CN111197093A - LAMP (loop-mediated isothermal amplification) detection primer group, kit and method for echinococcus caninum - Google Patents
LAMP (loop-mediated isothermal amplification) detection primer group, kit and method for echinococcus caninum Download PDFInfo
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Abstract
The invention provides an LAMP detection primer group, a kit and a method for echinococcus canicola. The primer group comprises: when the primer group is used for detection, the detection method has the advantages of high sensitivity, high efficiency, short reaction time, high specificity, simplicity and convenience in operation, simplicity in result judgment and the like, and is widely applied to a basal layer.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an LAMP (loop-mediated isothermal amplification) detection primer group, a kit and a method for echinococcus canicola.
Background
Echinococcosis, also known as echinococcosis, is a parasitic disease of zoonosis caused by echinococcus larvae-echinococcosis parasitizing in human and animal bodies. The adult echinococcus granulosus is parasitic in the small intestine of the final host canine, and after the body of the insect is mature, the egg and the mature gestational segment can be discharged out of the body along with the feces. After being eaten by the intermediate host (mainly rodents and some domestic animals such as sheep, cattle, horses, pigs, etc.), live eggs form larvae which parasitize the liver, lungs, abdominal cavity, etc., and dogs are infected by eating viscera with the larvae. Canines, the terminal host of echinococcosis, are the most important source of infection.
At present, the detection of echinococcus is mainly performed by a traditional conventional stool detection method, a stool antigen ELISA detection method and a PCR molecular biology detection method. The conventional excrement detection method is complicated and time-consuming, has low detection rate, cannot distinguish the ova of the echinococcus granulosus and echinococcus multilocularis from the excrement of the dog, and has the risk of infecting operators; the fecal antigen ELISA detection method and the PCR have good specificity and sensitivity, but are time-consuming, labor-consuming, complex in required instruments, and not beneficial to investigation of basic epidemiology and detection of a large number of samples. Therefore, it is of great significance to establish a rapid, simple and sensitive epidemiological investigation and on-site diagnosis method for the echinococcus granulosus.
Disclosure of Invention
In order to solve the technical problems, the invention provides an LAMP detection primer group, a kit and a method for echinococcus canicola.
In order to achieve the purpose, the invention adopts the following technical scheme that:
a group of LAMP detection primer sets for echinococcus canicola, which comprises: the lateral primer pair F3 and B3 combined with COX2 gene sequence of Echinococcus canicola, and the medial primer pair FIP and BIP combined with COX2 gene sequence of Echinococcus canicola, are characterized in that: the nucleotide sequences of F3 and B3 are shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of FIP and BIP are shown as SEQ ID NO.3 and SEQ ID NO. 4.
The LAMP detection primer for the echinococcus canicola is applied to the preparation of the LAMP kit or detection reagent for detecting the echinococcus canicola.
The invention also provides a detection kit containing the LAMP detection primer for the echinococcus canicola.
Preferably, the detection kit is a LAMP kit, and comprises the following components in addition to the primer set: a loop-mediated isothermal amplification buffer solution, a positive control, a negative control and/or a developing solution.
The positive control is an echinococcus canicola DNA positive sample or an echinococcus canicola DNA extracted sample separated domestically or contains a sequence shown in SEQ ID NO.5 or COX 2.
Preferably, the loop-mediated isothermal amplification buffer solution comprises Tris-HCl, KCl and (NH)4)2SO4、Tween-20、MgSO4Betaine (Betaine), deoxynucleotides (dNTPs), Bst DNA polymerase.
The color development liquid comprises SYBR Green I and Hydroxymethyl Naphthol Blue (HNB). Further, preferably, the amount of solute-containing substance per 2 μ l of color developing solution is: SYBR Green I0.16. mu. mol, hydroxymethyl naphthol blue 0.3. mu. mol.
Further, the invention also provides an LAMP detection method for rapidly detecting the echinococcus canicola by adopting the primers, which is characterized in that the LAMP detection primers of the echinococcus canicola are used for carrying out loop-mediated isothermal amplification reaction on the DNA of a sample to be detected from a diseased or dead animal, an amplification product is detected, and whether the diseased or dead animal carries the echinococcus canicola is determined; the conditions of the loop-mediated isothermal amplification reaction are as follows: 63 ℃ for 30-60 minutes.
The LAMP detection method can judge whether the sample contains Echinococcus canicola or not by instrument interpretation or direct examination, and the direct examination method comprises the following steps: during detection, DNA containing echinococcus canicola is simultaneously set as a positive control and a negative control for detection by water;
observing the solution after reaction, wherein the reaction tube with the positive control has bright green visible fluorescence, and the reaction tube with the negative control is still brown yellow non-fluorescent liquid; if the reaction tube filled with the sample to be detected is still brown yellow non-fluorescent liquid, the detection result of the echinococcus canicola in the sample to be detected is negative; if the reaction tube filled with the sample to be detected has bright green visible fluorescence, the detection result of the echinococcus canicola in the sample is positive.
Specifically, the LAMP detection method for rapidly detecting Echinococcus canicola by using the primers comprises the following steps:
s1, taking a sample to be detected of dog excrement, and extracting DNA;
s2, performing loop-mediated isothermal amplification, and amplifying the extracted DNA by adopting the primer group;
s3, judging the result: judging whether the sample contains echinococcus canicola by instrument interpretation or by direct inspection,
if the reaction tube filled with the sample to be detected is still brown yellow non-fluorescent liquid, the detection result of the echinococcus canicola in the sample to be detected is negative;
if the reaction tube filled with the sample to be detected has bright green visible fluorescence, the detection result of the echinococcus canicola in the sample is positive.
The LAMP detection method as described above, preferably, in step S2, in step S2, a positive control of Echinococcus canicola-containing DNA and a negative control of water detection are provided simultaneously. In the LAMP detection method as described above, preferably, in step S2, the loop-mediated isothermal amplification system is configured such that 2. mu.l of the DNA and 2. mu.l of a color developing solution are added to a reaction tube containing 23. mu.l of the LAMP reaction solution, and the amplification reaction is performed at 63 ℃ for 30 to 60 minutes.
In the LAMP detection method as described above, it is preferable that the 23. mu.l LAMP reaction solution contains Tris-HCl 0.5. mu. mol, KCl 0.25. mu. mol, (NH)4)2SO40.25μmol、Tween-200.025μL、MgSO40.2-20 mu mol, 20 mu mol of betaine, 0.035 mu mol of each of 4 deoxynucleotides, 16U of Bst DNA polymerase, 0.04-0.06 mu mol of each of FIP and BIP as an inner primer pair, and 0.004-0.008 mu mol of each of F3 and B3 as an outer primer pair.
Further, preferably, the LAMP reaction solution of 23 μ l can be prepared by using the following components: Tris-HCl 0.5. mu. mol, KCl 0.25. mu. mol, (NH)4)2SO40.25μmol、Tween-20 0.025μL、MgSO40.2. mu. mol, betaine 20. mu. mol, 4 deoxynucleotides 0.035. mu. mol each, Bst DNA polymerase 16U, the inner primer pair FIP and BIP 0.04. mu. mol each, the outer primer pair F3 and B3 0.004. mu. mol each.
The invention has the beneficial effects that:
the LAMP detection kit for echinococcus canicola provided by the invention can be used for detecting or screening whether the echinococcus canicola is carried in the samples of diseased or dead animals or in vitro samples or feeds and the like, is convenient to use, simple and convenient to operate and low in cost, and can be carried out under the required conditions only in a water bath kettle or a metal constant temperature heater. The detection method has the advantages of high sensitivity, high efficiency, short reaction time, high specificity, simple and convenient operation, good safety, simple result judgment and the like, and is widely applied to basic layers.
Drawings
FIG. 1 shows the sensitivity of the Echinococcus canicola LAMP detection method, N: negative control; the concentrations of the solutions 1 to 6 are 10 respectively0、101、102、103、104、105copise/ml。
FIG. 2 shows the sensitivity of the PCR detection method for Echinococcus canicola, wherein M: 100bp DNAladder DNAPLUS MASS (5. mu.l); n: negative control; strips 1-6: the content of template bacteria in LAMP reaction is 105、104、103、102、101、100copise/ml。
FIG. 3 shows the specificity of the Echinococcus canicola LAMP detection method, wherein M: 2K PlusDNA MARKER; 1-5 of the strip: negative control, positive control, dog roundworm, toxoplasma gondii, hookworm DNA, respectively.
FIG. 4 is the specificity of PCR detection of Echinococcus canicola, where M: 2K Plus DNAmarker; strips 1-5 are negative control, positive control, canine ascaris lumbricoides, toxoplasma gondii, hookworm DNA, respectively.
FIG. 5 is a visualized interpretation result of Echinococcus canicola LAMP detection clinical samples under sunlight.
FIG. 6 is a visualized interpretation result of Echinococcus canicola LAMP detection clinical samples under ultraviolet light.
Detailed Description
The following examples are intended to further illustrate the invention but should not be construed as limiting it. Modifications and substitutions may be made thereto without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
EXAMPLE 1 preparation of plasmid
The echinococcus canicola DNA is used, a genome COX gene sequence is obtained by amplification according to a PCR method, and then the genome COX gene sequence is connected with a T vector to form a plasmid of the needed MN gene, wherein one plasmid molecule corresponds to one copy of the COX gene. The plasmid is transferred into competent cells, and the competent cells are cultured under appropriate conditions, so that the plasmid can be replicated along with the propagation of the competent cells. Finally, the purified plasmid is extracted from the competent cells. The resulting plasmid can be determined by spectrophotometry and the copy number concentration calculated.
The plasmid was diluted to the desired concentration with double distilled water, and 2. mu.L of each concentration of plasmid was added for detection, each reaction containing 10. mu.L of plasmid5Copy/. mu.L, 104Copy/. mu.L, 103Copy/. mu.L, 102Plasmid samples at copy/. mu.L, 10 copies/. mu.L, 1 copy/. mu.L. (refer to molecular cloning guidelines, third edition, all reagents and equipment are commercially available.)
Example 2 Echinococcus canicola LAMP detection method
Aiming at the echinococcus canicola, the invention is designed for LAMP detection, and is verified by a large number of experiments, and finally the echinococcus canicola used for LAMP detection is determined to comprise two pairs of primers, wherein the two pairs of primers are mainly used for the echinococcus canicola which is shown as SEQ ID NO.5 (COX2 gene sequence) and serves as a conserved region of the echinococcus canicola which is separated domestically. Wherein, one pair of primers is an inner primer pair combined with COX2 gene sequence of Echinococcus canicola, and comprises an upstream primer (FIP) and a downstream primer (BIP); one pair of primers is a lateral primer pair combined with the COX2 gene sequence of the echinococcus canicola, and comprises an upstream primer (F3) and a downstream primer (B3). The specific nucleotide sequence is as follows:
F3(SEQ ID NO.1)：5'-GCTGTTAAATTTGGTAGTGAGAA-3'
B3(SEQ ID NO.2)：5'-CTACCACAAAATAATCCCCAAT-3'
FIP(SEQ ID NO.3)：5'-TCAAGGCACACAAAACTAATACAACTTTTTCAGATTATAGAGTTGGTATGAAC-3'
BIP(SEQ ID NO.4)：5'-TTCTGAGACTATTAAGGTGGTTGGTTTTTCGGAAACGAATCATAGCCA-3'。
the detection kit of the LAMP detection primer for the echinococcus granulosus comprises the primer, a loop-mediated isothermal amplification buffer solution, a positive control, a negative control and a developing solution, wherein the loop-mediated isothermal amplification buffer solution comprises Tris-HCl, KCl, (NH)4)2SO4、Tween-20、MgSO4An aqueous solution of betaine, deoxynucleotide, Bst DNA polymerase; the amount of solute-containing substance per 2. mu.l of color developing solution was: SYBR Green I0.16. mu. mol, hydroxymethyl naphthol blue 0.3. mu. mol.
When the kit is used for detection, the method comprises the following detection steps:
(1) extraction of DNA
A sample of dog feces was taken and DNA was extracted using a kit (Transzol (TransGen) DNA extraction kit used in the present invention).
(2) Loop-mediated isothermal amplification
① mu.l of dog feces DNA sample was added to a reaction tube containing 23. mu.l of LAMP reaction solution.
② and placing in a water bath or a metal constant temperature heater at 63 deg.C (all reaction temperatures are 63 deg.C) for 60 min, and taking out.
(3) Determination of results
The sample can be judged whether to contain echinococcus canicola by direct inspection. And if the reaction tube filled with the sample to be detected is still brown yellow non-fluorescent liquid, the detection result of the echinococcus canicola in the sample to be detected is negative. If the reaction tube filled with the sample to be detected has bright green visible fluorescence, the detection result of the echinococcus canicola in the sample is positive. Wherein, 23 μ l of LAMP reaction solution, the amount of the solute substance is as follows:
Tris-HCl 0.5μmol、KCl 0.25μmol、(NH4)2SO40.25μmol、Tween-200.025μL、MgSO40.2-20 mu mol, 20 mu mol of betaine, 0.035 mu mol of 4 deoxynucleotides respectively, 16U of Bst DNA polymerase, 0.04-0.06 mu mol of upstream primer and downstream primer of the inner primer pair respectively, and 0.004-0.008 mu mol of upstream primer and downstream primer of the outer primer pair respectively.
In order to ensure the accuracy of the result, a positive control and a negative control are set in the detection, wherein the positive control is detected by using DNA containing echinococcus canicola as a template, and the negative control is detected by using water as a template. And when judging the result: observing the solution after reaction, wherein the reaction tube with the positive control has bright green visible fluorescence, and the reaction tube with the negative control is still brown yellow non-fluorescent liquid; if the reaction tube filled with the sample to be detected is still brown yellow non-fluorescent liquid, the detection result of the echinococcus canicola in the sample to be detected is negative; if the reaction tube filled with the sample to be detected has bright green visible fluorescence, the detection result of the echinococcus canicola in the sample is positive.
Example 3 detection of Echinococcus granulosus Canine
In this example, 23. mu.L of the reaction solution for loop-mediated isothermal amplification contained: 4 μ L125 mmol/L LTris-HCl, 2 μ L100mmol/L MgSO4，2μL 125mmol/L KCl，2μL 125mmol/L(NH4)2SO40.4. mu.L of 50mol/LBetaine, 0.025. mu.L of Tween-20, 100mmol/L of dNTPs each 0.35. mu.L, 2. mu.L of 12U/. mu.L of Bst DNA polymerase large fragment (New England Biolabs), 1. mu.L of 40mmol/L FIP, 1. mu.L of 40mmol/L BIP, 1. mu.L of 5mmol/LF3, 1. mu.L of 5mmol/LB3, sterile double distilled water to make up to 23. mu.L, and template 2. mu.L. Respectively detecting the plasmid samples with the concentrations, wherein the detection steps are as follows:
(1) loop-mediated isothermal amplification
mu.L of each 10 of example 1 was added to 3 reaction tubes each containing 23. mu.L of LAMP reaction solution4Copy/μ L of plasmid sample as test group 1; mu.L of each 10 of example 1 was added to 3 reaction tubes each containing 23. mu.L of LAMP reaction solution3Copy/μ L plasmid sample as detection group 2; mu.L of step one was added to each of 3 reaction tubes containing 23. mu.L of LAMP reaction solution to give 102Copy/μ L plasmid sample as detection group 3; mu.L of plasmid samples of 10 copies/. mu.L obtained in example 1 were added to 3 reaction tubes each containing 23. mu.L of LAMP reaction solution, and used as a detection group 4; mu.L of the plasmid sample of 1 copy/. mu.L obtained in example 1 was added to each of 3 reaction tubes containing 23. mu.L of LAMP reaction solution, and used as a detection group 5; adding Echinococcus canicola DNA positive samples into 3 reaction tubes filled with 23 mu L of LAMP reaction solution respectively to serve as positive control groups; to 3 reaction tubes each containing 23. mu.L of LAMP reaction solution, 2. mu.L of sterile double distilled water was added as a negative control group.
The reaction tube was placed in a water bath at 63 ℃ for 60 minutes and then taken out.
(2) Direct inspection of
The results show that the three reaction tubes of the positive control group all have bright green visible fluorescence, and the three reaction tubes of the negative control group all are brown yellow non-fluorescent liquid. The three reaction tubes of the detection group 1 all have bright green visible fluorescence, the three reaction tubes of the detection group 2 all have bright green visible fluorescence, the three reaction tubes of the detection group 3 all have bright green visible fluorescence, the three reaction tubes of the detection group 4 all have bright green visible fluorescence, and the three reaction tubes of the detection group 5 all are brown yellow non-fluorescent liquid.
Example 4 sensitivity detection
Continuously diluting 10 times of echinococcus canicola COX gene plasmid DNA with known content by 10 times5～100The copied plasmid is used as a template, and the LAMP amplification system adopted by the method is 23 mu L of loop-mediated isothermal amplification reaction liquid and comprises the following steps: mu.L of 16U/. mu.L Bst DNA polymerase large fragment (New England Biolabs); 12.5ul LAMP buffer: 40mmol/L Tris-HCl (pH8.8), 16mmol/LMgSO4，20mmol/L KCl，20mmol/L(NH4)2SO41.6mol/L Betaine, 0.2% Tween-20, 2.5mmol/L dNTPs; 1 μ L40 mmol/L FIP, 1 μ L40 mmol/L BIP, 1 μ L5mmol/L F3, 1 μ L5mmol/L B3; adding sterilized double distilled water to make up to 23 μ L; template 2. mu.L was added.
Detection was performed while DNA from known Echinococcus canicola concentration gradients was detected by PCR detection (PCR program see below).
PCR procedure:
F：5'-GCTGTTAAATTTGGTAGTGAGAA-3'，
R：5'-CTACCACAAAATAATCCCCAAT-3'
25 μ L reaction: mu.l template, 2 × EasyTaq PCR Supermix (Whole gold organism), 0.5. mu.l upstream and downstream primers, made up to 25. mu.l in double distilled water.
Reaction procedure: denaturation at 94 deg.C for 5 min; 30 cycles of 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1 min; finally, extension is carried out for 10min at 72 ℃.
The sensitivity of PCR detection is determined by 1% agarose gel electrophoresis, the interpretation of the instrument is known,the lowest detection of Echinococcus canicola by LAMP is 101The copy results are shown in FIG. 1, and the lowest detection of Echinococcus canicola by PCR is 103Copy results are shown in FIG. 2, and the sensitivity of LAMP detection to Echinococcus canicola is 100 times higher than that of PCR.
Example 5 specific assay
By using the LAMP amplification system and method in example 3, Echinococcus granulosus, Echinococcus multilocularis, Taenia canicola, and Taenia vesiculosus DNA were subjected to LAMP detection using water as a negative control and the plasmid prepared in example 1 as a positive control, and the detection was performed by using these as templates and the PCR detection method in example 4. The results of the LAMP detection method are shown in FIG. 3, and the results of the PCR detection method are shown in FIG. 4, which shows that the method of the present invention has strong specificity, which is the same as the current PCR specificity.
Example 6 clinical examination in this example 23. mu.L of the loop-mediated isothermal amplification reaction solution included: mu.L of 16U/. mu.L BstDNA polymerase large fragment (New England Biolabs); 12.5. mu.l LAMP buffer: 40mmol/L Tris-HCl (pH8.8), 16mmol/L MgSO4，20mmol/L KCl，20mmol/L(NH4)2SO41.6mol/L Betaine, 0.2% Tween-20, 2.5mmol/L dNTPs; 1 μ L40 mmol/L FIP, 1 μ L40 mmol/L BIP, 1 μ L5mmol/L F3, 1 μ L5mmol/L B3; sterilized double distilled water was added to make up to 23. mu.L. Then 1. mu.L SYBR Green I and 1. mu.L Hydroxymethyl Naphthol Blue (HNB) are added into the reaction system. When the result is judged, if the result is observed under ultraviolet rays, the color of the positive result is green, and the color of the negative result is purple. 120 samples of clinical collection were tested for DNA extraction. The result shows that the number of positive samples detected by LAMP and PCR methods is 86 parts and 65 parts respectively, and the detection rate of the LAMP method is obviously higher than that of the PCR method. The visual color development results of the LAMP method for detecting samples are shown in FIG. 5 and FIG. 6.
The LAMP detection method does not need an expensive PCR instrument, only needs a common metal or water bath, can judge the detection result by observing visible fluorescence, and is simple and convenient to operate.
Compared with the PCR detection method, the method can be applied to the instant detection of the echinococcus canicola on the primary site.
Sequence listing
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gtgttatatt gtggcggttt gtgtatttat tgtttgtttt gtgtatgttt tgttgtgttg 300
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ggtatgaact gttattccta ctgtggttgt attagttttg tgtgccttga aagttaaatt 420
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Claims (10)
1. A group of LAMP detection primer sets for echinococcus canicola, which comprises: the lateral primer pair F3 and B3 combined with COX2 gene sequence of Echinococcus canicola, and the medial primer pair FIP and BIP combined with COX2 gene sequence of Echinococcus canicola, are characterized in that: the nucleotide sequences of F3 and B3 are shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of FIP and BIP are shown as SEQ ID NO.3 and SEQ ID NO. 4.
2. The application of the LAMP detection primer for Echinococcus canicola of claim 1 in preparation of an LAMP kit or a detection reagent for detecting Echinococcus canicola.
3. A detection kit containing the LAMP detection primer for Echinococcus canicola as defined in claim 1.
4. The kit of claim 3, further comprising: a loop-mediated isothermal amplification buffer solution, a positive control, a negative control and/or a developing solution.
5. The kit of claim 4, wherein the loop-mediated isothermal amplification buffer comprises Tris-HCl, KCl, (NH)4)2SO4、Tween-20、MgSO4An aqueous solution of betaine, deoxynucleotide, Bst DNA polymerase;
the color development liquid comprises SYBR Green I and hydroxymethyl naphthol blue.
6. The LAMP detection method for rapidly detecting the echinococcus canicola by adopting the primer set according to claim 1 is characterized in that the LAMP detection primer set for the echinococcus canicola is used for carrying out a loop-mediated isothermal amplification reaction on a sample DNA to be detected to detect an amplification product; the conditions of the loop-mediated isothermal amplification reaction are as follows: 63 ℃ for 30-60 minutes.
7. The LAMP detection method according to claim 6, characterized in that it comprises the following steps:
s1, taking a sample to be detected of dog excrement, and extracting DNA;
s2, carrying out loop-mediated isothermal amplification reaction on the DNA of a sample to be detected by using the LAMP detection primer group of Echinococcus canicola;
s3, judging the result: if the reaction tube filled with the sample to be detected is still brown yellow non-fluorescent liquid, the detection result of the echinococcus canicola in the sample to be detected is negative;
if the reaction tube filled with the sample to be detected has bright green visible fluorescence, the detection result of the echinococcus canicola in the sample is positive.
8. The LAMP detection method according to claim 7, wherein in step S2, a positive control of Echinococcus canicola DNA and a negative control of water detection are set simultaneously.
9. The LAMP detection method according to claim 7, wherein in step S2, the amplification system of the loop-mediated isothermal amplification is such that 2. mu.l of the DNA is added to a reaction tube containing 23. mu.l of the LAMP reaction solution; the 23 mul LAMP reaction solution comprises Tris-HCl 0.5 mul mol, KCl 0.25 mul mol and (NH)4)2SO40.25μmol、Tween-20 0.025μL、MgSO40.2-20 mu mol, 20 mu mol of betaine, 0.035 mu mol of each of 4 deoxynucleotides, 16U of Bst DNA polymerase, 0.04-0.06 mu mol of each of FIP and BIP as an inner primer pair, and 0.004-0.008 mu mol of each of F3 and B3 as an outer primer pair.
10. The LAMP detection method according to claim 9, characterized in that the 23. mu.l of LAMP reaction solution comprises: Tris-HCl 0.5. mu. mol, KCl 0.25. mu. mol, (NH)4)2SO40.25μmol、Tween-20 0.025μL、MgSO40.2. mu. mol, betaine 20. mu. mol, 4 deoxynucleotides 0.035. mu. mol each, Bst DNA polymerase 16U, the inner primer pair FIP and BIP 0.04. mu. mol each, the outer primer pair F3 and B3 0.004. mu. mol each.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2019101532499 | 2019-02-28 | ||
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