CN106939356B - Detection primer group, detection kit and detection method for rapidly detecting bee filovirus - Google Patents
Detection primer group, detection kit and detection method for rapidly detecting bee filovirus Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a detection primer group, a detection kit and a detection method for rapidly detecting bee filovirus. The invention designs and screens a specific detection primer group, a detection kit containing the detection primer group and a detection method for determining whether the bee filovirus exists in a sample to be detected by using the detection kit through loop-mediated isothermal amplification aiming at the specific target gene of the bee filovirus ATPase. The detection kit and the detection method have the advantages of high sensitivity, strong specificity, good accuracy and short detection time, only take 4 hours from sample treatment to report results, do not need a PCR instrument and an electrophoresis instrument, have simple operation process and have higher specificity than other PCR technologies. Is particularly suitable for bee pathogen investigation and disease prevention and control.
Description
The technical field is as follows:
the invention belongs to the field of biological detection, and particularly relates to a detection primer group, a detection kit and a detection method for rapidly detecting bee filovirus.
Background art:
the Italian bee filovirus AmFV (AmFV) is the only dsDNA Virus, the genome of the AmFV reaches 498500bp, the genome DNA and nucleoprotein are folded and cyclized to form a rod-shaped Virus with the length of 450 × 170nm, and the rod-shaped Virus is similar to the baculovirus of invertebrate according to genome analysis.
The LAMP technology is a novel nucleic acid amplification technology developed by Notomi and the like in the past 2000, has short detection time, high sensitivity and specificity and simple operation, and can complete detection only by a common water bath. In recent years, the kit has been widely used for detecting various pathogens. It is characterized by that it designs 4-6 specific primers for 6 regions of target gene to be tested, and under the action of BstDNA polymerase it can make specific high-effective quick nucleic acid amplification in the isothermal condition of about 65 deg.C within 1h, and can be up to 109The copy and amplification result can be directly observed by magnesium pyrophosphate precipitation or judged by adding a color developing agent. AmFV virus is the only double-stranded DNA bee virus discovered at present, and the research and development of related LAMP technology are not available.
The invention content is as follows:
the invention aims to provide a detection primer group, a detection kit and a detection method for rapidly detecting the bee filovirus, which have the advantages of rapidness, good specificity and high sensitivity.
The first purpose of the invention is to provide a detection primer group for detecting the bee filovirus by using loop-mediated isothermal amplification, which is characterized by comprising the following primers:
the upstream outer primer F3: 5'-TGGATCGTCGGGATCGTC-3' (shown in SEQ ID NO. 1);
downstream outer primer B3: 5'-AACTCGGGAAACAGTGGC-3' (shown in SEQ ID NO. 2);
an upstream inner primer FIP: 5'-GTGACTCAAACAGACTCGCGGTCTGCTTCATCTGAACCGTCT-3' (shown as SEQ ID NO. 3);
the downstream inner primer BIP: 5'-GCGGGACCTCCTATTTCGCTGGTCAACTTGGCCCGTAACC-3' (shown in SEQ ID NO. 4).
The second object of the present invention is to provide a method for detecting a bee filovirus for non-disease diagnosis and treatment purposes, which comprises extracting total DNA from a sample, and selectively amplifying the sample DNA by the loop-mediated isothermal amplification method using the above detection primer set to confirm the presence or absence of an amplification product.
The sample is preferably a bee or bee product. The bee can be larva, pupa, imago or tissue thereof. The bee product can be honeycomb material, pollen, etc.
The selective amplification of the sample DNA by using the detection primer group through the method of loop-mediated isothermal amplification is specifically that the loop-mediated isothermal amplification system has the total volume of 25 mu L and comprises 2.5 mu L of 10 × Thermopol reaction buffer solution, 0.4 mu L of 10mM dNTPs, 0.5 mu L of 10 mu M upstream outer primer F3, 0.5 mu L of 10 mu M downstream outer primer B3, 1 mu L of 40 mu M upstream inner primer FIP, 1 mu L of 40 mu M downstream inner primer BIP and 0.6 mu L of 100mM MgSO 100412.5 mu L of 2M betaine, 4 mu L of sterilized double distilled water, 1 mu L of 8U/mu L Bst DNA polymerase and 1 mu L of sample DNA template, wherein the reaction condition is incubation for 45-60 min at the temperature of 60-65 ℃.
And (3) determining whether the amplification product exists or not by utilizing electrophoresis detection, fluorescence color development detection or turbidity detection, preferably performing fluorescence color development detection, specifically stopping the reaction at 80 ℃ for 1-2min, adding SYBR Green I color developing agent into an amplification reaction tube, and observing the result after 1-5 min, wherein if the color is orange, the sample bee filovirus is negative, no bee filovirus exists, and if the color is Green, the sample bee filovirus is positive and contains bee filovirus.
The 10 × Thermopol reaction buffer is a material of the prior art, commercially available from reagent companies, and contains 200mmol/L Tris-HCl (pH8.8), 100mmol/L KCl (KCl), and 100mmol/L ammonium sulfate ((NH)4)2SO4) 20mmol/L magnesium sulfate (MgSO)4) And 1% Triton X-100 (Tttonx-100), the balance being water.
The third purpose of the invention is to provide a detection kit for the bee filovirus, which comprises a loop-mediated isothermal amplification reagent and a detection primer group, and is characterized in that the detection primer group consists of the following primers:
the upstream outer primer F3: 5'-TGGATCGTCGGGATCGTC-3' (shown in SEQ ID NO. 1);
downstream outer primer B3: 5'-AACTCGGGAAACAGTGGC-3' (shown in SEQ ID NO. 2);
an upstream inner primer FIP: 5'-GTGACTCAAACAGACTCGCGGTCTGCTTCATCTGAACCGTCT-3' (shown as SEQ ID NO. 3);
the downstream inner primer BIP: 5'-GCGGGACCTCCTATTTCGCTGGTCAACTTGGCCCGTAACC-3' (shown in SEQ ID NO. 4).
The invention designs and screens a specific detection primer group, a detection kit containing the detection primer group and a detection method for determining whether the bee filovirus exists in a sample to be detected by using the detection kit through loop-mediated isothermal amplification aiming at the ATPase specific target gene of the bee filovirus. The detection kit and the detection method have the advantages of high sensitivity, strong specificity, good accuracy and short detection time, only take 4 hours from sample treatment to report results, do not need a PCR instrument and an electrophoresis instrument, have simple operation process and have higher specificity than other PCR technologies. Is particularly suitable for bee pathogen investigation and disease prevention and control.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
1. Bee tissue DNA extracting solution
The bee tissue DNA extracting solution is a purchased insect tissue DNA extracting kit of KAPA company.
2. LAMP rapid detection series reagent
(1) The loop-mediated isothermal amplification reaction solution contained 10 × Thermopol reaction buffer, 300. mu.M dNTPs (mixture of four deoxynucleotides), 4-6mL magnesium sulfate (MgSO 2)4) 0.1-0.3. mu.M outer primer (F3/B3), 1-2. mu.M inner primer (FIP/BIP), 0.8-1M betaine.
Wherein the 10 × Thermopol buffer solution contains 200mM Tris-HCl (pH8.8), 100mM KCl (KCl), and 100mM ammonium sulfate ((NH)4)2SO4) 20mM magnesium sulfate (MgSO)4) And 1% Triton X-100 (Tttonx-100), the balance being water.
Detection primer set:
upstream outer primer F3(5 '-3'): TGGATCGTCGGGATCGTC (shown in SEQ ID NO. 1);
downstream outer primer B3(5 '-3'): AACTCGGGAAACAGTGGC (shown in SEQ ID NO. 2);
upstream inner primer FIP (5 '-3'): GTGACTCAAACAGACTCGCGGTCTGCTTCATCTGAACCGT CT (shown in SEQ ID NO. 3);
downstream inner primer BIP (5 '-3'): GCGGGACCTCCTATTTCGCTGGTCAACTTGGCCCGTAACC (shown as SEQ ID NO. 4).
(2) Bst DNA polymerase: each microliter containing 8 units of activity (8U/. mu.L)
(3) Color developing agent: 10% of fluorescent dye SYBR GREEN I
The optimal composition of 23. mu.L of the loop-mediated isothermal amplification (LAMP) reaction solution per tube is 2.5. mu.L of 10 × Thermopol reaction buffer, 0.4. mu.L of 10mM dNTPs, 0.5. mu.L of 10. mu.M upstream outer primer F3, and 0.5. mu.L of 10. mu.M downstream outer primer F3Primer B3, 1. mu.L of 40. mu.M upstream inner primer FIP, 1. mu.L of 40. mu.M downstream inner primer BIP, 0.6. mu.L of 100mM MgSO412.5. mu.L of 2M betaine, 4. mu.L of sterilized double distilled water ddH2O。
Example 1: detection of AmFV in Apis italica larva tissue
1. Apis italica larva tissue treatment
10mg of Italian bee larva tissue was taken and mashed.
2. Extraction of sample tissue DNA
Adding 50 mu L of insect tissue DNA extracting solution into the sample, mixing uniformly, carrying out water bath at 95 ℃ for 10min, centrifuging at 12000r/min for 5min, and taking the supernatant for later use to obtain the template DNA to be detected.
3. LAMP amplification
25 μ L of loop-mediated isothermal amplification (LAMP) reaction solution per tube comprises 2.5 μ L of 10 × Thermopol reaction buffer, 0.4 μ L of 10mM dNTPs, 0.5 μ L of 10 μ M upstream outer primer F3, 0.5 μ L of 10 μ M downstream outer primer B3, 1 μ L of 40 μ M upstream inner primer FIP, 1 μ L of 40 μ M downstream inner primer BIP, and 0.6 μ L of 100mM MgSO 100. mu.M412.5 uL of 2M betaine, 1 uL of 8U/. mu.L Bst DNA polymerase, 1 uL of template DNA to be detected and 4 uL of sterilized double distilled water ddH2And O, incubating for 60min at the temperature of 60 ℃ in a constant-temperature water bath, terminating the reaction for 1-2min at the temperature of 80 ℃, and taking out the reaction tube.
No DNA was added as a negative control, and DNA from Italian bee larva tissue carrying AmFV virus was added as a positive control.
4. Visualization of color development agent
Adding 1 mu L of color-developing agent (10% SYBR Green I color-developing agent) into the reaction tube, directly observing the color change by naked eyes, wherein the color of the reaction tube of the sample to be detected is changed into Green, the color of the reaction tube of the negative control is changed into orange, and the color of the reaction tube of the positive control is changed into Green, thereby indicating that the sample to be detected (bee tissue) contains AmFV. The result is consistent with the sequencing result of the common PCR product, and the data of the method is proved to be reliable.
Example 2: AmFV detection of healthy bee samples without pathological symptoms
1. Apis italica larva tissue treatment
10mg of Italian bee larva tissue was taken and mashed.
2. Extraction of sample tissue DNA
Adding 50 mu L of insect tissue DNA extracting solution into the sample, mixing uniformly, carrying out water bath at 95 ℃ for 10min, centrifuging at 12000r/min for 5min, and taking the supernatant for later use to obtain the template DNA to be detected.
3. LAMP amplification
25 μ L of loop-mediated isothermal amplification (LAMP) reaction solution per tube comprises 2.5 μ L of 10 × Thermopol reaction buffer, 0.4 μ L of 10mM dNTPs, 0.5 μ L of 10 μ M upstream outer primer F3, 0.5 μ L of 10 μ M downstream outer primer B3, 1 μ L of 40 μ M upstream inner primer FIP, 1 μ L of 40 μ M downstream inner primer BIP, and 0.6 μ L of 100mM MgSO 100. mu.M412.5 uL of 2M betaine, 1 uL of 8U/. mu.L Bst DNA polymerase, 1 uL of template DNA to be detected and 4 uL of sterilized double distilled water ddH2And O, incubating for 60min at the temperature of 60 ℃ in a constant-temperature water bath, terminating the reaction for 1-2min at the temperature of 80 ℃, and taking out the reaction tube.
No DNA was added as a negative control, and DNA from Italian bee larva tissue carrying AmFV virus was added as a positive control.
4. Visualization of color development agent
Adding 1 mu L of color-developing agent (10% SYBR Green I color-developing agent) into the reaction tube, directly observing the color change by naked eyes, wherein the color of the reaction tube of the sample to be detected is changed into Green, the color of the reaction tube of the negative control is changed into orange, and the color of the reaction tube of the positive control is changed into Green, thereby indicating that the sample to be detected (bee tissue) contains AmFV. The result is consistent with the sequencing result of the common PCR product, and the data of the method is proved to be reliable.
Example 3: experiment of specificity
Collecting 17 parts of bee samples (other bee viruses are shown in table 1) carrying the AmFV and not carrying the AmFV but carrying other bee viruses, respectively taking 10mg of corresponding bee sample tissues, mashing, adding 50 mu L of insect tissue DNA extracting solution, mixing uniformly, carrying out water bath at 95 ℃ for 10min, centrifuging at 12000r/min for 5min, and taking the supernatant for later use to obtain the template DNA to be detected.
25 μ L of loop-mediated isothermal amplification (LAMP) reaction solution, each tube, consists of 2.5 μ L of 10 × Thermopol reaction buffer solution, 0.4 μ L of 10mM dNTPs, and 0.5 μ L of 10 μ M upstream outer primerF3, 0.5. mu.L of 10. mu.M downstream outer primer B3, 1. mu.L of 40. mu.M upstream inner primer FIP, 1. mu.L of 40. mu.M downstream inner primer BIP, 0.6. mu.L of 100mM MgSO412.5. mu.L of 2M betaine, 1. mu.L of 8U/. mu.LBst DNA polymerase, 1. mu.L of template DNA to be detected and 4. mu.L of sterilized double distilled water ddH2And O, incubating for 50min at 63 ℃ in a constant-temperature water bath, terminating the reaction for 1-2min at 80 ℃, and taking out the reaction tube.
mu.L of color-developing agent (10% SYBR Green I color-developing agent) was added to the reaction tube, and the color change was directly observed with naked eyes, and if the color was orange, the sample AmFV was negative and no AmFV was present, and if the color was Green, the sample AmFV was positive and contains AmFV. The results are shown in Table 1.
Table 1: test results and test samples of different positive viruses
Note: +: AmFV is positive; -: AmFV negative
As can be seen from Table 1, the LAMP method of the present invention has good specificity, only AmFV amplification is positive, and other non-AmFV viruses are negative. This result is consistent with the results of sequencing of common PCR products.
Example 4: sensitivity test
AmFV positive sample DNA was diluted to 2.0 × 100GE/μL、2.0×101GE/μL、2.0×102GE/μL、2.0×103The sensitivity of the method was verified by performing 4 GE/. mu.L concentration gradients (GE: Genome Equisages viral Genome Equivalents) simultaneously with quantitative PCR assay and LAMP amplification (method same as example 1). 2 experiments were repeated with a sensitivity of 2.0 × 101GE/. mu.L, sensitivity of 1 experiment result 1.32 × 102GE/. mu.L, therefore, it was determined that the LAMP method of the present invention had a minimum detection limit of 2.0 × 101GE/μL～1.32×102GE/mu L, and has higher sensitivity.
Sequence listing
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<120> detection primer group, detection kit and detection method for rapidly detecting bee filovirus
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Claims (4)
1. A detection primer group for detecting the bee filovirus by using loop-mediated isothermal amplification is characterized by comprising the following primers:
the upstream outer primer F3: 5'-TGGATCGTCGGGATCGTC-3', respectively;
downstream outer primer B3: 5'-AACTCGGGAAACAGTGGC-3', respectively;
an upstream inner primer FIP: 5'-GTGACTCAAACAGACTCGCGGTCTGCTTCATCTGAACCGTCT-3', respectively;
the downstream inner primer BIP: 5'-GCGGGACCTCCTATTTCGCTGGTCAACTTGGCCCGTAACC-3' are provided.
2. A method for detecting bee filovirus for non-disease diagnosis and treatment purposes, comprising extracting total DNA from a sample, and selectively amplifying the DNA of the sample by loop-mediated isothermal amplification using the set of detection primers of claim 1 to confirm the presence or absence of amplification products.
3. The detection method according to claim 2, wherein the selective amplification of the sample DNA using the detection primer set of claim 1 is performed by the loop-mediated isothermal amplification method, wherein the loop-mediated isothermal amplification system comprises a total volume of 25. mu.L, including 2.5. mu.L of 10 × Thermopol reaction buffer, 0.4. mu.L of 10mM dNTPs, 0.5. mu.L of 10. mu.M upstream outer primer F3, 0.5. mu.L of 10. mu.M downstream outer primer B3, 1. mu.L of 40. mu.M upstream inner primer FIP, 1. mu.L of 40. mu.M downstream inner primer BIP, and 0.6. mu.L of 100mM MgSO 3. mu.M412.5 mu L of 2M betaine, 4 mu L of sterilized double distilled water, 1 mu L of 8U/mu L Bst DNA polymerase and 1 mu L of sample DNA template, wherein the reaction condition is incubation for 45-60 min at the temperature of 60-65 ℃.
4. The detection kit for the bee filovirus comprises a loop-mediated isothermal amplification reagent and a detection primer group, and is characterized in that the detection primer group consists of the following primers:
the upstream outer primer F3: 5'-TGGATCGTCGGGATCGTC-3', respectively;
downstream outer primer B3: 5'-AACTCGGGAAACAGTGGC-3', respectively;
an upstream inner primer FIP: 5'-GTGACTCAAACAGACTCGCGGTCTGCTTCATCTGAACCGTCT-3', respectively;
the downstream inner primer BIP: 5'-GCGGGACCTCCTATTTCGCTGGTCAACTTGGCCCGTAACC-3' are provided.
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| CN106939356A (en) | 2017-07-11 |
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