CN106755392B - qPCR (quantitative polymerase chain reaction) method for rapidly and quantitatively detecting coelomacter in algae culture - Google Patents
qPCR (quantitative polymerase chain reaction) method for rapidly and quantitatively detecting coelomacter in algae culture Download PDFInfo
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Abstract
The invention firstly provides the application of coerulea CoI gene in a molecular marker of a qPCR method for rapid quantitative detection of harmful coerulea in algae culture, a DNA molecular marker and a specific primer group shown as SEQ ID No. 1-3; and then providing a qPCR (quantitative polymerase chain reaction) method for rapidly and quantitatively detecting the coelomacter harms in the algae culture, wherein the specific primer group is adopted to carry out fluorescence quantitative PCR on the genome DNA of the sample to be detected. The invention has the technical effects that a molecular marker is found, a specific primer is designed, and the coelomacter can be effectively identified under the condition that other polluted protozoa exist in the algae culture process. The sensitivity and specificity of detection are greatly improved, the test time is shortened, the operation is simplified, and the fluorescence detection result is analyzed by computer software, so that the pollution caused by the product post-treatment process is avoided, and the method is more objective, sensitive and accurate than the conventional PCR.
Description
Technical Field
The invention relates to the technical field of microbial molecular detection, in particular to a qPCR method for early monitoring coelomyces in large-scale cultivation of algae.
Background
Coelomacter is an important contaminant protozoan present in large-scale cultivation of algae, and is a broad class of freshwater rotifers. The algae can be completely destroyed within 2 to 4 days after being infected, so that 'soup soaking' is caused, and great harm is brought to the large-scale culture of the algae. It is therefore of great importance for the early monitoring and determination of molecular identification methods for the presence of coelomycetes in algal cultures.
At present, the research on the coelomacter is limited, only the classification, diversity, community structure and the like of the coelomacter are researched, but the research on the harm caused by the coelomacter to large-scale algae cultivation is hardly carried out, dormant cysts exist when the environmental conditions are not favorable for production and propagation, the existence of the dormant cysts is difficult to observe in a microscope for large-scale algae cultivation, the traditional morphological observation sensitivity is low, and the research can be completed on the premise that the number of protozoa exists. The direct DNA sequencing and Polymerase Chain Reaction (PCR) have low detection efficiency, and can not be detected under the condition of very low DNA content, and the judgment of actual conditions is often influenced due to the phenomenon of false positive of a detection result. When the method is adopted to detect the coelomacter, the existence quantity is large enough to cause complete failure of the large-scale culture of the algae. Therefore, methods for early monitoring and molecular identification of the rotifers are of particular importance.
With the development of molecular biology detection technology, the detection means of protozoa has been developed rapidly, and the Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR, qPCR) has been widely applied in the molecular detection of protozoa and the pathogen detection of shellfish due to the characteristics of high detection speed, high sensitivity and strong specificity. Protozoan detection sensitivity on shellfish to reach 30 copies. Therefore, a detection means such as fluorescent quantitative PCR, which has high sensitivity, strong specificity and high detection speed, is urgently required for the detection of the coelomacter.
Disclosure of Invention
The invention aims to provide a fluorescence quantitative PCR detection method for coelomacter gondii in order to rapidly monitor the existence of pollutants in the early stage of algae culture. Compared with the traditional PCR method, the method has higher sensitivity and simple and convenient operation, can detect a large amount of samples and quantify the coelomacter, thereby providing an effective method for detecting and monitoring the pollution of the coelomacter in the early stage of algae culture.
The first technical purpose of the invention is to provide the application of the coelomacter CoI gene in the molecular marker of the qPCR method for the rapid quantitative detection of harmful coelomacter in algae culture. Due to the large similarity of protozoan gene sequences, the direct DNA sequencing and Polymerase Chain Reaction (PCR) are often used, which results in low detection efficiency and false positive detection results. After repeated research, the invention discovers that qPCR is carried out by using the specific region segment of the coe gene of the coe rotifer as a molecular marker, so that the coe rotifer can be effectively identified under the condition that other polluted protozoa exist in the algae culture process. Meanwhile, the invention takes the CoI gene as a molecular marker and also has the following characteristics: (1) the CoI gene can be distinguished by using a very short sequence as a molecular marker, and the related sequences of other molecular markers are relatively long, relatively complex and easy to misjudge; (2) the CoI gene can accurately distinguish species differences and has specificity; (3) the CoI gene exists in mitochondria, and a plurality of mitochondria exist in each biological cell, can be amplified during detection and has high sensitivity.
The second technical purpose of the invention is to provide a DNA molecular marker, which comprises three sections of specific region sequences of coeruleus CoI genes, wherein the sequences are respectively shown as SEQ ID No. 1-3.
Then, a set of primer sets is provided, which comprises three pairs of primers respectively used for specific amplification sequences SEQ ID No.1-3, and the sequences of the respective upstream and downstream primers are as follows (shown as SEQ ID No. 4-9):
(1)CoI-1F：5’-TAATGTTAGGGGTTGCTGAT-3’；
CoI-1R：5’-ATGAAGTTACTAATAAAATAGCTGT-3’；
(2)CoI-2F：5’-TTATCTTCTATTCTGGATGCTG-3’；
CoI-2R：5’-TTGGTATAATACAGGATTGCC-3’；
(3)CoI-3F：5’-GTTCTCGTACAACAAAAATGAT-3’；
CoI-3R：5’-TTGGTATAATACAGGATTGCC-3’。
the molecular marker or the primer can be applied to a qPCR (quantitative polymerase chain reaction) method for quickly and quantitatively detecting the coelomacter harms in the algae culture.
Further, the invention provides a rapid quantitative detection qPCR method for the harmful coelomacter in algae culture, which adopts the specific primer group to carry out fluorescent quantitative PCR detection on the genome DNA of a sample to be detected. Compared with the traditional PCR method, the method has higher sensitivity and simple and convenient operation, can detect a large amount of samples and quantify the coelomacter, thereby providing an effective method for detecting and monitoring the pollution of the coelomacter in the early stage of algae culture.
The quick quantitative detection qPCR method also comprises the following steps: the specific primer group is adopted to clone the gene containing the target amplification fragment into a plasmid PGEM-T, and the constructed recombinant plasmid PGEMT-CoI is used as a quantitative standard substance for detecting the concentration of the coelomacter.
The rapid quantitative detection qPCR method comprises the following specific primers: the concentration of the upstream primer and the concentration of the downstream primer are both 10 mu mol/L, and the final reaction concentration is 0.25 mu mol/L.
The fluorescent dye adopted by the fluorescent quantitative PCR is preferably SYBR Green dye, and the fluorescent detection wavelength is 470-514 nm.
Preferably, the reaction system of the fluorescent quantitative PCR is: the 20. mu.L reaction system contained: 2 XLightCycler 480SYBR Green I Master 10. mu.L, 10. mu. mol/L forward primer 0.5. mu.L, 10. mu. mol/L reverse primer 0.5. mu.L, DNA template 1. mu.L, water was added to a total volume of 20. mu.L.
The reaction procedure of the fluorescent quantitative PCR is as follows: 5min at 95 ℃ for 1 cycle; 10s at 95 ℃, 20s at 56 ℃ and 30s at 72 ℃ for 40 cycles.
The method for judging the result of the fluorescent quantitative PCR comprises the following steps:
(1) the Ct value of the positive control should be less than 36.0 and the Ct value of the negative control should be greater than 39.0. The positive template is plasmid quantitative standard PGEMT-CoI-1, PGEMT-CoI-2 and PGEMT-CoI-3, and the negative template is sterile double distilled water.
(2) If the Ct value is less than 36.0 and a typical amplification curve is presented, a positive result is judged, and the detection sample contains the coelomacter.
(3) If the Ct value is larger than 39.0 or no amplification signal exists, the negative result is judged, and the fact that the coelomacter does not exist in the detection sample is indicated.
(4) If the Ct value is between 36.0 and 39.0, the result is regarded as a suspicious result, and the sample needs to be checked repeatedly once.
If the result is still within this range, a negative result is judged.
And (4) calculating the content of the coelomacter in the sample with positive result according to the Ct value of the sample and the established quantitative standard curve.
The calculation formula is as follows:
the sample contained the number of individuals (cells/ml) of the luminal rotifers ═ conc/N, where N represents the copy number of the CoI gene per luminal rotifer.
The main technical effect of the invention is to find a molecular marker and design a specific primer, which can effectively identify the coelomacter under the condition that other polluted protozoa exist in the algae culture process. The adopted fluorescent quantitative PCR technology skillfully utilizes the advantages of efficient DNA amplification of the PCR technology, high specificity of specific primers and sensitivity and real-time quantification of the spectrum technology, overcomes some defects of conventional PCR qualitative detection, greatly improves the sensitivity and specificity of detection, shortens the test time, simplifies the experimental operation, and avoids pollution caused by the product post-treatment process due to the analysis of the fluorescent detection result by computer software, thereby being more objective, sensitive and accurate compared with the conventional PCR.
The method of the invention can also be designed into a kit for the early rapid identification of the coelomacter. The method has the characteristics of low requirement on samples, high efficiency, accurate detection, time and labor saving, cost saving, obvious effect and the like. The kit comprises the specific primer group.
Specifically, the kit can comprise a detection reagent and a genomic DNA extraction reagent. The detection reagent comprises a fluorescent dye, a specific primer and a positive standard substance. The genomic DNA extraction reagent preferably comprises a High Pure Template Preparation Kit from Roche.
The detection method provided by the invention has the characteristics of strong specificity and high sensitivity for the detection of the coelomacter, and the copy number of the mitochondrial CoI gene of a single coelomacter 3881 +/-341 copies/coelomacter is determined by the method. The lowest concentration which can be detected by the three pairs of primers in the method can respectively reach 47.99 copies/mu L, 45.35 copies/mu L and 8.97 copies/mu L, and the method has important significance for early monitoring of algae culture pollutants.
The sensitivity, stability and specificity of the method and the effectiveness of the environmental sample are comprehensively evaluated as follows:
(1) sensitivity: plasmid quantitative standards PGEMT-CoI-1, PGEMT-CoI-2 and PGEMT-CoI-3 were diluted 10 with sterile double distilled water, respectively10、109、108、107、106、105、104、103、102、101、100And (4) copies/mu L, detecting by adopting an optimized system, and verifying the sensitivity of the established method. The detection method is respectively 101～109copi es/reaction、102～109copies/reaction、101～109The high-sensitivity fluorescent probe has good linear relation in the range of copies/action orders, the correlation systems are respectively R1-1.00, R2-0.99 and R3-0.99, the detection ranges can respectively reach 9, 8 and 9 orders, and the sensitivity can reach below 10, 100 and 10 copies.
(2) Stability: we evaluated both from batch-to-batch and from intra-batch repeats. In-batch repeatability: selecting the same sample, setting 5 parallel reaction tubes to react simultaneously, and checking the variation coefficient of the Ct value, wherein the variation Coefficient (CV) of the Ct value detected repeatedly in batches is 1.43%. Batch-to-batch repeatability: selecting 2 recombinant plasmid standards with different concentrations, repeatedly testing for 3 times in a week, and testing the variation coefficient of the Ct value; the Coefficient of Variation (CV) of Ct values for the inter-batch reproducibility test was 1.76% and 1.93%, respectively. The detection method established by the invention has good stability and repeatability.
(3) Specificity: 22 samples were selected for detection in this study: 3 rotifer samples, 8 ciliate samples, 3 amoeba samples, 3 flagellates samples and 5 algae samples. And (3) detecting by adopting a PCR method, wherein the detection result shows that other samples except the coelomacter gondii are negative results.
Drawings
FIG. 1 is a standard curve for absolute quantification of CoI gene of Rotifer coeruleus, y1 ═ 3.3903 × log (conc) +41.70 (note: conc indicates the standard plasmid copy number in the reaction system).
FIG. 2 is a standard curve for absolute quantification of CoI gene of Rotifer coeruleus, y2 ═ 3.5882 Xlog (conc) +42.63 (note: conc indicates the standard plasmid copy number in the reaction system).
FIG. 3 is a standard curve for absolute quantification of coeruleus CoI gene, y3 ═ 3.2738 × log (conc) +39.12 (note: conc indicates the standard plasmid copy number in the reaction system).
FIG. 4 is a graph of fluorescence signals of the first pair of primers for the sensitive detection of fluorescence quantitative PCR, with the standard concentrations from left to right being 109～101copies/μL。
FIG. 5 is a graph of the fluorescence signals of the second pair of primers for the sensitive detection of the fluorescent quantitative PCR, with the respective standard concentrations from left to right being 109～102copies/μL。
FIG. 6 is a graph of the fluorescence signals of the sensitivity detection of the third pair of primers for fluorescence quantitative PCR, with the respective standard concentrations from left to right being 109～101copies/μL。
FIG. 7 is an agarose gel electrophoresis of the PCR product of the first primer specificity verification, M: 100bp DNA Ladder; 1: branchionus plicis; 2: lecane sp.; 3: philidina sp; 4: tetrahymena sp.; 5: euplotes sp.; 6: gastrostyla sp.; 7: sterkiella sp; 8: paramecium sp; 9: vorticilla sp; 10: (ii) a Cydium marinum; 11: colpoda sp; 12: a nucleic acid sequence; 13: hartmann sp.; 14: mayorella sp.; 15: poterioochromonas malh mensis; 16: eeuglena sp; 17: bodonidae sp; 18 Haematocuclcus p luvialis.
FIG. 8 is an agarose gel electrophoresis of the PCR product of the second primer pair specificity verification, M: 100bp DNA Ladder; 1: branchionus plicis; 2: lecane sp.; 3: philidina sp; 4: tetrahymena sp.; 5: euplotes sp.; 6: gastrostyla sp.; 7: sterkiella sp; 8: paramecium sp; 9: vorticilla sp; 10: (ii) a Cydium marinum; 11: colpoda sp; 12: a nucleic acid sequence; 13: hartmann sp.; 14: mayorella sp.; 15: poterioochromonas malh mensis; 16: eeuglena sp; 17: bodonidae sp; 18 Haematocuclcus p luvialis.
FIG. 9 is an agarose gel electrophoresis of the PCR product of the third primer specificity verification, M: 100bp DNA Ladder; 1: branchionus plicis; 2: lecane sp.; 3: philidina sp; 4: tetrahymena sp.; 5: euplotes sp.; 6: gastrostyla sp.; 7: sterkiella sp; 8: paramecium sp; 9: vorticilla sp; 10: (ii) a Cydium marinum; 11: colpoda sp; 12: a nucleic acid sequence; 13: hartmann sp.; 14: mayorella sp.; 15: poterioochromonas malh mensis; 16: eeuglena sp; 17: bodonidae sp; 18 Haematocuclcus p luvialis.
FIG. 10 is a graph showing the fluorescence signals detected by 1-5 coelomacters in the first primer pair, from left to right, 5-1 coelomacters, respectively.
FIG. 11 is a graph showing the fluorescence signals detected by the second pair of primers, 1-5 coelomacters, 5-1 coelomacters from left to right, respectively.
FIG. 12 is a graph showing the fluorescence signals detected by the third pair of primers, 1-5 coelomacters, 5-1 coelomacters from left to right, respectively.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1.
Designing a primer, and the steps are as follows:
molecular barcode methods are often used to identify microbial contamination, and currently one gene present in all eukaryotes is mainly studied: a portion of the cytochrome oxidase I (CoI cytochrome c oxidase I) gene. The DNA sequence can accurately distinguish species differences, and has the specificity of each species, just like the ID card number of the species. During the algae cultivation, the pollution of different kinds of microorganisms can occur, and in order to clarify the information of the pollutants, the coeruleus CoI gene is taken as a priority for monitoring for the first time.
Using the 119 pieces of coelomacter CoI gene sequence obtained in GenBank, and
the CoI gene sequences of 43 protozoa obtained from GenBank are analyzed by bioinformatics comparison, sequence specificity regions are selected, Primer 5.0 software is applied to design three pairs of PCR primers, the target amplified fragments are 344bp, 360bp and 193bp respectively, and the amplified sequences are shown as SEQ ID No. 1-3:
SEQ ID No.1：
AATGTTAGGGGTTGCTGATATAGCTTTCCCTCGTATGAATAATCTTTCTTTTTGGCTACTAATTCCTTCTTTTTTATTTTTGCTTTTATCTTCTATTTTAGATGCTGGTGTTGGTACTGGGTGAACAGTTTACCCTCCTCTTTCTGATGCTAAGTTTCATTCTGGTATTTCTGTAGATTTAGCTATTTTTAGATTACACTTAGCTGGGGTGTCTTCTATTCTAGGAAGAATTAATTTTTTAACAACTATTATTTGTTCTCGAACAACTAAAATGGTTTCTATGGATCGCCTACCTTTAATGATTTGAGCTTTGGCTGTGACAGCTATTTTATTAGTAACTTCAT；
SEQ ID No.2：
TTGGTATAATACAGGATTGCCACCACCAGCAGGATCAAAGAAAGAAGTGTTAAAATTTCGGTCAGTTAAAAGCATAGTAATAGCCCCAGCTAAAACAGGCAATCTTGTGACCAATAAAATAGCAGTCACAGCCAAAGCTCAAATCATTAAAGGTAGGCGATCCATAGAAACCATTTTAGTTGTTCGAGAACAAATAATAGTTGTTAAAAAATTAATTCTTCCTAGAATAGAAGACACCCCAGCTAAGTGTAATCTAAAAATAGCTAAATCTACAGAAATACCAGAATGAAACTTAGCATCAGAAAGAGGAGGGTAAACTGTTCACCCAGTACCAACACCAGCATCCAGAATAGAAGATAA；
SEQ ID No.3：
TTGGTATAATACAGGATTGCCACCACCAGCAGGATCAAAGAAAGAAGTGTTAAAATTTCGGTCAGTTAAAAGCATAGTAATAGCCCCAGCTAAAACAGGCAATCTTGTGACCAATAAAATAGCAGTCACAGCCAAAGCTCAAATCATTAAAGGTAGGCGATCCATAGAAATCATTTTTGTTGTACGAGAACAA。
the primer sequences are respectively (shown as SEQ ID No. 4-9):
the upstream primer CoI-1F: 5'-TAATGTTAGGGGTTGCTGAT-3', respectively;
the downstream primer CoI-1R: 5'-ATGAAGTTACTAATAAAATAGCTGT-3', respectively;
the upstream primer CoI-2F: 5'-TTATCTTCTATTCTGGATGCTG-3', respectively;
the downstream primer CoI-2R: 5'-TTGGTATAATACAGGATTGCC-3', respectively;
the upstream primer CoI-3F: 5'-GTTCTCGTACAACAAAAATGAT-3', respectively;
the downstream primer CoI-3R: 5'-TTGGTATAATACAGGATTGCC-3', respectively;
species information for protozoan CoI sequences obtained from GenBank is shown in table 1:
table 1: species information of protozoan CoI sequences obtained from GenBank
Example 2.
Construction and preparation of plasmid quantitative standard
The experimental steps are as follows:
1. construction of quantitative standards
Cloning by a PCR method to obtain a CoI gene segment containing a target gene sequence, recombining the CoI gene segment into a vector PGEM-T, and determining a DNA sequence. The constructed recombinant plasmids were designated PGEMT-CoI-1, PGEMT-CoI-2 and PGEMT-CoI-3 as quantitative standards.
2. Preparation of quantitative standards
The plasmid PGEMT-CoI was extracted and purified with an Omega plasmid extraction kit, and the copy number was converted from the concentration and mass of the plasmid determined by Nanodrop8000 and the Avogastron constant.
The calculation formula is as follows:
the average molecular weight of one base pair is 660 g/mol;
the total length of the plasmid is the total length of the vector plus the length of the insert;
n: represents the Avogastron constant (6.02X 10)23copies/mol)。
Example 3.
Extraction of genomic DNA
The experimental steps are as follows:
1. collection of luminal rotifers
After washing and starving the coelomacter obtained from the algae culture system for 72 hours, the individual or a plurality of individuals were separated under a dissecting mirror, and the separated individuals were dispensed into 1.5ml EP (eppendorf) tubes, and then the number of the separated individuals was confirmed under the dissecting mirror. Quickly freezing with liquid nitrogen for 5min, placing at 95 deg.C, bathing for 5min, and repeating for 3 times.
2. Extraction of genomic DNA
Comparing the extraction efficiency of the DNA of the coelomacter DNA extracted by different DNA extraction kits, the method described by the three kits DNeasy Blood & Tissue Kit (QIAGEN), DNeasy Plant Mini Kit (QIAGEN) and High Pure Template Preparation Kit (Roche), CTAB method, bead beating method and the patent application named as 'a method for rapidly extracting genomic DNA of single rotifer' is respectively adopted, and the extraction efficiency of obtaining the High Pure Template Preparation Kit (Roche) is the highest.
DNA was extracted from the collected coelomacter axostomatus using a High Pure Template prediction Kit (Roche), and the concentration and mass of the DNA were measured using Nanodrop 8000. And selecting DNA with the best concentration and quality for storage and standby.
Example 4.
Establishment and optimization of fluorescent quantitative PCR detection method
The experimental steps are as follows:
1. establishment of absolute quantitative standard curve
1) The plasmid PGEMT-CoI was extracted and purified with an Omega plasmid extraction kit, and the copy number was converted from the concentration and mass of the plasmid determined by Nanodrop8000 and the Avogastron constant.
2) Quantitative PCR reaction condition optimization by comparing the quantitative PCR primers with different concentrations in the reaction system with experimental results of supermix of different companies and different annealing temperatures, annealing times and the like in the reaction program, a reaction system and reaction conditions are selected, wherein the reaction system and reaction conditions have high reaction sensitivity, low background fluorescence signals, a typical S-type amplification fluorescence signal curve and reaction efficiency close to 1. The fluorescent quantitative PCR instrument used in the invention is LightCycle of Roche@96. The fluorescence detection program is set to collect the fluorescence signal at the end of the third step of each cycle, with the detection wavelength of 470-514 nm.
The quantitative PCR cycle is carried out in three steps, annealing and extension being carried out separately.
Screening of a reaction system:
| reagent | Screening conditions |
| supermix | Roche, Bio-Rad, kang are |
| Quantitative PCR primerArticle (A) | 0.3μmol/L、0.25μmol/L、0.2μmol/L、0.15μmol/L |
Screening of reaction conditions:
| condition | Screening conditions |
| Annealing temperature | 52℃、54℃、56℃、58℃ |
| Annealing time | 10s、20s、30s |
The optimized quantitative PCR procedure was:
the optimized quantitative PCR reaction system (20. mu.L reaction system) is:
3) absolute quantitative amplification standard curve establishment
The quantitative standards PGEMT-CoI-1, PGEMT-CoI-2 and PGEMT-CoI-3 were diluted in sterile double-distilled water in 10-fold gradient. The plasmid concentration after dilution was 10 respectively10、109、108、107、106、105、104、103、102、101、100And (4) reactions are carried out by adopting the optimized system and conditions. After the detection, a standard curve was plotted using the software (LightCycler 96SW 1.1) of the fluorescent PCR kit (FIG. 1, FIG. 2, FIG. 3).
The three pairs of primers of the detection method are respectively 101～109copies/reaction、102～109copies/reaction、101～109The high-sensitivity fluorescent probe has good linear relation in the range of copies/interaction orders, the correlation systems are respectively R1-1.00, R2-0.99 and R3-0.99, the detection ranges can respectively reach 9, 8 and 9 orders, and the sensitivity can reach less than 10, 100 and 10 copies (figure 4, figure 5 and figure 6).
Formula for calculating copy number of single coelomacter CoI gene in detection reaction:
(a, b are taken from the standard curve y ═ ax + b)
The formula for calculating the concentration of the coelomacter in the sample is as follows:
sample number of luminal rotifers (cells/ml) ═ conc/N (note: N represents copy number of CoI gene per luminal rotifer)
2. Stability analysis
The stability of the established fluorescent quantitative PCR reaction system is evaluated from two aspects of batch repeatability and batch repeatability.
1) And (3) carrying out batch repeatability inspection:
the same specimen was selected and subjected to nucleic acid extraction according to the methods described in examples 2 and 3.5 parallel reaction tubes are arranged by adopting an optimized system and conditions for carrying out reaction simultaneously, and the variation coefficient of the Ct value is tested by utilizing a statistical method. The Coefficient of Variation (CV) of Ct values for the in-batch reproducibility test was 1.43%.
2) And (3) batch-to-batch repeatability test:
2 recombinant plasmid standards with different concentrations are selected, the reaction system and the conditions described in the embodiment 4 are adopted, repeated detection is carried out for 3 times in a week, and the coefficient of variation of the Ct value is tested by a statistical method. The Coefficient of Variation (CV) of Ct values for the inter-batch reproducibility test was 1.76% and 1.93%, respectively. The detection method established by the invention has good stability and repeatability.
3. Specificity analysis
To test the applicability of the established fluorescent quantitative PCR method, 22 samples were selected for detection in this study: 3 rotifer samples, 8 ciliate samples, 3 amoeba samples, 3 flagellates samples and 5 algae samples. The PCR method is adopted for detection, and the detection result shows that the other samples except the coelomacter gondii are negative results (figure 7, figure 8 and figure 9).
4. Determination of detection results and quantification of Rotifer Coccida
1) The Ct value of the positive control should be less than 36.0 and the Ct value of the negative control should be greater than 39.0. The positive template is plasmid quantitative standard PGEMT-CoI-1, PGEMT-CoI-2 and PGEMT-CoI-3, and the negative template is sterile double distilled water.
2) If the Ct value is less than 36.0 and a typical amplification curve is presented, a positive result is judged, and the detection sample contains the coelomacter.
3) If the Ct value is larger than 39.0 or no amplification signal exists, the negative result is judged, and the fact that the coelomacter does not exist in the detection sample is indicated.
4) If the Ct value is between 36.0 and 39.0, the result is regarded as a suspicious result, and the sample needs to be checked repeatedly once.
If the result is still within this range, a negative result is judged.
And (4) calculating the content of the coelomacter in the sample with positive result according to the Ct value of the sample and the established quantitative standard curve.
The calculation formula is as follows:
sample number of luminal rotifers (cells/ml) ═ conc/N (note: N represents copy number of CoI gene per luminal rotifer)
Example 5.
Determination of mitochondrial CoI Gene copy number
The experimental steps are as follows:
1-5 coelomacters were collected in 1.5ml EP tubes. The nucleic acid was extracted according to the methods described in examples 2 and 3. And carrying out reaction by adopting an optimized system and conditions. After the detection is finished, the CoI gene copy numbers of coeruleus insects with different quantities are respectively calculated by combining the standard curve according to the method described in the embodiment 4, and then the mitochondrial CoI gene copy number of a single coeruleus insect is calculated. The detection results are shown in Table 2, and the fluorescence signals detected by different primers are shown in FIGS. 10, 11 and 12.
The calculation formula is as follows:
mitochondrial CoI gene copy number (copies/cell) ═ conc × V/M for single coelomacter (note: V represents the ratio of the volume solubilized at the time of DNA extraction to the volume of DNA used in the experiment, M represents the number of brachionus plicatilis)
TABLE 2 fluorescent quantitative PCR results of different numbers of coelomacters
The method determines that the copy number of the mitochondrial CoI gene of a single coelomacter griseus is 3881 +/-341 copies/coepies. The method has the characteristics of strong specificity and high sensitivity for the detection of the coelomacter, the lowest concentration which can be detected by the three pairs of primers in the method can respectively reach 47.99 copies/mu L, 45.35 copies/mu L and 8.97 copies/mu L, and the method has important significance for the early monitoring of the algae culture pollutants.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (11)
1. A primer group is characterized by comprising three pairs of primers which are respectively used for specific amplification sequences SEQ ID No.1-3, and the upstream and downstream primer sequences of each pair are as follows:
（1）CoI-1F：5’- TAATGTTAGGGGTTGCTGAT -3’；
CoI-1R：5’- ATGAAGTTACTAATAAAATAGCTGT -3’；
（2）CoI-2F：5’- TTATCTTCTATTCTGGATGCTG -3’；
CoI-2R：5’- TTGGTATAATACAGGATTGCC -3’；
（3）CoI-3F：5’- GTTCTCGTACAACAAAAATGAT -3’；
CoI-3R：5’- TTGGTATAATACAGGATTGCC-3’。
2. the primer group of claim 1, wherein the primer group is used in qPCR (quantitative polymerase chain reaction) method for rapid quantitative detection of coelomyces gondii in algae culture.
3. A qPCR method for rapid quantitative detection of coelomacter in algae culture is characterized in that the primer group of claim 1 is adopted to perform fluorescent quantitative PCR detection on genomic DNA of a sample to be detected, any pair of primers in the primer group of claim 1 is adopted to clone a gene containing a target amplification fragment into a plasmid PGEM-T, a recombinant plasmid is constructed to serve as a quantitative standard substance for detecting the concentration of the coelomacter, and the judgment method of the detection result is as follows:
(1) the Ct value of the positive control is less than 36.0, and the Ct value of the negative control is more than 39.0; the positive standard substance is recombinant plasmid, and the negative standard substance is sterile double distilled water;
(2) if the Ct value is less than 36.0 and a typical amplification curve is presented, determining a positive result, indicating that the detection sample contains the coelomacter;
(3) if the Ct value is more than 39.0 or no amplification signal exists, determining as a negative result, and indicating that the detection sample does not contain the coelomacter;
(4) if the Ct value is between 36.0 and 39.0, the result is regarded as a suspicious result, and the sample needs to be checked repeatedly once; if the result is still within the range, judging as a negative result;
and (4) calculating the content of the coelomacter in the sample with positive result according to the Ct value of the sample and the established quantitative standard curve.
4. The rapid quantitative detection qPCR method according to claim 3, wherein the concentration of each upstream primer and each downstream primer in the primer group is 10 μmol/L, and the final reaction concentration is 0.25 μmol/L.
5. The method of claim 3, wherein the fluorescent dye used in the quantitative fluorescence PCR is SYBR Green dye, and the fluorescence detection wavelength is 470-514 nm.
6. The rapid quantitative detection qPCR method according to claim 3, wherein the reaction system of the fluorescence quantitative PCR is as follows: 2 XLightCycler 480SYBR Green I Master 10. mu.L, 10. mu. mol/L forward primer 0.5. mu.L, 10. mu. mol/L reverse primer 0.5. mu.L, DNA template 1. mu.L, water was added to a total volume of 20. mu.L.
7. The rapid quantitative detection qPCR method according to claim 3, wherein the reaction procedure of the fluorescence quantitative PCR is as follows: 5min at 95 ℃ for 1 cycle; 10s at 95 ℃, 20s at 56 ℃ and 30s at 72 ℃ for 40 cycles.
8. A qPCR kit comprising the primer set of claim 1.
9. The kit of claim 8, wherein: comprises a detection reagent and a genome DNA extraction reagent.
10. The kit of claim 9, wherein: the detection reagent comprises a fluorescent dye, the positive standard of claim 3.
11. The kit of claim 9, wherein: the genomic DNA extraction reagent comprises a High Pure Template Preparation Kit of Roche.
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