CN113801944A - Primer, probe, kit and application for detecting taenia bovis - Google Patents
Primer, probe, kit and application for detecting taenia bovis Download PDFInfo
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Abstract
The invention discloses a primer, a probe, a kit and application for detecting taenia bovis, and belongs to the technical field of biological engineering. The primers can amplify the conserved region in the bovine taenia mitochondrial cytochrome C oxidase 1 gene, and the probe is hybridized with the conserved region of the bovine taenia mitochondrial cytochrome C oxidase 1 gene amplified by the primers. The primers, the probes and the kit are used for detecting the taenia bovis, have high sensitivity and strong specificity, and are suitable for popularization and application.
Description
Technical Field
The invention belongs to the technical field of biological engineering, and particularly relates to a primer, a probe, a kit and application for detecting tapeworm.
Background
Taenia bovis (Taenia sapinata), also known as beef tapeworm, obesity tapeworm or hamate tapeworm, is a zoonosis parasite of the genus Taenidae (Talaphiida) of the order Torulata (Cestoda) of the phylum Platyhelminths (Plastylenchs). It is human as its only terminal host; cattle and buffalo are the most important intermediate hosts; wild goats, boars, reindeer, llamas, hornhorses, foxes, sheep, and the like may also be used as intermediate hosts.
The taenia bovis is an intestinal taeniasis caused by the adult taeniasis bovis parasitizing the small intestine of a human body, is mainly prevalent in regions eating beef, particularly in regions with a habit of eating beef, and most of the common regions have sporadic cases.
The cysticercosis cellulosae is also called as cysticercosis bovis, and the pathogen is the larva of the taenia bovis, namely the cysticercosis cellulosae (which can parasitize all the muscle tissues and organs such as lung, liver, brain and the like of a cow, and causes mechanical damage and even neurological symptoms, so that the development of animal husbandry is influenced, and great economic loss is caused.
The existing methods for detecting the taenia bovis comprise smear detection, immunological detection and conventional PCR detection, but the detection specificity and sensitivity of the existing methods are to be improved. Therefore, how to provide a product with high sensitivity and strong specificity for detecting the taenia bovis is a problem to be solved urgently in the field.
Disclosure of Invention
The invention discloses a primer, a probe and a kit for detecting taenia bovis, which are used for detecting the taenia bovis and have high sensitivity and strong specificity.
In order to achieve the purpose, the invention adopts the following technical scheme:
the primers for detecting the taenia bovis comprise a forward primer and a reverse primer,
the forward primer and the reverse primer are used for amplifying a conserved region in the bovine taenia mitochondrial cytochrome C oxidase 1 gene.
Mitochondrial Cytochrome C oxidase 1 (COX 1) is a rate-limiting enzyme at the end of a respiratory chain, participates in important physiological processes such as energy supply, apoptosis, metabolism, active oxygen generation and the like, and a primer is designed by utilizing a gene conserved region of the Cytochrome C oxidase 1 for amplification detection of the taenia bovis, so that the sensitivity is high and the specificity is strong.
Preferably, the forward primer nucleotide sequence is: 5'-CGTTTAAATGCTTYAAGTGCR TG GTT-3', SEQ ID NO: 3;
the reverse primer nucleotide sequence is: 5'-CTACTTGAAAATAATGAYGACGACAAA G-3' and SEQ ID NO: 4.
The application of the primer for detecting the taenia bovis Hemsl in preparing a product for detecting the taenia bovis Hemsl.
A probe for detecting a Taenia bovis bacterium, which hybridizes to a conserved region of the Taenia bovis mitochondrial cytochrome C oxidase 1 gene amplified by the primer of claim 1 or 2.
Preferably, the probe nucleotide sequence is 5'FAM-GTTCGGTTACTATGATAATAGG AGTACCAACAGG-MGB 3' and SEQ ID NO: 5.
The application of the primer for detecting the taenia bovis Hemsl in preparing a product for detecting the taenia bovis Hemsl.
The kit for detecting the taenia bovis comprises a primer for detecting the taenia bovis.
Preferably, the kit for detecting taenia bovis further comprises the probe for detecting taenia bovis.
Preferably, the kit for detecting taenia bovis further comprises a standard substance, and the standard substance is a plasmid containing the taenia bovis mitochondrial cytochrome C oxidase 1 gene.
The kit can be used for detecting the crowds in the epidemic area of the taenia bovis seu Bubali disease or the crowds with the history of the epidemic area, and can also be used for strengthening quarantine inspection, preventing the beef and products thereof containing the taenia bovis seu Bubali disease from flowing into the market, further controlling and preventing the propagation of the beef and products thereof in time, and being beneficial to the development of animal husbandry and the improvement of economy.
A method for detecting the taenia bovis is to extract a sample DNA to be detected as a template and use the kit to carry out real-time fluorescence quantitative PCR detection.
In conclusion, the primer, the probe and the kit are used for detecting the taenia bovis, have high sensitivity and strong specificity, and are suitable for popularization and application.
Drawings
FIG. 1 shows the results of conventional PCR detection of Taenia bovis;
among them, lane 1 is DNA of healthy tissue, and lane 2 is DNA of Taenia bovis.
FIG. 2 shows the Real-time PCR standard curve.
FIG. 3 shows the results of Real-time PCR sensitivity experiments;
wherein, 1, 2: 0.1 ng; 3. 4: 10 pg; 5. 6: 1 pg; 7. 8: 100 fg; 9. 10: 10 fg; 11. 12: 1 fg; 13: 0.1fg Taenia bovis DNA.
FIG. 4 shows the results of conventional PCR sensitivity experiments;
wherein, 1: h2O; 2-9: 0.1ng, 10pg, 1pg, 100fg, 10fg, 1fg, 0.1fg and 0.01fg of Taenia bovis DNA, respectively.
FIG. 5 shows the results of Real-time PCR assay clinical samples.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following examples, the experimental procedures without specifying the specific conditions were generally carried out by the methods described in "molecular biology laboratory Manual of Fine text" (F.M. Osber, R.E. Kingston, J.G. Sedman, et al, Mashimi, Shujiong, et al, Beijing: scientific Press, 2004).
Example 1 establishment of Real-time PCR detection method for Taenia bovis
1. Preparation of a Taenia bovis DNA sample
The method comprises the following steps of transferring about 20mg of clinically collected and separated taenia bovis adult tablets into a precooled mortar, slowly pouring a proper amount of liquid nitrogen, quickly and forcefully grinding into homogenate, extracting the genome DNA of an insect body according to the instruction of a tissue DNA extraction kit, measuring the concentration and the purity, and establishing a detection template for the method.
2. Cloning of target gene and construction of recombinant positive plasmid
According to the sequence (AB271695.1) of the taenia bovis mitochondrial COX1 gene logged in GenBank, specific primers P1: 5'-ATATTTACTTTAGATCATAAGCGG-3' (SEQ ID NO.1) and P2: 5'-ACGAGAAAATATATTAGTCATAAA-3' (SEQ ID NO.2) are designed as upstream and downstream primers, and conventional PCR amplification is carried out by using the taenia bovis DNA extracted in the step 1 as a template.
Conventional PCR reaction system:
a total volume of 25. mu.L containing 2.5. mu.L of 10 XPCR buffer, 2.5. mu.L of 2mM dNTP, 0.2. mu.L of 0.5U AmpliTaq Gold Taq DNA polymerase (Applied Biosystems, USA), 1. mu.L of 10. mu. M P1 primer, 1. mu.L of 10. mu. M P2 primer, 1. mu.L (50ng) of template DNA, and 16.8. mu.L deionized water.
Conventional PCR reaction conditions:
pre-denaturation at 94 ℃ for 3 min; at 94 ℃ for 45s, at 52 ℃ for 45s and at 72 ℃ for 1min, for 35 cycles; after the circulation is finished, the extension is carried out for 10min at 72 ℃.
The PCR product was detected by 1% agarose gel electrophoresis, and the result is shown in FIG. 1, wherein 513bp target fragment was specifically amplified, and the target fragment was not amplified in the DNA control group extracted from healthy tissue.
And (3) recovering and purifying the specific amplification product by using a DNA gel recovery kit, connecting the purified fragment to a PMD18-T vector, transforming the positive plasmid into DH5 alpha receptor peptide cells, screening by using a resistance culture medium, and selecting a positive clone. And (3) inoculating the positive clone to a liquid LB culture medium, extracting a plasmid, determining a plasmid sequence, and comparing and verifying the plasmid sequence with a COX1 gene of Taenia bovis in an NCBI database to confirm that the recombinant positive plasmid is successfully constructed.
3. Establishment of Real-time PCR detection method for taenia bovis and preparation of standard curve
Design of Real-time PCR primers and probes:
primer P3:5'-CGTTTAAATGCTTYAAGTGCRTGGTT-3' (SEQ ID NO. 3);
primer P4:5'-CTACTTGAAAATAATGAYGACGACAAAG-3' (SEQ ID number 4);
probe (Probe): 5'FAM-GTTCGGTTACTATGATAATAGGAGTACCAAC AGG-MGB 3' (SEQ ID NO. 5);
the amplified fragment size was 128 bp.
And (3) taking the recombinant positive plasmid constructed in the step (2) as a standard substance, diluting 8 gradients by 10 times of deionized water as a template, and carrying out amplification detection by using the Real-time PCR primer and the probe.
Real-time PCR reaction system:
10 mu L of Premix Ex Taq (Probe qPCR), 1 mu L (200nM) of each of the upstream and downstream primers (P3, P4), 1 mu L (400nM) of the Probe, 1 mu L (50ng) of the template DNA, and adding deionized water to make the system reach 20 mu L;
real-time PCR reaction conditions:
pre-denaturation at 95 ℃ for 10 min; 95 ℃ for 15s, 60 ℃ for 60s, 40 cycles.
The Real-time PCR reaction was performed on an Applied Biosystems 7500 fluorescent Real-time quantitative PCR instrument, the results were analyzed by fluorescent Real-time quantitative PCR software, the obtained Real-time PCR standard curve is shown in FIG. 2, and the correlation parameter R of the standard curve2The amplification efficiency E of 0.9903 is preferably 1.02.
Example 2 application of Real-time PCR detection method of Taenia bovis
Real-time PCR specificity assay
Trichina, trypanosoma, coccidia and cryptosporidium are all provided by the animal parasite emphasis laboratory of the department of agriculture. All polypide DNAs were extracted as templates, and COX1 target gene was amplified using the P3 primer, P4 primer, probe, Real-time PCR system and reaction conditions in example 1, and the specificity was observed.
The result shows that the Real-time PCR detection method for the taenia bovis, which is established in the example 1, has strong specificity only to the taenia bovis DNA and has no specific amplification to other parasites.
Real-time PCR sensitivity test
And purifying DNA extracted from the Taenia bovis Seu Bubali, diluting by 8 gradients in a double ratio, performing contrast detection by respectively adopting two methods of conventional PCR and Real-time PCR under the condition of consistent template amount, and observing the lower limit of detection.
Conventional PCR reaction system:
a total volume of 25. mu.L, contained 2.5. mu.L of 10 XPCR buffer, 2.5. mu.L of 2mM dNTP, 0.2. mu.L of 0.5U AmpliTaq Gold Taq DNA polymerase (Applied Biosystems, USA), 1. mu.L of 10. mu. M P3 primer, 1. mu.L of 10. mu. M P4 primer, 1. mu.L (50ng) of template DNA, and 16.8. mu.L deionized water.
Conventional PCR reaction conditions:
pre-denaturation at 94 ℃ for 3 min; at 94 ℃ for 45s, at 52 ℃ for 45s and at 72 ℃ for 1min, for 35 cycles; after the circulation is finished, the extension is carried out for 10min at 72 ℃.
The Real-time PCR method includes the primer P3, the primer P4, the probe, the Real-time PCR reaction system and the reaction conditions in example 1.
As a result, as shown in FIGS. 3 and 4, the initial concentration was 0.1ng, 8 gradients were diluted 10-fold (0.1ng, 10pg, 1pg, 100fg, 10fg, 1fg, 0.1fg and 0.01fg), the lower detection limit of Real-time PCR was 1fg (FIG. 3, 2 replicates per sample), and the lower detection limit of conventional PCR was 100fg (FIG. 4), whereby it was found that the sensitivity of Real-time PCR was 100 times that of conventional PCR.
3. Clinical detection of animal infection with taenia bovis
And (3) taking the extracted taenia bovis DNA as a positive control and the DNA of healthy animal tissues as a negative control, and respectively detecting the sample by adopting a conventional PCR (polymerase chain reaction) method and a Real-time PCR method.
The positive samples of the conventional PCR detection are subjected to Real-time PCR detection, the Real-time PCR method can detect the infection of the taenia bovis from the low-concentration template (figure 5), and all curves in figure 5 are respectively randomly extracted positive samples with different strengths.
Various modifications to the above-described embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The primer for detecting the taenia bovis is characterized in that,
comprises a forward primer and a reverse primer,
the forward primer and the reverse primer are used for amplifying a conserved region in the bovine taenia mitochondrial cytochrome C oxidase 1 gene.
2. The primer for detecting Taenia bovis as claimed in claim 1,
the nucleotide sequence of the forward primer is as follows: 5'-CGTTTAAATGCTTYAAGTGCRTGGTT-3', SEQ ID NO: 3;
the reverse primer nucleotide sequence is as follows: 5'-CTACTTGAAAATAATGAYGACGACAAAG-3' and SEQ ID NO: 4.
3. The use of the primer for detecting taenia bovis as claimed in claim 1 or 2 in the preparation of a product for detecting taenia bovis.
4. A probe for detecting Taenia bovis is characterized in that,
the probe hybridizes to a conserved region of the bovine taenia mitochondrial cytochrome C oxidase 1 gene amplified by the primer of claim 1 or 2.
5. The probe for detecting Taenia bovis as claimed in claim 4,
the probe nucleotide sequence is 5'FAM-GTTCGGTTACTATGATAATAGGAGTACCAACAGG-MGB 3' and SEQ ID NO: 5.
6. The use of the primer for detecting taenia bovis as claimed in claim 4 or 5 in the preparation of a product for detecting taenia bovis.
7. A kit for detecting taenia bovis is characterized in that,
comprising the primer for detecting taenia bovis as claimed in claim 1 or 2.
8. The kit for detecting Taenia bovis as claimed in claim 7,
further comprising the probe for detecting Taenia bovis as claimed in claim 4 or 5.
9. The kit for detecting Taenia bovis as claimed in claim 8,
the kit also comprises a standard substance, wherein the standard substance is a plasmid containing the taenia nivalis mitochondrial cytochrome C oxidase 1 gene.
10. A method for detecting taenia bovis is characterized in that,
extracting DNA of a sample to be tested as a template, and performing real-time fluorescence quantitative PCR detection by using the kit of claim 8 or 9.
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|---|---|---|---|---|
| WO1997036007A1 (en) * | 1996-03-25 | 1997-10-02 | The Regents Of The University Of California | Specific dna probes for the identification of the taenia solium and taenia saginata species |
| WO2005090989A1 (en) * | 2004-03-17 | 2005-09-29 | Japan Science And Technology Agency | Agent and method for examining infection with adult taeniasis solium and taeniarhynchus saginatus |
| CN102424837A (en) * | 2011-12-09 | 2012-04-25 | 中国农业科学院兰州兽医研究所 | Kit for identifying and distinguishing types of human taeniasis |
| CN108342490A (en) * | 2018-04-17 | 2018-07-31 | 湖南农业大学 | A kind of primer sets, kit and its application method of detection Spirometra mansoni |
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- 2021-09-27 CN CN202111137214.XA patent/CN113801944A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997036007A1 (en) * | 1996-03-25 | 1997-10-02 | The Regents Of The University Of California | Specific dna probes for the identification of the taenia solium and taenia saginata species |
| WO2005090989A1 (en) * | 2004-03-17 | 2005-09-29 | Japan Science And Technology Agency | Agent and method for examining infection with adult taeniasis solium and taeniarhynchus saginatus |
| CN102424837A (en) * | 2011-12-09 | 2012-04-25 | 中国农业科学院兰州兽医研究所 | Kit for identifying and distinguishing types of human taeniasis |
| CN108342490A (en) * | 2018-04-17 | 2018-07-31 | 湖南农业大学 | A kind of primer sets, kit and its application method of detection Spirometra mansoni |
Non-Patent Citations (4)
| Title |
|---|
| JEROEN H ROELFSEMA ET AL.: "Novel PCRs for differential diagnosis of cestodes", 《EXP PARASITOL》, vol. 161, pages 1 - 2 * |
| LEIGH CUTTELL ET AL.: "Bovine cysticercosis--development of a real-time PCR to enhance classification of suspect cysts identified at meat inspection", 《VET PARASITOL》, vol. 194, no. 1, pages 65 - 69, XP028584915, DOI: 10.1016/j.vetpar.2013.02.018 * |
| ROSANA W S POON ET AL.: "Molecular identification of cestodes and nematodes by cox1 gene real-time PCR and sequencing", 《DIAGN MICROBIOL INFECT DIS》, vol. 89, no. 3, pages 187 * |
| 陈峥宏 等: "用cox1基因片段的PCR鉴别亚洲带绦虫和牛带绦虫", 《贵阳医学院学报》, vol. 34, no. 3, pages 240 * |
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