CN111705141B - Method for identifying conch in the shape of conch by fluorescence quantitative PCR detection - Google Patents
Method for identifying conch in the shape of conch by fluorescence quantitative PCR detection Download PDFInfo
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Abstract
The invention provides a method for identifying concha haliotidis by fluorescent quantitative PCR detection, which belongs to the technical field of pest detection and identification, and uses specific primers and probes to identify molecular characteristics of concha haliotidis, wherein the sequences of the primers and the probes are shown as SEQ ID NO. 1-3. The method can rapidly and accurately detect and identify the conch in the shape of the emulsion, has the advantages of strong specificity, high sensitivity, short time consumption, accurate result judgment and simple and convenient operation, does not need post-treatment of PCR, and can effectively avoid false positive and cross contamination. The invention provides a new technical means for rapid and accurate identification of the auricularia auricula-judae in the aspects of checking goods such as incoming and outgoing grains, seedlings, wood, flowers and the like, agricultural production, disease prevention and control and scientific research, and has important practical significance in the aspects of formulating technical trade barrier measures, protecting domestic agricultural production safety, human and animal health and the like.
Description
Technical Field
The invention relates to a method for identifying the conch in the form of emulsion by fluorescent quantitative PCR detection, which belongs to the technical field of pest detection and identification and is suitable for rapid and accurate identification of the conch in the fields of inspection of goods such as incoming and outgoing grains, seedlings, wood, flowers and the like, agricultural production, disease prevention and control and scientific research.
Background
Milky ear screwOtala lactea(Muller, 1774) belonging to the genus Mollusca, the phylum Mollusca, gastrodia, the phylum Plmonata, the order Stylomatophara, the family Dasnailaceae, the genus EarpiumOtalaSchumacher, 1817, is a pest that is extremely serious to agricultural production and human and animal health. The conch in the form of emulsion is produced in the eastern coast of Mediterranean sea, and is spread to many countries and regions in the world, so that the agricultural and forestry production is seriously endangered, the great economic loss is caused, and the parasitic diseases caused by the zoonotic diseases are spread, so that the health of people and livestock is threatened. In 2017, the national port quarantine organization intercepts the milky auricular snail from spanish inbound goods for the first time, and risk analysis shows that the snail belongs to a particularly dangerous species, can be suitable for most areas of China, and has extremely high invasion risk. In order to prevent invasion of harmful organisms and protect national biological safety, the national quality inspection general office of 11 months in the same year issues a warning report (quality inspection police [ 2017 ] No. 39) about first intercepting of spanish goods in import. The department of agricultural department planting industry management in 2018 issues ' letters on dynamically adjusting the ideas of the plant quarantine pests of the inbound plants of China ' (agricultural plant protection [ 2018 ]. No. 1), and the auricularia auricula-judae supplement is listed in ' the plant quarantine pests directory of the inbound plants of the people's republic of China '. The milky ear snail has very similar appearance to similar seed-dispersed snail, cover snail, worm snail, bright snail, etc. and is difficult to distinguish from shell form alone. Moreover, the shells of the conch in the shape of a milky ear are often subjected to variation due to the influence of external geographic environment, climate factors and the like, so that the identification is more difficult. For historical reasons, the plant protection system of the domestic agricultural and forestry universities does not have courses in the aspect of harmful snails or professional research forces at present, and although some sporadic reports exist in the agricultural harmful snail research field, the plant protection system is basically blank. Due to lack of effectiveness of the milky conchThe rapid detection and identification method of (1) often has the phenomena of false report and false report in the inspection of the incoming and outgoing species, and seriously damages national biological safety and human and animal health. The technical problems of incapability of detecting, inaccurate detection and low detection speed of the milky auricular snail are to be solved. With the rapid development of molecular biology technology, fluorescent dye is added into a reaction system by using a fluorescent quantitative PCR technology, and the DNA amplification amount is reflected by the transmission of fluorescent signals, so that rapid and accurate detection and identification can be realized. To date, no fluorescent quantitative PCR technique for detection and identification of the conch in the form of a cream has been found. If a specific primer and a fluorescent quantitative PCR amplification method for a probe can be designed, a new technical means is provided for rapid and accurate identification of the conch in the shape of a cream ear, and the difficult problem is solved. The invention establishes a technology for detecting and identifying the conch in the form of emulsion by using a fluorescent quantitative PCR method, so as to prevent invasion of harmful organisms, maintain national biological safety and protect agriculture and forestry production and human and animal health.
Disclosure of Invention
Aiming at the defects of the traditional morphology and the common PCR identification technology, the invention provides a detection method which has the advantages of good accuracy, high sensitivity, strong specificity, short time consumption and simple operation, and is applied to the rapid detection and identification of the conch in the form of a milky ear.
The invention provides a method for identifying concha arcae by fluorescent quantitative PCR detection, which is characterized in that specific primers and fluorescent probes are used for identifying molecular characteristics of concha arcae, and the primers and the fluorescent probes are as follows:
the upstream primer is OLF: 5'-TTCCGTGGATTTGGCTATTT-3' the number of the individual pieces of the plastic,
the downstream primer is OLR: 5'-TGGTAATAGCACCCGCCAAA-3' the number of the individual pieces of the plastic,
the fluorescent probe is OLP: the fluorescent reporter group marked at the 5 '-FAM-CGGTCACCAGGAGTCACTCTAGAACG-BHQ 1-3', the fluorescent quenching group marked at the 3 '-end is FAM and the fluorescent quenching group marked at the 5' -end is BHQ1,
the concentrations of the primers and the probes are 10 mu mol/L.
The total volume of the fluorescent quantitative PCR reaction system is 25. Mu.L, wherein 2X Premix Ex TaqTM PCR (Probe qPCR) is 12.5. Mu.L, and each 1. Mu.L of the upstream primer, the downstream primer and the Probe100 ng/. Mu.L of DNA template 3. Mu.L, ROXII 0.5. Mu.L, ddH 2 O 7μL。
The fluorescent quantitative PCR reaction comprises the following steps: pre-denaturation at 94℃for 5min;94 ℃ for 10s,60 ℃ for 32s and 40 cycles; the reaction was ended.
After the fluorescent quantitative PCR reaction is finished, according to the amplification curve judgment result, the judgment method comprises the following steps: under the conditions that the blank control and the negative control have no Ct value and no amplification curve, when a typical amplification curve appears in a sample to be detected and the Ct value is less than or equal to 35, judging that the sample to be detected is the conch in the shape of a milky ear; when the Ct value is equal to 40, it is judged as a non-milky conch. If Ct is greater than 35 and less than 40, the test should be repeated: when the Ct value is equal to 40, judging that the conch is a non-milky conch; when Ct is less than 40, the test piece is judged as a conch in the form of a milky ear.
The invention has the remarkable advantages that: the method can conveniently, rapidly and accurately detect and identify the conch in the shape of the emulsion. The invention provides a new technical means for rapid and accurate identification of the conch in the aspects of inspection of the goods in and out of the environment, agricultural production, disease prevention and control and scientific research, and has important significance for protecting the agriculture and forestry production and the health of people and livestock and maintaining the national biological safety.
The method for identifying the conch in the form of the emulsion by fluorescence quantitative PCR detection provided by the invention has the following beneficial effects:
1. the specificity is strong: the adopted fluorescent primers and probes are specific primers and probes designed for the COI gene sequence of the conch, and a pair of specific primers and a specific probe can perform double control on a target, so that the specificity is strong.
2. The sensitivity is high: the detection sensitivity of the conch emulsion can reach 10 pg/mu L on the DNA level.
3. The result judgment is accurate and visual: the specific primer and probe for detecting and identifying the concha haliotidis designed by the invention only detects the concha haliotidis, 8 representative snails, namely, similar species of concha haliotidis, bulk snail, forest shallot snail, garden shallot snail, cover snail, pizza tea snail, worm Yi snail and Liang snail, are tested and verified, and the PCR amplification result can be directly displayed on a computer without other extra steps, so that the result judgment is accurate and visual.
4. The operation is simple and convenient: the method does not need complex instruments or special reagents, can react and detect by only one fluorescent quantitative PCR instrument, and is simple and convenient to operate.
5. The practicability is good: when the traditional taxonomic method is difficult to identify the conch in the emulsion, the method is complemented by a molecular biological technology, so that the fluorescent quantitative PCR method for detecting the conch in the emulsion is obtained with high speed, accuracy and high sensitivity. Therefore, the method has good practicability and can meet the requirements of quick and reliable detection and identification of the conch in the shape of a milky ear.
Drawings
FIG. 1 shows the results of a fluorescent quantitative PCR primer specificity assay for Oncomelania. Wherein: curve 1 is a milky conchOtala lactea(Muller, 1774); curve 2 is a conch with a spot earOtala punctata(Muller, 1774); curve 3 is a bulk snailHelix aspersaThe method comprises the steps of carrying out a first treatment on the surface of the Curve 4 is forest shallot snailCepaea nemoralisThe method comprises the steps of carrying out a first treatment on the surface of the Curve 5 is garden onion snailCepaea hortensisThe method comprises the steps of carrying out a first treatment on the surface of the Curve 6 is a cover big snailHelix pomatiaThe method comprises the steps of carrying out a first treatment on the surface of the Curve 7 is pizza snailTheba pisanaThe method comprises the steps of carrying out a first treatment on the surface of the Curve 8 is the worm Yi's snailEobania vermiculataThe method comprises the steps of carrying out a first treatment on the surface of the Curve 9 is a bright big snailHelix lucorumThe method comprises the steps of carrying out a first treatment on the surface of the Curve 10 is a blank.
FIG. 2 shows the results of the fluorescent quantitative PCR detection sensitivity test for the conch in the form of a cream. Wherein curve 1 is 100 ng/. Mu.L; curve 2 is 10 ng/. Mu.L; curve 3 is 1 ng/. Mu.L; curve 4 is 100 pg/. Mu.L; curve 5 is 10 pg/. Mu.L; curve 6 is 1 pg/. Mu.L; curve 7 is 10 fg/. Mu.L; curve 8 is 10 fg/. Mu.L; curve 9 is 1 fg/. Mu.L; curve 10 is a blank.
Detailed Description
Example 1 fluorescent quantitative PCR primer specificity assay for Emulation conch
1. Preparation of materials
The test snails were as follows: a milky conch; spot ear snails; bulk snail; forest shallot snail; garden onion snail; cover big snail; pizza tea snail; worm snail; bright big snail.
The snail line is obtained by domestic investigation and collection or intercepted in national port quarantine, confirmed by a customs administration national mollusc quarantine identification key laboratory, and stored at-20 ℃ for standby.
2. Establishment of fluorescent quantitative PCR method
2.1, designing and synthesizing a primer: based on the COI gene sequence of the conch, soft Primer Express3 is designed by using a Primer to assist in designing and analyzing the Primer, and the Primer is synthesized by Shanghai Biotechnology service Co-Ltd after the specificity of the Primer is tested by NCBI Blast. The optimal primer finally determined by repeated comparison test is as follows:
the upstream primer is OLF: 5'-TTCCGTGGATTTGGCTATTT-3' the number of the individual pieces of the plastic,
the downstream primer is OLR: 5'-TGGTAATAGCACCCGCCAAA-3' the number of the individual pieces of the plastic,
the fluorescent probe is OLP: the fluorescent reporter group marked at the 5 '-FAM-CGGTCACCAGGAGTCACTCTAGAACG-BHQ 1-3', the fluorescent quenching group marked at the 3 '-end is FAM and the fluorescent quenching group marked at the 5' -end is BHQ1.
2.2, DNA extraction: TIANGEN tissue genomic DNA extraction kit extraction, DNA was extracted according to instructions for use. The concentration of DNA was determined by nucleic acid protein analyzer, and finally the DNA was diluted to 100 ng/. Mu.L with TE solution and stored at-20℃for further use.
2.3, fluorescent quantitative PCR amplification reaction system: the total volume was 25. Mu.L, of which 2X Premix Ex TaqTM PCR (Probe qPCR) was 12.5. Mu.L, 1. Mu.L each of the upstream and downstream primers and probes, 100 ng/. Mu.L of DNA template 2. Mu.L, ROXII 0.5. Mu.L, ddH 2 O7. Mu.L. And (3) uniformly mixing, and then placing the mixture into a fluorescent quantitative PCR amplification instrument for amplification, wherein the concentration of the primer and the probe is 10 mu mol/L.
2.4, the two-step amplification reaction procedure of fluorescent quantitative PCR is as follows: pre-denaturation at 95 ℃ for 5min;95 ℃ 10s,60 ℃ 35s,40 cycles; the reaction was ended.
2.5, specificity test results: only the conch emulsion showed a typical amplification curve on the fluorescent quantitative PCR instrument, and the Ct value was less than 35, and no typical amplification curve was observed for other samples, indicating that the method can be used for specific detection of conch emulsion (FIG. 1).
Example 2: fluorescent quantitative PCR detection sensitivity test for conch in the form of emulsion
The emulsion conch DNA stock solution (100 ng/. Mu.L) extracted in example 1 was diluted to different concentration gradients of 10 ng/. Mu.L, 1 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L, 1 pg/. Mu.L, 100 fg/. Mu.L, 10 fg/. Mu.L and 1 fg/. Mu.L using a 10-fold concentration serial dilution method.
Fluorescent quantitative PCR amplification reaction system: the total volume was 25. Mu.L, of which 2X Premix Ex TaqTM PCR (Probe qPCR) was 12.5. Mu.L, 1. Mu.L each of the upstream and downstream primers and probes, 100 ng/. Mu.L of DNA template 2. Mu.L, ROXII 0.5. Mu.L, ddH 2 O7. Mu.L. And (3) uniformly mixing, and then placing the mixture into a fluorescent quantitative PCR amplification instrument for amplification, wherein the concentration of the primer and the probe is 10 mu mol/L.
The fluorescent quantitative PCR two-step amplification reaction procedure is: pre-denaturation at 95 ℃ for 5min;95 ℃ 10s,60 ℃ 35s,40 cycles; the reaction was ended.
Sensitivity test results: the amplification curve still appears when the DNA concentration is 10 pg/. Mu.L, which shows that the method has higher sensitivity, and the detection sensitivity can reach 10 pg/. Mu.L (FIG. 2).
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
SEQUENCE LISTING
<110> Fuzhou customs technical center
<120> method for identifying conch in the form of conch by fluorescent quantitative PCR
<130> 3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> artificial sequence
<400> 1
<210> 2
<211> 20
<212> DNA
<213> artificial sequence
<400> 2
<210> 3
<211> 26
<212> DNA
<213> artificial sequence
<400> 3
cggtcaccag gagtcactct agaacg 26
Claims (1)
1. A method for identifying the conch in the form of emulsion by fluorescent quantitative PCR detection is characterized in that: the method is characterized in that a specific primer and a fluorescent probe are used for identifying the molecular characteristics of the conch in the shape of a lactylose, and the primer and the fluorescent probe are as follows:
the upstream primer is OLF: 5'-TTCCGTGGATTTGGCTATTT-3' the number of the individual pieces of the plastic,
the downstream primer is OLR: 5'-TGGTAATAGCACCCGCCAAA-3' the number of the individual pieces of the plastic,
the fluorescent probe is OLP: the fluorescent reporter group marked at the 5 '-FAM-CGGTCACCAGGAGTCACTCTAGAACG-BHQ 1-3', the fluorescent quenching group marked at the 3 '-end is FAM and the fluorescent quenching group marked at the 5' -end is BHQ1,
the concentration of the primer and the probe is 10 mu mol L;
the total volume of the fluorescent quantitative PCR reaction system is 25 mu L, wherein 2X Premix Ex TaqTM PCR (Probe qPCR) is 12.5 mu L,1 mu L of each of the upstream primer, the downstream primer and the Probe, 100ng mu L of the DNA template is 2 mu L, ROXII is 0.5 mu L, and ddH2O is 7 mu L; the fluorescent quantitative PCR reaction comprises the following steps: pre-denaturation at 95 ℃ for 5min;95 ℃ for 10s,60 ℃ for 32s,40 cycles; ending the reaction;
after the fluorescent quantitative PCR reaction is finished, according to the amplification curve judgment result, the judgment method comprises the following steps: under the conditions that the blank control and the negative control have no Ct value and no amplification curve, when a typical amplification curve appears in a sample to be detected and the Ct value is less than or equal to 35, judging that the sample to be detected is the conch in the shape of a milky ear; when the Ct value is equal to 40, judging that the conch is a non-milky conch; when the Ct value is greater than 35 and less than 40, the test is re-performed: when the Ct value is equal to 40, judging that the conch is a non-milky conch; when Ct is less than 40, the test piece is judged as a conch in the form of a milky ear.
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| CN107058592A (en) * | 2017-06-19 | 2017-08-18 | 福建出入境检验检疫局检验检疫技术中心 | A kind of method that fluorescence quantitative PCR detection identifies the white snail in Mediterranean |
| CN109182555A (en) * | 2018-10-25 | 2019-01-11 | 福建出入境检验检疫局检验检疫技术中心 | A kind of method of fluorescence quantitative PCR detection identification forest green onion snail |
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| CN107058592A (en) * | 2017-06-19 | 2017-08-18 | 福建出入境检验检疫局检验检疫技术中心 | A kind of method that fluorescence quantitative PCR detection identifies the white snail in Mediterranean |
| CN109182555A (en) * | 2018-10-25 | 2019-01-11 | 福建出入境检验检疫局检验检疫技术中心 | A kind of method of fluorescence quantitative PCR detection identification forest green onion snail |
Non-Patent Citations (4)
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| Jeannette Kneubühler等.Anatomical and phylogenetic investigation of the genera Alabastrina Kobelt, 1904, Siretia Pallary, 1926, and Otala Schumacher, 1817 (Stylommatophora, Helicidae).《ZooKeys》.2019,第843卷第1-37页. * |
| 杨海芳 ; 杨姗萍 ; 王沛 ; 赵菊鹏 ; 陈振韬 ; 周卫川 ; .乳状耳形螺的检疫鉴定与风险管理措施.植物保护.2012,第38卷(第03期),第108-111页. * |
| 王沛 ; 赵菊鹏 ; 周卫川 ; 陈元晓 ; 张卫红 ; .乳状耳形螺在中国的适生区预测与危险性评价.植物检疫.2018,(第04期),第70-73页. * |
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