CN115820877A - Primer, probe and kit for real-time detection of acrossocheilus pachyrhizi based on fluorescence PCR - Google Patents
Primer, probe and kit for real-time detection of acrossocheilus pachyrhizi based on fluorescence PCR Download PDFInfo
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
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- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The invention discloses a primer and a probe for detecting acrossocheilus crassipes in real time based on fluorescence PCR, wherein an upstream primer of the primer is HF6, and a 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 in SEQ ID No. 3. The real-time detection method for the acrossocheilus pachychiatus 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 the level of 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 acrossocheilus crassipes in real time based on fluorescence PCR.
Background
The thick lip bare heavy lip fish (Gymnodiptychus pachycheilus) belongs to Cypriniformes (Cypriniformes), cyprinidae (Cyprinidae), schizothoracinae (Schizothoracinae) and Gymnodiptychus (Gymnodiptychus), and is a special species in China. The lips are developed, fat and fleshy, the lower lip is divided into left and right leaves, the surface of the lower lip is obviously wrinkled, only the shoulder belt and the base part of the hip fin are provided with a small amount of scales, and the rest parts are exposed without scales, so the fish is named as the thick-lip naked heavy-lipped fish. According to related investigation data, the thick-lip bare lipped fries are mainly distributed at the upstream of a yellow river water system and the upstream of a Yangtze river valley Jinshajiang water system Yajiangjiang. The distribution range belongs to a plateau aquatic ecosystem, has the characteristics of simple structure and low productivity, and is easily influenced by the outside. In addition, the plateau fishes such as the thick-lip bare-heavy-lip fishes have the characteristics of slow growth, long resource supplement period, high adaptability and dependence on the environment and the like, and the disturbance of an ecological system can cause influence or even damage to the wild resource quantity of the plateau fishes to different degrees. In recent years, with the increase of human activities, including wading engineering construction, over-fishing, water environment pollution and the like, and the influence of global climate change factors, the wild resource amount of the bare lipped labeo has been seriously declined, and the wild labeo is listed as a national secondary protection animal in 2021. Therefore, it is necessary to establish a rapid and accurate identification method for the thick-lip bare-heavy-lip fish.
Due to the limitation of professional backgrounds of investigators and biological characteristics of species, the accuracy and the information quantity of biological information acquisition of part of biological groups are limited, for example, the acrossochekia crassipes inhabit on the bottom layer of a water body mostly, so that the investigators are difficult to perceive and the identification accuracy is influenced. In addition, the Yangtze river basin is completely forbidden for ten years in 2020, so that the direct monitoring and evaluation of the population of the thick-lip bare-weight hemibarbus labeo fries is increasingly difficult. The environmental DNA technology is a novel species monitoring technology with high sensitivity, low cost and no damage, and can quickly detect target species only by directly extracting DNA from a water environment sample. So far, no patent literature and non-patent literature relevant to real-time fluorescence identification of environmental DNA of the acrossocheilus crassipes are disclosed.
Identifying and distinguishing the thick-lip bare heavy-lip fishes in the fishes living in the Yazhenjiang river. The method is limited by the existing observation and sampling technology, the protective biological information of the thick-lip bare-heavy-lip fish is quite limited, and the distribution range and population condition of the thick-lip bare-heavy-lip fish are not clear. And the variety of the elegans is more, the difference of the class molecular level of the closer geographical distribution is also smaller, which causes certain difficulty to the design of the real-time fluorescence specificity Taqman probe.
Disclosure of Invention
The invention aims to provide a primer and a probe for detecting the acrossocheilus crassipes in real time based on fluorescence PCR.
The invention also aims to provide a detection kit for the acrossocheilus pachyrhizi.
The purpose of the invention is realized by the following technical scheme:
a primer and a probe for real-time detection of acrossocheilus pachyrhizi based on 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 in 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 invention discloses a primer and a probe for detecting lip bald bare-headed hemibarbus labeo in real time based on fluorescence PCR, which are designed according to a gene sequence of 12SrRNA in mitochondrial DNA (mtDNA) of the lip bald bare-headed hemibarbus labeo.
A detection kit for a thick-lip naked heavy-lip fish is characterized by comprising the following probes and primers: the upstream primer HF6 has the nucleotide sequence shown in SEQ ID No. 1; the downstream primer HR6 nucleotide sequence 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 acrossocheilus crassipes based on the fluorescent PCR primers and the probes comprises the steps of extracting DNA of the acrossocheilus crassipes and carrying out fluorescent PCR amplification, and is characterized in that: the fluorescence PCR amplification system is preferably 20 mu L: comprises the upstream primer HF6/HR6 of 0.4. Mu.L, the H-MGB6 probe of 0.4. Mu.L and the DNA template of 2.0. Mu.L.
Further preferably, the PCR reaction conditions, the two-step amplification reaction procedure: pre-denaturation at 95 ℃ for 30sec; then, the reaction was circulated 40 times at 95 ℃ for 5sec and at 60 ℃ for 30 sec.
The invention has the following beneficial effects:
the invention provides a real-time fluorescence PCR detection method for detecting the acromion labialis recklessly and a primer and a probe for detection based on environmental DNA, which are used for detecting the specific plateau fish acromion labialis recklessly in China, and establishes the real-time fluorescence PCR detection method for the acromion labialis recklessly according to the gene sequence design specific primer and probe of 12SrRNA in the mtDNA of the acromion labialis recklessly. 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 fluorescent PCR detection can reach the level of pg level.
The difference of the thick lip bare lip fish and the carpidae fish with similar geographic population on the molecular level is smaller, false positive amplification can occur when the probe is selected poorly, the design of the probe needs to be highly conservative, and the thick lip bare lip fish can not be matched when one base is different, so that certain difficulty is caused to the design of the real-time fluorescence specific probe of the thick lip bare lip fish, and the amplification efficiency of the same probe is different when different primers are selected; at the same time, it is also necessary to ensure stable amplification of the target fragment and that the primers themselves do not generate hairpin structures and dimers. And because the probe has high sensitivity, aerosol pollution is very easily generated in the qPCR experiment process, so that false positive is generated in the experiment result, and the experiment difficulty is greatly increased. Finally, a large number of amplification experiments prove that the combination of the specific primer HF6/HR6 and the probe H-MGB6 of the acrossocheilus crassipes has the characteristics of good specificity and high sensitivity, the DNA of the acrossocheilus crassipes can be specifically detected, the acrossocheilus crassipes and other cyprinid fishes can be effectively distinguished, and the minimum detected DNA concentration can reach 2.6 multiplied by 10 -6 ng/uL. The specificity is dual guaranteed by the primer and the probe, a new method is provided for identifying the thick-lip bare heavy-lipped fish, a fish body sample does not need to be collected, the damage to the fish body can be avoided, the detection accuracy is high, the analysis is simple and rapid, and the method can be used for rapidly identifying the thick-lip bare heavy-lipped fish.
Drawings
FIG. 1: the primer HF6/HR6 and the probe H-MGB6 of the invention are positioned on the mtDNA of the acrossocheilus crassipes.
FIG. 2: the invention adopts a primer HF6/HR6 and a probe H-MGB6 to perform real-time fluorescence PCR amplification on the acrossocheilus crassipes and the blank control.
FIG. 3: the invention applies a primer HF6/HR6 and a probe H-MGB6 to the results of real-time fluorescence PCR amplification of samples and blank samples of acrossocheilus crassipes and other species.
FIG. 4: the invention relates to a fluorescence signal curve diagram measured by real-time fluorescence PCR after 10-fold gradient dilution of plasmid constructed by DNA of a thick lip naked heavy lipped fish sample.
FIG. 5: the invention relates to a fluorescence signal curve graph (Log scale) determined by real-time fluorescence PCR after 10-fold gradient dilution of a plasmid constructed by DNA of a thick lip naked heavy lipped sample.
Detailed Description
Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed under conventional conditions, e.g., sambrook et al: the 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 environmental DNA of the acrossocheilus macrobrachium comprises the following steps:
1. primers and probes were designed.
The following primers HF6/HR6 and probe H-MGB6 (primers and probes synthesized by Biotechnology, shanghai, ltd.) were designed based on the gene sequence of 12SrRNA in mitochondrial DNA (mtDNA) of Gymnodiptympana pachychei.
The sequence of the primer is as follows: the upstream primer HF6 has the nucleotide sequence shown in SEQ ID No. 1; a 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, the fluorescent group is connected to the 5 'terminal of the probe, and the quenching group is connected to the 3' terminal.
The detection principle of the primers and the probes is shown in FIG. 1. Probe H-MGB6 is an oligonucleotide probe with a fluorophore attached to the 5 'end of the probe and a quencher at the 3' end. When the probe is complete, the fluorescent signal emitted by the reporter group is absorbed by the quenching group; during PCR amplification, a primer HF6/HR6 and the mtDNA of the acroleis crassipes complete thermal cycle of high-temperature denaturation and low-temperature renaturation extension, the polymerase chain reaction rule is observed, meanwhile, the added probe H-MGB6 is complementary with the mtDNA of the acroleis crassipes, and in the low-temperature renaturation extension stage, the probe H-MGB6 is degraded by enzyme digestion through 5'-3' exonuclease activity of Taq enzyme in a reaction system, so that a report fluorescent group and a quenching fluorescent group are separated, a fluorescence monitoring system can receive a fluorescence signal, namely, one fluorescence molecule is formed when each DNA chain is amplified, and the accumulation of the fluorescence signal and the formation of a PCR product are completely synchronous.
2. Extracting DNA of the thick-lip naked heavy-lip fish.
Placing muscles of Gymnodiptychus pachycheilus in mortar, adding nitrogen, grinding into powder, and grinding
Cell/Tissue DNA Kit Cell/Tissue DNA extraction Kit (Shishin Sheng Biotechnology (Shanghai) Co., ltd.) extracts sample DNA, and the bare lipped to-be-tested lip fish is taken from an elegance water power station proliferation and releasing station in the area of Ganzui in Sichuan province.
3. And (3) real-time fluorescence PCR detection.
Real-time fluorescent PCR amplification system 20. Mu.L (Baozi physician technology (Beijing) Co., ltd., probe qPCR Mix, with UNG): probe qPCR Mix with 10. Mu.L of UNG, 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 enzyme-free water (DNase/RNase-free ddH) 2 O) to 20. Mu.L. Real-time fluorescent PCR amplification was performed on a Bio-rad CFX Connect PCR instrument. Opening 'Bio-Rad CFX Maestro', setting PCR reaction conditions, and carrying out a two-step amplification reaction program: pre-denaturation at 95 ℃ for 30sec; then, the reaction was circulated 40 times at 95 ℃ for 5sec and at 60 ℃ for 30 sec. And (3) clicking to operate, carrying out real-time fluorescence PCR reaction, finishing the reaction for about 1h, saving a file, and opening analysis software.
After real-time fluorescent PCR amplification, the specific primer HF6/HR6 and the probe H-MGB6 of the acrossocheilus macrobrachium result in a fluorescent growth curve for 1 sample of the acrossocheilus macrobrachium to be tested, a strong fluorescent signal is increased, positive amplification is shown (the CT value is less than 35), and the sample amplification curve refers to FIG. 2. The tested thick lip bare and heavy lip fish sample has positive amplification, and the blank control does not detect a fluorescent signal and shows negative.
Example 2
The method for verifying the specificity and testing the sensitivity of the primer HF6/HR6 and the probe H-MGB6 of the acrossocheilus pachychiatus comprises the following steps: 1. and (4) extracting DNA.
Putting muscles of the thick-lip bare heavy-lip fish and other fishes (the samples and sources are shown in table 1) into a mortar, adding liquid nitrogen, grinding into powder,
Cell/Tissue DNA Kit Cell/Tissue DNA extraction Kit (Hippocampus Biotech Co., ltd.) extracts sample DNA.
TABLE 1 detailed information of the samples tested
| Serial number | Numbering | Seed name | Producing area | Quantity (strip) |
| 1 | H | Thick lip bare heavy lip fish | Sichuan province Ganzui region | 1 |
| 2 | X | Schizothorax gracilis | Sichuan province Ganzui region | 1 |
| 3 | JTB | Erythroculter ilishaeformis | Beiqing Beibei region | 1 |
| 4 | TY | Copper fish | Beiqing Beibei region | 1 |
| 5 | YZY | Myxocyprinus asiaticus | Beiqing Beibei region | 1 |
| 6 | HHF | Megalobrama amblycephala | Beiqing Beibei region | 1 |
| 7 | SJ | Saurogobio ventralis | Beiqing Beibei region | 1 |
| 8 | ZHDCB | Chinese spinibarbus | Beiqing Beibei region | 1 |
2. And (3) verifying the specificity of the primers HF6/HR6 and the probe H-MGB6 of the acrossocheilus pachyma.
Through real-time fluorescence PCR amplification, a fluorescence growth curve appears on a sample of the pellegelia pachyrhizi to be tested by using a specific primer HF6/HR6 and a probe H-MGB6, strong fluorescence signals are increased, positive amplification is shown, the amplification curve refers to a figure 2, 7 (sample numbers are shown in a table 1) pellegelia lenok, a culter ilishaeformis, a copper fish, a myxocyprinus asiaticus, a megalobrama amblycephala, a snakegobio and a Sinonorbus sinobrevicilus and a blank control to be tested do not detect fluorescence signals, the amplification curve refers to a figure 3, and the amplification curve is negative. Therefore, the real-time fluorescence PCR detection shows that the acrossocheilus macrobrachium is detected, and the primer HF6/HR6 and the probe H-MGB6 can specifically detect the acrossocheilus macrobrachium.
3. And (3) testing the sensitivity of the specific primer HF6/HR6 and the probe H-MGB6 of the acrossocheilus craibianus.
And (3) carrying out 10-time gradient dilution on the plasmid constructed by the DNA of the thick-lip naked heavy-lip fish sample, and carrying out detection limit test, wherein the initial concentration of the plasmid is 260 ng/mu L. The real-time fluorescence PCR determination (the reaction system and the reaction conditions are the same as the test sample) shows that the fluorescence signal curves are shown in FIGS. 4 and 5, and the Ct values are respectively: 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 the Ct value of No. 9 is 36.41>35, it cannot be determined whether it is positive. Therefore, the detection limit DNA concentration of the specific probe H-MGB6 of the bare lipped labeo-gobio is 260 multiplied by 10 -8 ng/. Mu.L, i.e. 2.6X 10 -6 ng/μL。
The lowest DNA concentration detected by real-time fluorescence PCR can reach pg level, and the sensitivity is equivalent to that of nested PCR. Therefore, the real-time fluorescence PCR detection method can improve the detection sensitivity of the environmental DNA of the acrossocheilus crassipes and achieve the unexpected effect. The difference of the molecular level of the Yaxijiang fishes is small, which causes certain difficulty in the design of the real-time fluorescence specificity Taqman probe for the thick-lip naked heavy-lip fishes. 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, so that the detection accuracy and the sensitivity are high.
Claims (5)
1. A primer and a probe for real-time detection of acrossocheilus pachyrhizi based on 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 in SEQ ID No. 3.
2. The primers and probes as claimed in 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. A detection kit for a thick-lip naked heavy-lip fish is characterized by comprising the following probes and primers: the upstream primer HF6 has the nucleotide sequence shown in SEQ ID No. 1; the downstream primer HR6 nucleotide sequence is shown as SEQ ID No. 2; the nucleotide sequence of the probe H-MGB6 is shown in SEQ ID No. 3.
4. The real-time detection method for the acrossocheilus macrobrachium based on the fluorescent PCR primer and the probe as claimed in claim 1 or 2 comprises the steps of extracting DNA of the acrossocheilus macrobrachium and carrying out fluorescent PCR amplification, and is characterized in that: the fluorescence PCR amplification system is preferably 20 mu L: comprises the upstream primer HF6/HR6 of 0.4. Mu.L, the H-MGB6 probe of 0.4. Mu.L and the DNA template of 2.0. Mu.L.
5. The real-time detection method of claim 4, wherein: the PCR reaction is a two-step amplification reaction program: pre-denaturation at 95 ℃ for 30sec; then, the reaction was circulated 40 times at 95 ℃ for 5sec and at 60 ℃ for 30 sec.
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| CN102783442A (en) * | 2012-08-23 | 2012-11-21 | 甘肃省水产研究所 | Artificial breeding method of Gymnodiptychus pachycheilus |
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