CN115820877B - Primer, probe and kit for detecting naked thick-lip and heavy-lip fish in real time based on fluorescence PCR - Google Patents
Primer, probe and kit for detecting naked thick-lip and heavy-lip fish in real time based on fluorescence PCR Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The invention discloses a primer and a probe for detecting naked lip fish with thick lip in real time based on fluorescent PCR, wherein the upstream primer of the primer is HF6, and the nucleotide sequence of the upstream primer is shown as SEQ ID No. 1; the downstream primer is HR6, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2; the probe is H-MGB6, and the nucleotide sequence of the probe is shown as SEQ ID No. 3. The real-time detection method of the naked thick-lip and heavy-lip fish is established, the detection time is greatly shortened, the detection can be completed within 6 hours, the monitoring result is accurate, the sensitivity is high, and the minimum DNA concentration of the real-time fluorescence PCR detection can reach pg level.
Description
Technical Field
The invention belongs to the technical field of molecular biological detection, and particularly relates to a primer, a probe and a kit for detecting naked lip fish on the basis of fluorescence PCR (polymerase chain reaction) in real time.
Background
The naked cheilis (Gymnodiptychus pachycheilus) belongs to the order of Cyprinimes, cyprinidae, schizophreniform, and Gymnilis, and is a special species of China. The lips are developed, the lips are rich and fleshy, the lower lips are divided into left and right leaves, the surfaces of the lips are provided with obvious folds, only the shoulder straps and the base parts of the hip fins are provided with a small amount of scales, and the other parts are exposed without scales, so that the fish with thick lips and bare heavy lips is named. According to related investigation data, the naked thick-lip heavy-lip fish is mainly distributed on the upstream of a yellow river system and the upstream of a Yangtze river basin YangSha river system. The distribution range belongs to a high-raw water ecological system, has the characteristics of simple structure and low productivity, and is easily influenced by the outside. In addition, the plateau fishes such as the bare thick-lip and heavy-lip fishes have the characteristics of slow growth, long resource supplementing period, high adaptation and dependence on habitat and the like, and the disturbance of an ecological system can influence and even destroy the wild resource quantity of the plateau fishes to different degrees. In recent years, with the increase of human activities, including wading engineering construction, excessive fishing, water environment pollution and the like, and affected by global climate change factors, the wild resource amount of the naked thick-lip and heavy-lip fish has severely degenerated, and is listed into the national second-class protection animals in 2021. Therefore, it is necessary to establish a rapid and accurate identification method for the naked thick-lip bald fish.
Due to the limitations of professional backgrounds of researchers and biological characteristics of species, the accuracy and information quantity of biological information acquisition of partial biological groups are limited, for example, thick-lip bare and heavy-lip fishes mostly inhabit the bottom of a water body, so that the researchers are difficult to perceive, and the identification accuracy is affected. And the general forbidding of ten years from the year 2020 in Yangtze river basin makes it more difficult to directly monitor and evaluate the bare and heavy lip fingerling population of thick lip. The environmental DNA technology is a novel high-sensitivity, low-cost and damage-free species monitoring technology, and can rapidly detect target species only by directly extracting DNA from a water environment sample. No disclosures of patent literature and non-patent literature related to real-time fluorescent identification of the environmental DNA of naked thick-lip balms have been found to date.
And identifying and distinguishing the thick-lip bare and heavy-lip fish in the fish upstream of the elegance hulling river. The biological information of the protection of the bare thick-lip bald fish is quite limited by the prior observation and sampling technology, and the distribution range and population condition of the bare thick-lip bald fish are not clear. The variety of the elegance hulling Jiang Yulei is more, the difference of the molecular level of the variety with relatively close geographic distribution is also relatively small, and the design of the real-time fluorescence specificity Taqman probe is difficult to a certain extent.
Disclosure of Invention
The invention aims to provide a primer and a probe for detecting naked thick-lip bald fish in real time based on fluorescent PCR.
The invention further aims at providing a detection kit for the bare thick-lip and heavy-lip fish.
The invention aims at realizing the following technical scheme:
a primer and a probe for detecting naked thick lip fish in real time based on fluorescent PCR are characterized in that: the upstream primer of the primer is HF6, and the nucleotide sequence of the upstream primer is shown as SEQ ID No. 1; the downstream primer is HR6, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2; the probe is H-MGB6, and the nucleotide sequence of the probe is shown as SEQ ID No. 3.
The 5 'end and the 3' end of the probe are respectively connected with a fluorescent group and a quenching group.
The primer and the probe for detecting the naked thick lip fish in real time based on fluorescent PCR are designed according to the gene sequence of 12SrRNA in mitochondrial DNA (mtDNA) of the naked thick lip fish.
The kit for detecting the naked thick-lip bald fish is characterized by comprising the following probes and primers: the nucleotide sequence of the upstream primer HF6 is shown as SEQ ID No. 1; the nucleotide sequence of the downstream primer HR6 is shown as SEQ ID No. 2; the nucleotide sequence of the probe H-MGB6 is shown as SEQ ID No. 3.
The real-time detection method for the naked thick-lip bald fish based on the fluorescent PCR primer and the probe comprises the steps of extracting DNA of the naked thick-lip bald fish and carrying out fluorescent PCR amplification, and is characterized in that: the fluorescent PCR amplification system is preferably 20 mu L: the primer HF6/HR6 was 0.4. Mu.L each, the H-MGB6 probe was 0.4. Mu.L, and the DNA template was 2.0. Mu.L.
Further preferably, the PCR reaction conditions described above, a two-step amplification reaction procedure: pre-denaturation at 95℃for 30sec; then, the mixture was circulated 40 times at 95℃for 5sec and 60℃for 30 sec.
The invention has the following beneficial effects:
the existing bare thick-lip fish identification is completely judged manually, and the identification accuracy is affected. The detection time is greatly shortened, the detection can be completed within 6 hours, the monitoring result is accurate, the sensitivity is high, and the minimum DNA concentration of the real-time fluorescence PCR detection can reach pg level.
The difference of the molecular level of the naked heavy lip fish with the thick lip and the carp fish with similar geographical population is small, false positive amplification can occur due to poor probe selection, the design of the probe needs to be highly conserved, the two probes can not be matched due to different bases, the design of the real-time fluorescence specific probe of the naked heavy lip fish with the thick lip is difficult, and the amplification efficiency of the same probe is different due to the selection of different primers; at the same time, stable amplification of the target fragment and no hairpin structure and dimer of the primer are ensured. In addition, the probe has high sensitivity, aerosol pollution is easy to generate in the qPCR experimental process, false positive appears in experimental results, and the experimental difficulty is greatly increased. Finally, a large number of amplification experiments prove that the combination of the specific primers HF6/HR6 and the probe H-MGB6 of the naked lip fish has the characteristics of good specificity and high sensitivity, can specifically detect the DNA of the naked lip fish, effectively distinguish the naked lip fish from other carps, and can achieve the lowest DNA concentration of 2.6x10 -6 ng/uL. The specificity is ensured by the primer and the probe, a novel method is provided for identifying the naked thick-lip heavy-lip fish, a fish body sample is not required to be collected, damage to the fish body can be avoided, the detection accuracy is high, the analysis is simple and quick, and the method can be used for quickly identifying the naked thick-lip heavy-lip fish.
Drawings
Fig. 1: the primers HF6/HR6 and probe H-MGB6 of the present invention were located on mtDNA of naked thick-lip bald fish.
Fig. 2: the invention uses the primer HF6/HR6 and the probe H-MGB6 to carry out real-time fluorescence PCR amplification results on the naked thick lip fish and the blank control.
Fig. 3: the invention uses the primer HF6/HR6 and the probe H-MGB6 to amplify the samples of the naked lip fish and other species and blank real-time fluorescence PCR.
Fig. 4: the plasmid constructed by the DNA of the naked thick-lip and heavy-lip fish sample is subjected to 10-time gradient dilution and then is subjected to a fluorescent signal graph determined by real-time fluorescent PCR.
Fig. 5: the plasmid constructed by the DNA of the naked thick lip balm sample is subjected to 10-time gradient dilution and then is subjected to real-time fluorescence PCR (polymerase chain reaction) determination to obtain a fluorescence signal curve graph (Log scale).
Detailed Description
The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by conventional conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.
Example 1
The real-time fluorescence PCR detection method of the naked heavy lip fish environmental DNA comprises the following steps:
1. primers and probes were designed.
The following primers HF6/HR6 and probe H-MGB6 (primers and probes were synthesized by Biotechnology (Shanghai) Co., ltd.) were designed based on the gene sequence of 12SrRNA in mitochondrial DNA (mtDNA) of Alaska thickia.
The sequence of the primer is as follows: the nucleotide sequence of the upstream primer HF6 is shown as SEQ ID No. 1; downstream primer HR6: the nucleotide sequence is shown as SEQ ID No. 2; probe H-MGB6: the nucleotide sequence is shown as SEQ ID No.3, a fluorescent group is connected at the 5 'end of the probe, and a quenching group is connected at the 3' end.
The detection principle of the primer and the probe is shown in FIG. 1. Probe H-MGB6 is an oligonucleotide probe, with a fluorescent group attached to the 5 'end of the probe and a quenching group at the 3' end. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; during PCR amplification, the primer HF6/HR6 and the mtDNA of the naked heavy lip fish complete the thermal cycle of high temperature denaturation and low Wen Fuxing extension and observe the polymerase chain reaction rule, and the added probe H-MGB6 is complementary with the mtDNA of the naked heavy lip fish, and in the low temperature renaturation extension stage, the 5'-3' exonuclease activity of Taq enzyme in the reaction system is used for carrying out enzyme digestion degradation on the probe H-MGB6 to separate a reporting fluorescent group from a quenching fluorescent group, so that a fluorescent monitoring system can receive a fluorescent signal, namely, one fluorescent molecule is formed every time one DNA chain is amplified, and the accumulation of the fluorescent signal and the complete synchronization of PCR product formation are realized.
2. Extracting DNA of the naked thick-lip bald fish.
Adding muscle of naked thick lip and heavy lip fish into a mortar, adding liquid nitrogen, grinding into powder, and usingCell/Tissue DNA Kit Cell/Tissue DNA extraction Kit (Hitachi Biotechnology (Shanghai) Co., ltd.) extracts sample DNA, and the test thick lip naked heavy lip fish is obtained from the power station proliferation and releasing station of the Ganmai area elegance rice huller in Sichuan province.
3. And (5) real-time fluorescence PCR detection.
Real-time fluorescent PCR amplification System 20. Mu.L (Bao Ri doctor Material technology (Beijing) Co., ltd., probe qPCR Mix, with UNG): probe qPCR Mix, with UNG 10. Mu.L, upstream and downstream primers HF6/HR6 (10. Mu. Mol/L) each 0.4. Mu.L, H-MGB6 Probe (10. Mu. Mol/L) 0.4. Mu.L, DNA template 2.0. Mu.L, sterile and asepsis water (DNase/RNase-free ddH 2 O) was added to 20. Mu.L. Real-time fluorescent PCR amplification was performed on a Bio-rad CFX Connect PCR instrument. The "Bio-Rad CFX Maestro" was turned on, the PCR reaction conditions were set, and the two-step amplification reaction procedure was followed: pre-denaturation at 95℃for 30sec; then, the mixture was circulated 40 times at 95℃for 5sec and 60℃for 30 sec. Clicking for operation, performing real-time fluorescence PCR reaction, finishing the reaction about 1h, saving a file, and opening analysis software.
The real-time fluorescence PCR amplification shows that the specific primers HF6/HR6 and the probe H-MGB6 of the naked heavy lip fish show fluorescence growth curves on 1 naked heavy lip fish sample to be tested, the strong fluorescence signal increase is shown as positive amplification (CT value is less than 35), and the sample amplification curve is shown as figure 2. Positive amplification of the test thick-lip naked heavy-lip fish sample occurs, and no fluorescent signal is detected by the blank control, so that the test thick-lip naked heavy-lip fish sample is negative.
Example 2
The specificity verification and sensitivity test of the naked heavy lip fish primer HF6/HR6 and the probe H-MGB6 comprise the following steps: 1. extracting DNA.
The naked thick lip and the muscles of other fishes (the samples and sources are shown in table 1) are taken and put into a mortar, added with liquid nitrogen and ground into powder,Cell/Tissue DNA Kthe it cell/tissue DNA extraction kit (Highway Biotechnology (Shanghai) Co., ltd.) extracts sample DNA.
TABLE 1 specific information on test sample
| Sequence number | Numbering device | Seed name | Production area | Quantity (stripe) |
| 1 | H | Bare thick-lip and heavy-lip fish | Sichuan Ganzi district | 1 |
| 2 | X | Schizothorax grahami | Sichuan Ganzi district | 1 |
| 3 | JTB | Erythroculter ilishaeformis (Bleeker) Bleeker | Chongqing city north medium area | 1 |
| 4 | TY | Copper fish | Chongqing city north medium area | 1 |
| 5 | YZY | Rouge fish | Chongqing city north medium area | 1 |
| 6 | HHF | Megalobrama amblycephala | Chongqing city north medium area | 1 |
| 7 | SJ | Gobio crocus (wall) | Chongqing city north medium area | 1 |
| 8 | ZHDCB | Chinese barb-shaped cyclocheilus | Chongqing city north medium area | 1 |
2. Specificity verification of the naked heavy lip fish primer HF6/HR6 and the probe H-MGB6.
The real-time fluorescence PCR amplification shows that the specific primers HF6/HR6 and the probe H-MGB6 of the naked red lip fish show fluorescence increase curves, the strong fluorescence signal increase is shown as positive amplification, the amplification curves refer to FIG. 2, the 7 (sample numbers refer to Table 1) of the naked red lip fish, the Erythroculter grahami, the copper fish, the carmine fish, the megalobrama amblycephala, the snake gobius and the Chinese barb cheilus and the blank control show no fluorescence signal, the sample amplification curves refer to FIG. 3. This shows that this time of real-time fluorescence PCR detected the naked thick-lip bald fish, indicating that primers HF6/HR6 and probe H-MGB6 can specifically detect the naked thick-lip bald fish.
3. Sensitivity test of the specific primers HF6/HR6 and probe H-MGB6 of the naked and heavy labial fish.
Plasmid constructed by the DNA of the naked thicklips heavy lip sample is subjected to 10-time gradient dilution, and detection limit test is carried out, wherein the initial concentration of the plasmid is 260 ng/. Mu.L. The fluorescence signal curves are shown in fig. 4 and 5, and Ct values are respectively: no.1, no. 8.44, no.2, no. 12.16, no.3, no. 16.04, no. 4, no. 19.51, no. 5, no. 22.84, no. 6, no. 26.21, no. 7, no. 29.72, no. 8, no. 33.14, no. 9, no. 36.41. Since Ct value of No. 9 is 36.41>35, it is impossible to determine whether it is positive. Therefore, the detection limit DNA concentration of the specific probe H-MGB6 of the naked thick lip bald fish is 260×10 -8 ng/. Mu.L, i.e. 2.6X10 -6 ng/μL。
The lowest DNA concentration detected by the real-time fluorescence PCR can reach pg level, and the sensitivity of the PCR is equivalent to that of the PCR. Therefore, the real-time fluorescence PCR detection method can improve the detection sensitivity of the naked thick lip bald environmental DNA and achieve unexpected effects. The molecular level difference of the elegance hulling fish is small, which causes a certain difficulty in designing the real-time fluorescence specificity Taqman probe of the naked thick-lip fish. Therefore, in the final determination method of the experiment, HF6/HR6 with high amplification efficiency and good repeatability is selected as an amplification primer, and a probe H-MGB6 with good specificity is screened. The primer and the probe are ensured in double, so that the detection accuracy and the sensitivity are high.
Claims (5)
1. A primer and a probe for detecting naked thick-lip bald fish based on real-time fluorescence PCR are characterized in that: the upstream primer of the primer is HF6, and the nucleotide sequence of the upstream primer is shown as SEQ ID No. 1; the downstream primer is HR6, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2; the probe is H-MGB6, and the nucleotide sequence of the probe is shown as SEQ ID No. 3.
2. The primer and probe of claim 1, wherein: the 5 'end and the 3' end of the probe are respectively connected with a fluorescent group and a quenching group.
3. The kit for detecting the naked thick-lip bald fish is characterized by comprising the following probes and primers: the nucleotide sequence of the upstream primer HF6 is shown as SEQ ID No. 1; the nucleotide sequence of the downstream primer HR6 is shown as SEQ ID No. 2; the nucleotide sequence of the probe H-MGB6 is shown as SEQ ID No. 3.
4. The method for detecting the naked thick-lip fish by using the primer and the probe according to claim 1 or 2, which comprises the steps of extracting DNA of the naked thick-lip fish and performing fluorescent PCR amplification, and is characterized in that: 20 mu L of the fluorescent PCR amplification system: the primers HF6/HR6 were included at 0.4. Mu.L each, the H-MGB6 probe at 0.4. Mu.L, and the DNA template at 2.0. Mu.L.
5. The method of detection according to claim 4, wherein: the PCR reaction is a two-step amplification reaction program: pre-denaturation at 95℃for 30sec; then, the mixture was circulated 40 times at 95℃for 5sec and 60℃for 30 sec.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102783442A (en) * | 2012-08-23 | 2012-11-21 | 甘肃省水产研究所 | Artificial breeding method of Gymnodiptychus pachycheilus |
| CN107557482A (en) * | 2017-10-26 | 2018-01-09 | 浙江海洋大学 | A kind of molecular biology method for the naked skin-carp meat in Xinjiang that quick discriminating south, North SinKiang are distributed |
| CN113667762A (en) * | 2021-08-23 | 2021-11-19 | 西南大学 | Procypris rabaudi real-time fluorescent PCR amplification primer, probe and detection method based on environmental DNA |
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| US20180037959A1 (en) * | 2016-08-08 | 2018-02-08 | Universiti Brunei Darussalam | Process using ZEN hydrolysis probe for detection of porcine contamination and a kit thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102783442A (en) * | 2012-08-23 | 2012-11-21 | 甘肃省水产研究所 | Artificial breeding method of Gymnodiptychus pachycheilus |
| CN107557482A (en) * | 2017-10-26 | 2018-01-09 | 浙江海洋大学 | A kind of molecular biology method for the naked skin-carp meat in Xinjiang that quick discriminating south, North SinKiang are distributed |
| CN113667762A (en) * | 2021-08-23 | 2021-11-19 | 西南大学 | Procypris rabaudi real-time fluorescent PCR amplification primer, probe and detection method based on environmental DNA |
Non-Patent Citations (2)
| Title |
|---|
| The complete mitochondrial genome of Gymnodiptychus pachycheilus (Teleostei, Cyprinidae, Schizothoracinae);Chunna Chen等;Mitochondrial DNA PART A;20140311;第27卷(第1期) * |
| 虎永彪,等.厚唇裸重唇鱼人工繁殖技术.中国水产.2014,第8卷第63-64页. * |
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