advantages of high sensitivity, strong specificity, short reaction time, convenient result interpretation, simple equipment requirement and the like. The detection result of 300 foot-and-mouth disease virus samples by Yamazaki and the like by adopting an RT-LAMP method shows that the sensitivity and the specificity of the method are respectively 98.0 percent and 98.1 percent, and false detection does not occur. The RT-LAMP method is used for detecting the virus, the operation is simple and convenient, suspected pathological tissue can be directly taken for gene amplification, and the complicated nucleic acid extraction step of the conventional PCR detection is omitted. The minimum detection amount is lower than that of the conventional PCR method, the sensitivity is nearly one order higher than that of the PCR, and the method is more suitable for diagnosing the early stage of virus infection and plays an increasingly important role in clinical early diagnosis.
Disclosure of Invention
The invention designs and develops a detection primer for detecting hantavirus, and aims to provide the detection primer which has accurate result, strong specificity, high sensitivity and quick and simple detection.
The invention designs and develops a detection kit for detecting hantavirus, and aims to provide the detection kit for detecting hantavirus.
The invention designs and develops a detection method for detecting hantavirus, and aims to provide a detection method for detecting hantavirus.
The technical scheme provided by the invention is as follows:
the detection primers are used for detecting hantavirus, and comprise detection primers of hantavirus, detection primers of hancheng virus and detection primers of pramalavirus;
the detection primer of the hantaan virus consists of nucleotides with base sequences shown by SEQ ID NO.1 in a sequence table to SEQ ID NO.4 in the sequence table; wherein, SEQ ID NO.1 is an upstream outer primer, SEQ ID NO.2 is a downstream outer primer, SEQ ID NO.3 is an upstream inner primer, and SEQ ID NO.4 is a downstream outer primer;
the detection primer of the Hancheng virus consists of nucleotides with base sequences shown by SEQ ID NO.5 in a sequence table to SEQ ID NO.8 in the sequence table; wherein, SEQ ID NO.5 is an upstream outer primer, SEQ ID NO.6 is a downstream outer primer, SEQ ID NO.7 is an upstream inner primer, and SEQ ID NO.8 is a downstream outer primer;
the detection primer of the promela virus consists of nucleotides with base sequences shown by SEQ ID NO.9 in a sequence table to SEQ ID NO.12 in the sequence table; wherein, SEQ ID NO.9 is an upstream outer primer, SEQ ID NO.10 is a downstream outer primer, SEQ ID NO.11 is an upstream inner primer, and SEQ ID NO.12 is a downstream outer primer.
The detection kit for detecting the hantavirus uses the detection primer for detecting the hantavirus.
Preferably, the kit further comprises a DNA polymerase, a template DNA, ddH2O, an indicator.
Preferably, the indicator is hydroxynaphthol blue.
The detection method for detecting the hantavirus uses the detection kit for detecting the hantavirus to detect by LAMP reaction.
Preferably, the method comprises the following steps:
step one, extracting plasmids of a sample to be detected;
step two, configuring an LAMP reaction system; the reaction system comprises detection primers of Hantaan virus, detection primers of Hancheng virus and detection primers of Primala virus;
performing amplification reaction, and detecting a sample to be detected; wherein, the color of the reaction system under natural light is changed from lake blue to sky blue, and the detection result is judged to be positive.
Preferably, the LAMP reaction system comprises: primer mixture, reaction buffer, DNA template, DNA polymerase and indicator.
Preferably, the LAMP reaction system is incubated at the constant temperature of 61-65 ℃ for 1 hour, and inactivated at 80 ℃ for 2 minutes after the reaction is finished; and
the indicator is hydroxynaphthol blue, and the set concentration is 100-200 mu mol/L.
Preferably, the sample to be tested has a concentration of 10-7～100Gradient copy number.
Compared with the prior art, the invention has the following beneficial effects:
1. the result is accurate, and the detection accuracy of the Hantaan virus, the Hancheng virus and the Primala virus can reach 100 percent;
2. the specificity is strong, only three subtypes, namely hantaan virus, hancheng virus and pumara virus can be detected, and Sinkiang hemorrhagic fever virus, Q fever virus, Marburg virus and Ebola virus which are related to the hantaan virus cannot be detected;
3. high sensitivity, and a detectable amount of 100～10-9；
4. The method is rapid and simple, and the result can be observed by naked eyes without electrophoretic detection (namely, the color is changed from lake blue to sky blue under natural light).
Drawings
FIG. 1 is a diagram showing the results of the 3% agarose gel electrophoresis of the present invention. (in the figure, M is a molecular weight marker, 1 is HTNV, 2 is SEOV, 3 is PUUV, 4 is XHFV, 5 is Marburg, 6 is Q Fever, 7 is Ebola, and 8 is a negative control).
FIG. 2 is a diagram showing the results of 3% agarose gel electrophoresis detection of the HTNV plasmid of the present invention. (in the figure, M is a molecular weight marker, 1-10 are 100～10-9Results of different gradient copy number detection).
FIG. 3 is a diagram showing the results of 3% agarose gel electrophoresis detection of the SEOV plasmid of the present invention. (in the figure, M is a molecular weight marker, 1-10 are 100～10-9Results of different gradient copy number detection).
FIG. 4 is a diagram showing the results of 3% agarose gel electrophoresis detection of the PUUV plasmid of the present invention. (in the figure, M is a molecular weight marker, 1-10 are 100～10-9Results of different gradient copy number detection).
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
Example 1 detection primer design and Synthesis
1. Material
LAMP DNA amplification kit, plasmid extraction kit, DNA Marker, Hantaan virus (HTNV), Hantaan virus (SEOV), pumara virus (PUUV), Xinjiang Hemorrhagic Fever Virus (XHFV), Q Fever virus (Q Fever), Marburg virus (Marburg) and Ebola virus (Ebola) plasmids are purchased from Shanghai (Producer) Biotech limited company.
2. Primer design and Synthesis
The invention provides a detection Primer group (comprising an upstream outer Primer (F3) of SEQ ID NO.1, a downstream outer Primer (B3) of SEQ ID NO.2, an upstream inner Primer (FIP) of SEQ ID NO.3, a downstream inner Primer (BIP) of SEQ ID NO. 4), a detection Primer group (comprising an upstream outer Primer (F3) of SEQ ID NO.5, a downstream outer Primer (B3) of SEQ ID NO.6, an upstream inner Primer (FIP) of SEQ ID NO.7, a downstream inner Primer (BIP) of SEQ ID NO.8 and a detection Primer group (PUUV) of SEQ ID NO. 78) of SEQ ID NO. 9) of Hantaan virus (HTNV) (comprising an upstream outer Primer (F3) of SEQ ID NO.1, a downstream outer Primer (B3) of SEQ ID NO. 2) of a sequence list), primers designed by using Primer Explorer V4 software according to a conserved region of HTNV (GenBank: KT885049.1), SEOV (GenBank), a detection Primer group (comprising an upstream outer Primer (PUUV) of SEQ ID NO. 78) of a detection Primer group of SEQ ID NO.9, The downstream outer primer (B3) of SEQ ID NO.10, the upstream inner primer (FIP) of SEQ ID NO.11, and the downstream inner primer (BIP) of SEQ ID NO. 12) to obtain a primer set, which was synthesized by a solid phase phosphoramidite triester method, the primer set being shown in tables 1 to 3.
TABLE 1HTNV detection primer sequences
TABLE 2SEOV test primer sequences
TABLE 3PUUV detection primer sequences
2. Detection kit for detection of LAMP of hantavirus: the method comprises the following steps: detection primer sets of Hantavirus (HTNV) (including an upstream outer primer (F3) of SEQ ID No.1, a downstream outer primer (B3) of SEQ ID No.2, an upstream inner primer (FIP) of SEQ ID No.3, a downstream inner primer (BIP) of SEQ ID No. 4) of hantavirus (SEOV)), detection primer sets of hantavirus (SEOV) (including an upstream outer primer (F3) of SEQ ID No.5, a downstream outer primer (B3) of SEQ ID No.6, an upstream inner primer (FIP) of SEQ ID No.7, a downstream inner primer (BIP) of SEQ ID No. 8) and a detection primer set of PUUV) (including an upstream outer primer (F3) of SEQ ID No.9, a downstream outer primer (B3) of SEQ ID No. 10), an upstream primer (FIP) of SEQ ID No.11, a downstream inner primer (BIP) of SEQ ID No. 12) of the sequence listing)); a DNA polymerase; template DNA; ddH2O; color indicator hydroxynaphthol blue (HNB).
3. Procedure of experiment
(1) Plasmid extraction
Step one, carrying out plasmid extraction on a plasmid extraction box, adding 1-5 ml of overnight cultured bacterial liquid into a centrifuge tube, centrifuging for 1min at 12000rpm (about 13400 Xg) by using a conventional desktop centrifuge, and absorbing the supernatant as much as possible (when the bacterial liquid is more, collecting bacterial precipitates into one centrifuge tube through multiple times of centrifugation);
step two, adding 250 mul of solution P1 into the centrifugal tube with the bacterial sediment, and completely suspending the bacterial sediment by using a pipette or a vortex oscillator; wherein the solution P1 is a mixed solution consisting of 50mM sucrose reagent, 25mM Tris-HCl buffer reagent with the pH value of 8.0 and 10mM EDTA;
step three, adding 250 mul of solution P2 into the centrifuge tube, and gently turning the centrifuge tube up and down for 6-8 times to fully crack the thalli; wherein the solution P2 is a mixed solution consisting of 0.2N NaOH and 1% SDS by mass fraction;
step four, adding 350 mu l of solution P3 into the centrifuge tube, immediately and gently turning up and down for 6-8 times, and fully mixing uniformly, wherein white flocculent precipitates appear; using a centrifuge, setting the rotating speed to be 12000rpm (-13400 Xg), and centrifuging for 10 minutes; wherein, the solution P3 is 3M potassium acetate solution, and the solution preparation process comprises the following steps: using 300ml of 5M potassium acetate reagent, 57.5ml of acetic acid solution was added, and ddH was further added2Diluting O to 500ml, and storing in an environment at 4 ℃ for later use;
transferring the supernatant collected in the last step into an adsorption column CP3 (the adsorption column is placed into a collection tube) by using a pipette, paying attention to the fact that the precipitate is not sucked out as much as possible, setting the rotation speed to be 12000rpm (13400 Xg) for centrifugation for 30-60 sec, pouring the waste liquid in the collection tube, and placing an adsorption column CP3 into the collection tube;
step six, optional steps: adding 500 mu l of deproteinized liquid PD into an adsorption column CP3, setting the rotating speed to be 12000rpm (-13400 Xg), centrifuging for 30-60 sec, pouring waste liquid in a collecting tube, and putting the adsorption column CP3 back into the collecting tube again;
step seven, adding 600 mul of rinsing liquid PW (please check whether absolute ethyl alcohol is added or not) into the adsorption column CP3, setting the rotating speed to be 12000rpm (13400 Xg) for centrifuging for 30-60 sec, pouring the waste liquid in the collecting pipe, and putting the adsorption column CP3 into the collecting pipe;
step eight, repeating the operation step seven;
putting the adsorption column CP3 into a collecting pipe, and centrifuging at 12000rpm (-13400 Xg) for 2min to remove the residual rinsing liquid in the adsorption column;
and step ten, placing the adsorption column CP3 in a clean centrifuge tube, dropwise adding 50-100 mu l of elution buffer EB into the middle part of the adsorption membrane, placing for 2min at room temperature, centrifuging for 2min at 12000rpm (13400 Xg), and collecting the plasmid solution in the centrifuge tube.
(2) LAMP reaction system
Step one, dissolving 2 × Lamp Master Mix and primers on ice, fully mixing, and then establishing a reaction system as shown in Table 4:
TABLE 4 reaction System
Step two, fully and uniformly mixing the reaction tubes, and then placing the mixture in a 65 ℃ water bath kettle for heat preservation for 30-60 min;
step three, inactivating the DNA Polymerase at 80 ℃ for 10 min;
step four, the reaction product can be detected by adopting the following methods:
hydroxynaphthol blue (HNB) method: adding a color indicator HNB into the LAMP reaction system, observing the color of the reaction tube before reaction by naked eyes to be lake blue, and indicating that the color of the LAMP reaction tube is changed into sky blue, wherein the existence of an amplification product is indicated; otherwise, no amplification is indicated;
agarose electrophoresis: 3-5 mul of amplification product is taken, and 1% agarose electrophoresis detection is carried out, so that LAMP characteristic maps consisting of color bands with different sizes can be seen.
4. Establishment of LAMP reaction conditions
(1) Indicator (HNB) concentration selection: adding a color indicator HNB (the color of the reaction tube before reaction is lake blue, and the color of the reaction tube after LAMP reaction is changed into sky blue) into the LAMP reaction system, setting 5 concentrations of 100, 125, 150, 175, 200 mu mol/L and the like, and determining the appropriate HNB concentration by taking the color difference as the standard after the LAMP reaction.
(2) Building an LAMP reaction system and optimizing temperature: LAMP reaction System (25. mu.L): primer mixture 5.0. mu.L (containing 0.8. mu.M each of F3 and B3 and 0.2. mu.M each of BIP and FIP), Master Mix 12.5. mu.L, DNA template 0.5. mu.L (containing 0.16U/. mu.L of DNA), HNB 1.0. mu.L (reaction concentration 150. mu. mol/L), ddH2O5. mu.L, DNA polymerase 1.0. mu.L; preparing 5 identical reaction systems, respectively incubating at 61, 62, 63, 64, and 65 deg.C for 1h, and inactivating at 80 deg.C for 2 min; the temperature selection is based on the amplification efficiency and the reaction time, and whether the color change result is consistent with the results of electrophoresis and LAMP turbidimeter is observed.
The reaction condition optimization result is as follows: experiments prove that the LAMP amplification efficiency is highest when the temperature is 64 ℃, the time is 60 minutes and the HNB concentration is 125 mu mol/L, the reaction process is shortest, the color change is most obvious, and the color is changed from lake blue to sky blue.
Example 3 specificity and sensitivity assays for Hantaan Virus detection kits
1. Specificity verification
Respectively taking the extracted HTNV, SEOV, PUUV, XHFV, Q Fever, Marburg and Ebola plasmids as templates, respectively taking the HTNV, SEOV and PUUV primers F3, B3, FIP and BIP to perform loop-mediated isothermal amplification, verifying the specificity of the method, and observing whether the color change is consistent with the determination result of a gel electrophoresis apparatus.
And (3) specific verification results: as shown in FIG. 1, HTNV, SEOV, PUUV, XHFV, Q Fever, Marburg and Ebola plasmids are respectively used as templates, and HTNV, SEOV and PUUV primers are respectively used for LAMP, and FIG. 2 shows that HTNV, SEOV and PUUV reaction tubes in different experimental groups are respectively changed from lake blue to sky blue, and XHFV, Q Fever, Marburg, Ebola and negative control are not changed in color, which indicates good specificity, and the result of gel electrophoresis detection (shown in FIG. 1) is consistent with that of HNB indicator.
2. Sensitivity verification
HTNV, SEOV and PUUV plasmids were diluted to 1mg/mL and 10 times0～10-9HTNV, SEOV and PUUV primers F3, B3, FIP and BIP are respectively used for loop-mediated isothermal amplification to evaluate the sensitivity of the method.
The result of the sensitivity verification: HTNV, SEOV and PUUV plasmids were diluted to 1mg/mL and 10 times0～10-9When the copy number of 10 gradients and the LAMP reaction result are obtained, the three viruses 10-9～100Color changes appear in the copy reaction tube, which shows that the sensitivity of the copy reaction tube can reach 10-9FIGS. 2-4 show that the gel electrophoresis detection results are consistent with the HNB indicator results.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
SEQUENCE LISTING
<110> Jilin medical college
<120> detection primer, detection kit and detection method for detecting hantavirus
<160> 12
<170> PatentIn version 3.3
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<211> 23
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<213> Artificial sequence
<220>
<223> an upstream outer primer artificially synthesized to be used as a coding sequence of a detection primer for obtaining a hantaan virus
<400> 1
gcactattat tatcagggga atc 23
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> downstream outer primer artificially synthesized for use as coding sequence of detection primer for obtaining Hantaan virus
<400> 2
aagaaaggga aagaaagacg aa 22
<210> 3
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223> an upstream inner primer artificially synthesized to be used as a coding sequence of a detection primer for obtaining a hantaan virus
<400> 3
taactgaccc acccctgagt gtatatcagg gcatgggaac 40
<210> 4
<211> 44
<212> DNA
<213> Artificial sequence
<220>
<223> downstream inner primer artificially synthesized for use as coding sequence of detection primer for obtaining Hantaan virus
<400> 4
atccgttgtg ggtgggttta ggatcaaatg aaatctacat ccat 44
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> an upstream outer primer artificially synthesized to be used as a coding sequence of a detection primer for obtaining Hancheng virus
<400> 5
atcataagca caatcactgc 20
<210> 6
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223> downstream outer primer artificially synthesized to be used as coding sequence of detection primer for obtaining Hancheng virus
<400> 6
atgaggtagt atatattgag gtaga 25
<210> 7
<211> 38
<212> DNA
<213> Artificial sequence
<220>
<223> an upstream inner primer artificially synthesized to be used as a coding sequence of a detection primer for obtaining Hancheng virus
<400> 7
taaggtgacc tggccctaag atcacgggta tacgggtg 38
<210> 8
<211> 44
<212> DNA
<213> Artificial sequence
<220>
<223> downstream inner primer artificially synthesized to be used as coding sequence of detection primer for obtaining Hancheng virus
<400> 8
tcgaatacta atcctactgt cctctaggtt gtgaggtagt ttgt 44
<210> 9
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223> an upstream outer primer artificially synthesized to be used as a coding sequence of a detection primer for obtaining a Primala virus
<400> 9
aagctgaatt cttaaagagg aat 23
<210> 10
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> an outer primer synthesized by the same method as a downstream primer for obtaining a sequence encoding a detection primer for a Primala virus
<400> 10
acctagttct gccattcc 18
<210> 11
<211> 47
<212> DNA
<213> Artificial sequence
<220>
<223> an upstream inner primer artificially synthesized to be used as a coding sequence of a detection primer for obtaining a Primala virus
<400> 11
atcagcttat caatgtcagc tacatagata tactttatgc agcgtca 47
<210> 12
<211> 44
<212> DNA
<213> Artificial sequence
<220>
<223> downstream inner primer artificially synthesized to be used as coding sequence of detection primer for obtaining the virus of Primala
<400> 12
tattgagtct cctaatgcac cctgcaacat aaatgcatgt tggt 44
Claims (4)
1. The detection primers for detecting the hantavirus are characterized by comprising detection primers of the hantavirus, detection primers of the hancheng virus and detection primers of the prama virus;
the detection primer of the hantaan virus consists of nucleotides with base sequences shown by SEQ ID NO.1 in a sequence table to SEQ ID NO.4 in the sequence table; wherein, SEQ ID NO.1 is an upstream outer primer, SEQ ID NO.2 is a downstream outer primer, SEQ ID NO.3 is an upstream inner primer, and SEQ ID NO.4 is a downstream outer primer;
the detection primer of the Hancheng virus consists of nucleotides with base sequences shown by SEQ ID NO.5 in a sequence table to SEQ ID NO.8 in the sequence table; wherein, SEQ ID NO.5 is an upstream outer primer, SEQ ID NO.6 is a downstream outer primer, SEQ ID NO.7 is an upstream inner primer, and SEQ ID NO.8 is a downstream outer primer;
the detection primer of the promela virus consists of nucleotides with base sequences shown by SEQ ID NO.9 in a sequence table to SEQ ID NO.12 in the sequence table; wherein, SEQ ID NO.9 is an upstream outer primer, SEQ ID NO.10 is a downstream outer primer, SEQ ID NO.11 is an upstream inner primer, and SEQ ID NO.12 is a downstream outer primer.
2. A detection kit for detecting Hantaan virus, wherein the detection primer for detecting Hantaan virus according to claim 1 is used.
3. The test kit for detecting hantavirus of claim 2, wherein the kit further comprises a DNA polymerase、ddH2O and an indicator.
4. The detection kit for detecting hantavirus according to claim 3, wherein the indicator is hydroxynaphthol blue.
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