CN114231650A - Primer probe composition, kit and method for detecting babesia - Google Patents
Primer probe composition, kit and method for detecting babesia Download PDFInfo
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- CN114231650A CN114231650A CN202111622228.0A CN202111622228A CN114231650A CN 114231650 A CN114231650 A CN 114231650A CN 202111622228 A CN202111622228 A CN 202111622228A CN 114231650 A CN114231650 A CN 114231650A
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly provides a primer probe composition, a kit and a method for detecting babesia. The invention provides a kit which takes a primer probe composition for detecting babesia as a main component, has short time consumption, high specificity, good repeatability and high sensitivity, and provides reliable experimental basis for diagnosing the babesia.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a primer probe composition, a kit and a method for detecting babesia.
Background
Babesia is a tick-borne hematological disease caused by a variety of babesia parasitizing in erythrocytes, and is clinically characterized by fever, mental depression, lethargy, jaundice, anemia, vomiting, dyspnea, etc., which cause great harm to the health of dogs. The conventional etiological diagnosis of the canine babesiosis is that a blood smear is a staining microscopic examination which is a main basis for making a diagnosis, but the microscopic examination has low sensitivity. The PCR technology provides a detection method with high sensitivity and good specificity for accurately diagnosing sick dogs and insect-carrying dogs in the early infection stage. And the PCR technology has the advantages of simple and convenient operation, easy reagent acquisition and high detection speed. The positive control and the negative control in the kit can effectively prevent the occurrence of false positive and false negative.
Disclosure of Invention
The invention aims to solve the problems of long operation time and low accuracy of babesia detection in the prior art.
To this end, the present invention provides a primer probe composition for detecting babesia, comprising:
a forward primer having the sequence: 5'-TTCAGCCTTGCGACCATACT-3', respectively;
a reverse primer having sequence 5'-GATACCGTCGTAGTCCTAACCA-3';
a probe, the sequence of which is: 5'-CCAGAACCCAAAGACTTTGATTTCTCTCAA-3' are provided.
Specifically, the probe is labeled with a fluorescent reporter group at the 5 'end and a fluorescent quencher group at the 3' end.
Specifically, the fluorescent reporter group comprises one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas RED or LC RED 460; the fluorescence quenching group comprises one of BHQ1, BHQ2 or BHQ 3.
Specifically, the PCR reaction solution includes any one of the primer probe compositions for detecting Babesia.
Specifically, the PCR reaction solution includes: 1-5mM MgCl220-50mM Tris, 100-500mg/l BSA, 10-100. mu.M dNTPs, 0.1-1.0. mu.M forward primer, 0.1-1.0. mu.M reverse primer, 0.1. mu.M probe.
Specifically, the kit for detecting babesia further comprises an enzyme mixed solution; the enzyme mixture comprises 1-5U/. mu.l of DNA polymerase and 1-5U/. mu.l of hot start modified antibody.
Specifically, the kit for detecting babesia further comprises a positive control and a negative control; the positive control is a babesia positive sample; the negative control was ultrapure water.
The invention also provides a method for detecting babesia, comprising the following steps:
(1) extracting DNA of a sample to be detected;
(2) carrying out fluorescent quantitative PCR reaction by taking DNA of a sample to be detected as a template, wherein the PCR reaction system comprises any one of the primer probe composition for babesia;
(3) after the PCR reaction is finished, the detection result is judged according to the Ct value.
Specifically, the PCR reaction procedure in step (2) is as follows: at 95 ℃ for 3 min; 94 ℃, 10s, 58 ℃, 20s, 40 cycles.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the kit taking the primer probe composition for detecting the babesia as the main component has the advantages of short time consumption, high specificity, good repeatability and high sensitivity, and provides reliable experimental basis for diagnosing the babesia.
The present invention will be described in further detail below with reference to the accompanying drawings.
Drawings
FIG. 1 shows the results of PCR reaction in example 2 of the present invention.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Although representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made thereto without departing from the scope of the invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and equivalents thereof.
The invention provides a primer probe composition for detecting babesia, which comprises the following components:
a forward primer having the sequence: 5'-TTCAGCCTTGCGACCATACT-3', respectively;
a reverse primer having sequence 5'-GATACCGTCGTAGTCCTAACCA-3';
a probe, the sequence of which is: 5'-CCAGAACCCAAAGACTTTGATTTCTCTCAA-3' are provided.
The 5 'end of the probe is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quenching group. Wherein the fluorescent reporter group comprises one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas RED or LC RED 460; the fluorescence quenching group comprises one of BHQ1, BHQ2 or BHQ 3.
The invention also provides a kit for detecting babesia, which comprises PCR reaction liquid, enzyme mixed liquid, positive control and negative control.
Wherein the PCR reaction solution comprises any one of the primer-probe compositions for detecting Babesia. Specifically, the PCR reaction solution includes: 1-5mM MgCl220-50mM Tris, 100-500mg/l BSA, 10-100. mu.M dNTPs, 0.1-1.0. mu.M forward primer, 0.1-1.0. mu.M reverse primer, 0.1. mu.M probe;
the enzyme mixture comprises 1-5U/. mu.l of DNA polymerase and 1-5U/. mu.l of hot start modified antibody.
The positive control is a clinically collected babesia positive sample; the negative control was ultrapure water.
The invention also provides a method for detecting babesia, comprising the following steps:
(1) extracting DNA of a sample to be detected;
(2) carrying out fluorescent quantitative PCR reaction by taking DNA of a sample to be detected as a template, wherein the PCR reaction system comprises any one of the primer probe composition for babesia; the PCR reaction program is: at 95 ℃ for 3 min; 94 ℃, 10s, 58 ℃, 20s, 40 cycles;
(3) after the PCR reaction is finished, the detection result is judged according to the Ct value.
The effects of the primer probe composition, the kit and the method for detecting babesia according to the present invention will be examined by the following specific examples.
Example 1:
the embodiment provides a kit for detecting babesia, which comprises the following components:
(1) PCR reaction solution:2mM MgCl250mM Tris, 500mg/l BSA, 100. mu.M dNTPs, 0.1. mu.M forward primer, 0.1. mu.M reverse primer, 0.1. mu.M probe.
The forward primer sequence is: 5'-TTCAGCCTTGCGACCATACT-3', respectively;
the reverse primer sequence is 5'-GATACCGTCGTAGTCCTAACCA-3';
the probe sequence is as follows: 5'-CCAGAACCCAAAGACTTTGATTTCTCTCAA-3' are provided.
The 5 'end of the probe is marked with FAM fluorescent reporter group, and the 3' end is marked with BHQ1 fluorescent quenching group.
(2) Enzyme mixture liquid: 5U/. mu.l of DNA polymerase and 1U/. mu.l of hot start antibody;
positive control: a clinically collected Babesia strong positive sample;
negative control: ultrapure water.
Example 2:
this example provides a method of detecting babesia using the kit of example 1, comprising the steps of:
(1) and taking out the kit from the refrigerator, fully melting and uniformly mixing the required reagents in the kit, and instantaneously centrifuging to remove the liquid attached to the tube wall.
(2) The number of reactions (n) required for the current experiment was counted, and the amounts of the various reagents required for the current experiment were calculated based on the reaction system shown below.
n is the number of negative controls (1T) + the number of positive controls (1T) + the amount of error retention (1T) + the number of samples
(3) Adding the reagent into a sterile centrifuge tube, fully and uniformly mixing, and then carrying out instantaneous centrifugation. The reagent was dispensed to the wells of the chip at a dispensing amount of 3.0. mu.L/well.
(4) And (3) performing DNA extraction by using a commercial nucleic acid extraction kit, and taking the DNA as a sample to be detected for later use.
(5) Respectively adding the processed sample to be detected, the positive control and the negative control into the chip reaction sample tube prepared in the step (3) by 2.0 mul respectively, enabling the reaction final volume to be 5 mul/hole, covering a PCR reaction cover, transferring to a Q3-plus minitype fluorescence quantitative PCR instrument for PCR amplification, wherein the reaction program is as follows: at 95 ℃ for 3 min; 94 ℃, 10s, 58 ℃, 20s, 40 cycles; detecting fluorescence selects FAM.
The threshold setting principle is that the threshold line just exceeds the highest point of the normal negative control.
The positive control Ct value is less than 38, and the negative control Ct value is not high; the fluorescence curve above the threshold should have a pronounced sigmoidal curve, otherwise the experiment should be considered invalid and errors in instrumentation, reagents, amplification conditions, etc. should be checked.
When the method provided by the invention is adopted to detect the babesia, the judgment standard is as follows:
1. and if the Ct of the detection sample is less than or equal to 38.0, the detection sample is judged to be positive.
2. And (4) recommending the redo for the detection sample with the Ct more than 38.0 and less than or equal to 40, wherein the sample with the Ct value less than 40 in the redo result is positive, and the sample with the Ct value less than 40 in the redo result is negative.
The above examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention, which is intended to be covered by the claims and any design similar or equivalent to the scope of the invention.
Claims (9)
1. A primer probe composition for detecting babesia, comprising:
a forward primer having the sequence: 5'-TTCAGCCTTGCGACCATACT-3', respectively;
a reverse primer having sequence 5'-GATACCGTCGTAGTCCTAACCA-3';
a probe, the sequence of which is: 5'-CCAGAACCCAAAGACTTTGATTTCTCTCAA-3' are provided.
2. The primer-probe composition for detecting babesia as claimed in claim 1, wherein: the 5 'end of the probe is marked with a fluorescence reporter group, and the 3' end of the probe is marked with a fluorescence quenching group.
3. The primer-probe composition for detecting babesia as claimed in claim 2, wherein: the fluorescent reporter group comprises one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas RED or LC RED 460; the fluorescence quenching group comprises one of BHQ1, BHQ2 or BHQ 3.
4. A kit for detecting babesia, characterized in that: comprises a PCR reaction solution; the PCR reaction solution comprises the primer probe composition for detecting Babesia according to any one of claims 1 to 3.
5. The kit for detecting babesia as set forth in claim 4, wherein the PCR reaction solution comprises: 1-5mM MgCl220-50mM Tris, 100-500mg/l BSA, 10-100. mu.M dNTPs, 0.1-1.0. mu.M forward primer, 0.1-1.0. mu.M reverse primer, 0.1. mu.M probe.
6. The kit for detecting babesia as set forth in claim 4, wherein: also comprises enzyme mixed liquor; the enzyme mixture comprises 1-5U/. mu.l of DNA polymerase and 1-5U/. mu.l of hot start modified antibody.
7. The kit for detecting babesia as set forth in claim 4, wherein: positive and negative controls are also included; the positive control is a babesia positive sample; the negative control was ultrapure water.
8. A method for detecting babesia comprising the steps of:
(1) extracting DNA of a sample to be detected;
(2) performing a fluorescent quantitative PCR reaction by using DNA of a sample to be detected as a template, wherein the PCR reaction system comprises the primer probe composition for babesia as claimed in any one of claims 1 to 3;
(3) after the PCR reaction is finished, the detection result is judged according to the Ct value.
9. The method for detecting ureaplasma urealyticum according to claim 8, wherein the PCR reaction procedure in the step (2) is: at 95 ℃ for 3 min; 94 ℃, 10s, 58 ℃, 20s, 40 cycles.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111622228.0A CN114231650A (en) | 2021-12-28 | 2021-12-28 | Primer probe composition, kit and method for detecting babesia |
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| CN202111622228.0A CN114231650A (en) | 2021-12-28 | 2021-12-28 | Primer probe composition, kit and method for detecting babesia |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219837A (en) * | 2014-06-04 | 2016-01-06 | 中国人民解放军军事医学科学院微生物流行病研究所 | A kind of method and dedicated kit thereof detecting babesia |
| US20190024189A1 (en) * | 2017-07-18 | 2019-01-24 | Roche Molecular Systems, Inc. | Compositions and methods for detection of babesia |
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2021
- 2021-12-28 CN CN202111622228.0A patent/CN114231650A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219837A (en) * | 2014-06-04 | 2016-01-06 | 中国人民解放军军事医学科学院微生物流行病研究所 | A kind of method and dedicated kit thereof detecting babesia |
| US20190024189A1 (en) * | 2017-07-18 | 2019-01-24 | Roche Molecular Systems, Inc. | Compositions and methods for detection of babesia |
Non-Patent Citations (2)
| Title |
|---|
| JING LI等: "Development of a pan-Babesia FRET-qPCR and a survey of livestock from five Caribbean islands", BMC VET RES, vol. 11, pages 246 * |
| 杨伦: "犬吉氏巴贝斯虫PCR方法的建立及其临床病理学变化的研究", 中国优秀硕士学位论文全文数据库农业科技辑, no. 4, pages 050 - 429 * |
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