CN112176074A - Real-time fluorescent PCR primer probe and method for detecting patinopecten yessoensis - Google Patents

Real-time fluorescent PCR primer probe and method for detecting patinopecten yessoensis Download PDF

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CN112176074A
CN112176074A CN202011213818.3A CN202011213818A CN112176074A CN 112176074 A CN112176074 A CN 112176074A CN 202011213818 A CN202011213818 A CN 202011213818A CN 112176074 A CN112176074 A CN 112176074A
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time fluorescence
fluorescence pcr
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patinopecten yessoensis
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王庆志
杨滴
李华琳
滕炜鸣
刘项峰
于佐安
谢玺
李大成
傅志宇
张明
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LIAONING OCEAN AND FISHERIES SCIENCE RESEARCH INSTITUTE
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Abstract

A real-time fluorescent PCR primer probe and a method for detecting Japanese scallops are disclosed, wherein the PCR primer probe comprises the following components: upstream primer P1: TGGCTTCTGCCTTTGTTGTTG, respectively; the downstream primer P2: GCAAAAGGGACAAGCCAAAA, respectively; the probe sequence with the fluorescent dye is as follows: AGATTCCCTCGGGTCAACGCTCTGA are provided. During detection, firstly, extracting the genomic DNA of a sample to be detected; and then taking the extracted DNA as a template, adding the primer probe and the enzyme reaction solution to perform real-time fluorescence PCR reaction, and recording the Ct value of the sample reaction. The advantages are that: the method has the advantages of good sensitivity and specificity, simple and quick operation and accurate and reliable detection result, provides a simple, effective and accurate detection method for the identification and detection of the patinopecten yessoensis, and provides a powerful basis for the production and seedling raising, scientific research and market consolidation of the patinopecten yessoensis.

Description

Real-time fluorescent PCR primer probe and method for detecting patinopecten yessoensis
Technical Field
The invention belongs to the technical field of shellfish identification detection, and relates to a primer and a probe for real-time fluorescence PCR detection and a method for identifying and detecting comb shells by using the primer and the probe.
Background
Patinopecten yessoensis (Patinopecten (Mizuhopecten) yessoensis) belongs to the phylum mollusca (Molluea), the class of Paciferogil (Lamellibranchia), the class of Pterimorpha (Pterimorpha), the order of Pteriidae (Pectinidae), the genus Patinopecten (Mizuhopecten). Patinopecten yessoensis is distributed in Korea, Russian far east coast, south water area of Qiandao island, North sea of Japan and North of Benzhou, and introduced into China in 1982. The method is mainly distributed in northern parts of the yellow sea and in sea areas such as Shandong, Liaoning and the like in China, and the Dalian and the Shandong and the like in the northern parts form a certain culture scale. The patinopecten yessoensis has large individual, high yield and delicious meat taste, and the adductor muscle contains rich nutrient substances, so that the patinopecten yessoensis has become one of the main cultured shellfish in China at present.
The traditional method for identifying Japanese scallops mainly relies on morphological criteria such as: shell color, byssus holes, radial ribs, spines, etc. These morphological features are highly plastic, are greatly affected by the environment, and have artificial subjective tendencies. Usually, the processed patinopecten yessoensis only leaves adductor muscle, and loses morphological characteristics; no discernible morphological characteristics are formed in the planktonic stage and juvenile stage of the Japanese scallop larvae. Therefore, the conventional morphological identification cannot accurately identify the Japanese scallop in these cases. Therefore, the development of an efficient and practical method for identifying the comb shells based on the molecular biology technology provides a powerful basis for the production and seedling culture, scientific research and market consolidation of the comb shells.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art and provides a real-time fluorescent PCR primer probe and a method for detecting comb shells.
The technical solution of the invention is as follows:
the real-time fluorescent PCR primer probe for detecting the comb shells comprises the following components:
upstream primer P1: TGGCTTCTGCCTTTGTTGTTG, respectively;
the downstream primer P2: GCAAAAGGGACAAGCCAAAA, respectively;
the probe sequence with the fluorescent dye is as follows: AGATTCCCTCGGGTCAACGCTCTGA are provided.
Furthermore, the fluorescent dye has a FAM label at the 5 'end and a TAMRA label at the 3' end.
The real-time fluorescent PCR method for detecting the comb shells by using the real-time fluorescent PCR primer probe comprises the following steps:
a. extracting the genomic DNA of a sample to be detected;
b. adding the primer probe of claim 1 and the enzyme reaction solution into the extracted DNA as a template to perform real-time fluorescence PCR reaction, and recording the Ct value of the sample reaction;
the real-time fluorescent PCR reaction system is as follows:
Figure BDA0002759658850000021
the real-time fluorescent PCR reaction conditions are as follows: 20s at 95 ℃ for 1 cycle; 95 ℃ for 3s, 60 ℃ for 30s, 40 cycles.
The real-time fluorescence PCR method is provided with a blank control, a negative control and a positive control, and the determination standard is as follows:
blank control: by ddH2Performing real-time fluorescence PCR reaction according to the real-time fluorescence PCR reaction system and the real-time fluorescence PCR reaction conditions by taking O as a template, and detecting that the Ct value is more than or equal to 40;
negative control: taking the genomic DNA of the non-patinopecten yessoensis species as a template, carrying out real-time fluorescence PCR reaction according to the real-time fluorescence PCR reaction system and the real-time fluorescence PCR reaction conditions, and detecting that the Ct value is more than or equal to 40;
positive control: taking the patinopecten yessoensis species genome DNA as a template, carrying out real-time fluorescence PCR reaction according to the real-time fluorescence PCR reaction system and the real-time fluorescence PCR reaction conditions, and detecting that the Ct value is less than or equal to 30;
judging that the patinopecten yessoensis gene of the sample to be detected is not detected when the detection Ct value of the patinopecten yessoensis gene is more than or equal to 40 and the negative control, positive control and blank control results are normal; judging that the patinopecten yessoensis gene detected by the sample is normal if the Ct value of the patinopecten yessoensis gene detection of the sample to be detected is less than or equal to 36 and the negative control, positive control and blank control results are normal; the Ct value of the gene detection of the patinopecten yessoensis of the sample to be detected is between 36 and 40, the real-time fluorescence PCR amplification is repeated, the Ct value of the result after the secondary amplification is larger than 40, and the negative control, positive control and blank control results are normal, and the sample is judged to have no patinopecten yessoensis gene; and (5) after the secondary amplification, determining that the CT value of the result is still less than 40, and the negative control, positive control and blank control results are normal, and determining that the patinopecten yessoensis gene is detected from the sample.
The invention has the beneficial effects that:
by utilizing a molecular biological means and detecting a specific fragment in a species DNA sequence, the defect of morphological identification is effectively solved, and an efficient solution is provided for the rapid identification and detection of the patinopecten yessoensis sample. Compared with the common PCR method, the real-time fluorescence PCR method has the advantages of simple and convenient operation, high speed, integration of product detection and quantitative analysis and the like.
The real-time fluorescent PCR primer probe for detecting the comb shells designed by the invention has good sensitivity and specificity. The real-time fluorescence PCR specific amplification method can be used for rapidly detecting whether the sample contains the Japanese scallop, exerts the advantages of the real-time fluorescence PCR detection technology, and provides a powerful basis for the production and seedling raising, scientific research and market consolidation of the Japanese scallop.
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FIG. 1 is a fluorescence PCR amplification chart of a positive sample of Japanese scallop detected by the embodiment of the present invention;
wherein: 1 is the adductor muscle of Japanese scallop, 2, 3 is the tissue sample of Japanese scallop.
Detailed Description
Example 1
1. Primers and probes used:
the COX I gene of comb shell mtDNA is selected as an object, real-time fluorescent PCR specific amplification primers and probes are designed through Primer Express 3.0 software, and the real-time fluorescent PCR primers and the probes used in the invention are synthesized by biological engineering (Shanghai) corporation.
The real-time fluorescent PCR primer and probe sequence is as follows:
upstream primer P1: TGGCTTCTGCCTTTGTTGTTG, respectively;
the downstream primer P2: GCAAAAGGGACAAGCCAAAA, respectively;
and (3) probe: AGATTCCCTCGGGTCAACGCTCTGA, the probe is labeled at the 5 'end with FAM, a reporter fluorescent dye, and at the 3' end with TAMRA, a quencher fluorescent dye.
2. Extracting the patinopecten yessoensis sample DNA:
placing Japanese scallop sample in mortar, adding liquid nitrogen, grinding, mixing, collecting liquid nitrogen mixture of 200mg sample, and adopting Promega
Figure BDA0002759658850000031
The RSC Tissue DNA Kit extraction Kit extracts sample DNA, dissolves the extracted DNA in 100 mu L TE, and is stored in a refrigerator at the temperature of 20 ℃ below zero for standby.
3. Establishing a real-time fluorescent PCR amplification reaction system
TABLE 1 real-time fluorescent PCR reaction System
Figure BDA0002759658850000032
Figure BDA0002759658850000041
4. Selecting corresponding fluorescence channels according to the operation requirements of the instrument, and setting the amplification reaction conditions at 95 ℃ for 20s1 cycles; and recording the Ct value of the sample reaction at 95 ℃ for 3s and 60 ℃ for 30s for 40 cycles.
And after the detection is finished, judging the result according to the amplification curve and the Ct value.
5. The determining the quality control indicator is:
blank control: by ddH2Performing real-time fluorescence PCR reaction by using O as a template according to the conditions of 3 and 4, and detecting that the Ct value is more than or equal to 40;
negative control: taking the genomic DNA of the non-patinopecten yessoensis species as a template, carrying out real-time fluorescence PCR reaction according to the conditions of 3 and 4, and detecting that the Ct value is more than or equal to 40;
positive control: taking the genomic DNA of the Japanese scallop as a template, carrying out real-time fluorescence PCR reaction according to the conditions of 3 and 4, and detecting that the Ct value is less than or equal to 30;
6. and (4) judging a result:
judging that the patinopecten yessoensis gene of the sample to be detected is not detected when the detection Ct value of the patinopecten yessoensis gene is more than or equal to 40 and the negative control, positive control and blank control results are normal;
judging that the patinopecten yessoensis gene detected by the sample is normal if the Ct value of the patinopecten yessoensis gene detection of the sample to be detected is less than or equal to 36 and the negative control, positive control and blank control results are normal;
the Ct value of the gene detection of the patinopecten yessoensis of the sample to be detected is between 36 and 40, the real-time fluorescence PCR amplification is repeated, the Ct value of the result after the secondary amplification is larger than 40, and the negative control, positive control and blank control results are normal, and the sample is judged to have no patinopecten yessoensis gene; and (5) after the secondary amplification, determining that the CT value of the result is still less than 40, and the negative control, positive control and blank control results are normal, and determining that the patinopecten yessoensis gene is detected from the sample.
7. And (3) detection result and analysis:
preferably, the quantitative fluorescence PCR instrument of this embodiment is Quant Studio 7Flex
In the specificity and sensitivity verification experiment, the fluorescence PCR reaction is repeated three times, the Ct value representing the result represents the number of cycles that the fluorescence signal in each reaction tube passes when reaching the set threshold value, and the average value of the three results is taken; SD represents the standard deviation.
Example 2
1) Specificity verification of primers and probes
The primers and probes designed in this example were specifically verified to show that the primers and probes have different specificities at different annealing temperatures. The results show that the annealing temperature is 60 ℃ and the specificity is optimal, at this time, only the target species of Japanese scallop is positively amplified, which is shown in Table 2
TABLE 2 primer Probe specificity verification
Figure BDA0002759658850000051
Note: real-time fluorescent PCR annealing temperature; 2- (c) -: ct value is lower than detection limit.
2) Primer and probe sensitivity verification
According to the COX I gene sequence of the patinopecten yessoensis, a plasmid standard product is constructed by cloning and sequencing, the plasmid DNA is subjected to gradient dilution, and the sensitivity of a primer and a probe is detected. The results show that as shown in Table 3, when the DNA concentration was diluted to 100 copies, the Ct value obtained was 34.5; when the DNA concentration was diluted to 50 copies, the Ct value was already below the limit of detection. The sensitivity of the primer and probe is about 100 copies.
Figure BDA0002759658850000052
Note: real-time fluorescent PCR annealing temperature; 2- (c) -: ct value is lower than detection limit.
3) Summary of the invention
In the embodiment 1 of the invention, the tissue samples of the adductor muscle of Patinopecten yessoensis and Patinopecten yessoensis to be detected are subjected to real-time fluorescence PCR detection after DNA is extracted. Through detection, the samples of the comb shell muscle of the comb shell and the comb shell tissue of the comb shell containing the gene of the comb shell show positive amplification curves as shown in figure 1, and the results of the embodiment show that the primer probe of the invention has good specificity.
The above description is only exemplary of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (5)

1. A real-time fluorescent PCR primer probe and a method for detecting Japanese scallops are characterized in that:
respectively as follows:
upstream primer P1: TGGCTTCTGCCTTTGTTGTTG, respectively;
the downstream primer P2: GCAAAAGGGACAAGCCAAAA, respectively;
the probe sequence with the fluorescent dye is as follows: AGATTCCCTCGGGTCAACGCTCTGA are provided.
2. The real-time fluorescent PCR primer probe and method for detecting Japanese scallops according to claim 1, wherein the real-time fluorescent PCR primer probe comprises the following components: the fluorescent dye has FAM mark at the 5 'end and TAMRA mark at the 3' end.
3. A real-time fluorescence PCR method for detecting Japanese scallops by using the real-time fluorescence PCR primer probe as claimed in claim 1, which is characterized in that:
the method comprises the following steps:
a. extracting the genomic DNA of a sample to be detected;
b. adding a real-time fluorescent PCR primer probe and an enzyme reaction solution into the extracted DNA serving as a template to perform real-time fluorescent PCR reaction, and recording the Ct value of the sample reaction;
the real-time fluorescent PCR reaction system is as follows:
Figure FDA0002759658840000011
4. the real-time fluorescence PCR method for detecting Japanese scallops according to claim 3, wherein: the real-time fluorescent PCR reaction conditions are as follows: 20s at 95 ℃ for 1 cycle; 95 ℃ for 3s, 60 ℃ for 30s, 40 cycles.
5. The real-time fluorescence PCR method for detecting Japanese scallops according to claim 4, wherein: the real-time fluorescence PCR method is provided with a blank control, a negative control and a positive control, and the determination standard is as follows:
blank control: by ddH2Performing real-time fluorescence PCR reaction according to the real-time fluorescence PCR reaction system and the real-time fluorescence PCR reaction conditions by taking O as a template, and detecting that the Ct value is more than or equal to 40;
negative control: taking the genomic DNA of the non-patinopecten yessoensis species as a template, carrying out real-time fluorescence PCR reaction according to the real-time fluorescence PCR reaction system and the real-time fluorescence PCR reaction conditions, and detecting that the Ct value is more than or equal to 40;
positive control: taking the patinopecten yessoensis species genome DNA as a template, carrying out real-time fluorescence PCR reaction according to the real-time fluorescence PCR reaction system and the real-time fluorescence PCR reaction conditions, and detecting that the Ct value is less than or equal to 30;
judging that the patinopecten yessoensis gene of the sample to be detected is not detected when the detection Ct value of the patinopecten yessoensis gene is more than or equal to 40 and the negative control, positive control and blank control results are normal; judging that the patinopecten yessoensis gene detected by the sample is normal if the Ct value of the patinopecten yessoensis gene detection of the sample to be detected is less than or equal to 36 and the negative control, positive control and blank control results are normal; the Ct value of the gene detection of the patinopecten yessoensis of the sample to be detected is between 36 and 40, the real-time fluorescence PCR amplification is repeated, the Ct value of the result after the secondary amplification is larger than 40, and the negative control, positive control and blank control results are normal, and the sample is judged to have no patinopecten yessoensis gene; and (5) after the secondary amplification, determining that the CT value of the result is still less than 40, and the negative control, positive control and blank control results are normal, and determining that the patinopecten yessoensis gene is detected from the sample.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112695105A (en) * 2021-02-18 2021-04-23 辽宁省海洋水产科学研究院 Real-time fluorescence PCR identification method of chlamys farreri
CN115807109A (en) * 2022-12-20 2023-03-17 中国海洋大学三亚海洋研究院 PCR technology-based scallop variety identification method and specific primers thereof
KR102586202B1 (en) * 2022-12-01 2023-10-10 순천향대학교 산학협력단 Primer set for species discrimination of Mizuhopecten yessoensis, Argopecten irradians and Chlamys farreri nipponensis and method of determining species of Mizuhopecten yessoensis, Argopecten irradians and Chlamys farreri nipponensis using the same
KR102592643B1 (en) * 2022-12-01 2023-10-24 순천향대학교 산학협력단 Primer set for species discrimination of Japanese seabass and Spotted seabass and method of determining species of Japanese seabass and Spotted seabass using the same

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103602738A (en) * 2013-11-12 2014-02-26 辽宁大学 Multiple PCR (Polymerase Chain Reaction) primer and method for quickly identifying variety of scallops
CN105256056A (en) * 2015-11-17 2016-01-20 万超 Scallop species specificity detection primer and application

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103602738A (en) * 2013-11-12 2014-02-26 辽宁大学 Multiple PCR (Polymerase Chain Reaction) primer and method for quickly identifying variety of scallops
CN105256056A (en) * 2015-11-17 2016-01-20 万超 Scallop species specificity detection primer and application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
于涛等: "栉孔扇贝、虾夷扇贝及其杂交子代线粒体COI和Cytb基因遗传多样性分析", 《海洋科学》, no. 03, 15 March 2016 (2016-03-15), pages 3 - 11 *
赵庆等: "利用DNA条形码COI基因分析我国重要贝类系统进化关系", 《中国水产科学》, no. 04, 15 July 2018 (2018-07-15), pages 145 - 155 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112695105A (en) * 2021-02-18 2021-04-23 辽宁省海洋水产科学研究院 Real-time fluorescence PCR identification method of chlamys farreri
KR102586202B1 (en) * 2022-12-01 2023-10-10 순천향대학교 산학협력단 Primer set for species discrimination of Mizuhopecten yessoensis, Argopecten irradians and Chlamys farreri nipponensis and method of determining species of Mizuhopecten yessoensis, Argopecten irradians and Chlamys farreri nipponensis using the same
KR102592643B1 (en) * 2022-12-01 2023-10-24 순천향대학교 산학협력단 Primer set for species discrimination of Japanese seabass and Spotted seabass and method of determining species of Japanese seabass and Spotted seabass using the same
CN115807109A (en) * 2022-12-20 2023-03-17 中国海洋大学三亚海洋研究院 PCR technology-based scallop variety identification method and specific primers thereof

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