CN111485034A - Fluorescent quantitative RT-PCR method for detecting fish viral nervous necrosis and corresponding kit - Google Patents
Fluorescent quantitative RT-PCR method for detecting fish viral nervous necrosis and corresponding kit Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitative RT-PCR method for detecting fish viral nervous necrosis and a corresponding kit. The invention skillfully applies specific gene detection to distinguish the fish viral nervous necrosis disease from other strains or viruses of other species, and obtains accurate genus information through comprehensive judgment. Compared with the existing mainstream detection kit, the kit for detecting the fish viral nervous necrosis disease has the advantages of high sensitivity, rapidness, convenience, good specificity, rigorous and accurate judgment and the like, and has good application prospect and market value.
Description
Technical Field
The invention relates to disease detection of marine fishes, belongs to the technical field of molecular biology, and particularly relates to a method for performing fluorescent quantitative RT-PCR detection on viral nervous necrosis of fishes through specific genes and a corresponding detection kit.
Background
The nervous necrosis virus (VNNV) belongs to the family of nodaviridae, and has a 20-face shape without envelope, the diameter of the virus particle is generally 25-34nm, the genome of the virus particle consists of two single-stranded RNAs, one RNA encodes non-structural protein, and the other encodes main structural protein. Pathological examination of the diseased fish can show that a large number of vacuoles are formed in spinal cord, central nervous system and retina tissues, and the diseased fish is degraded neurologically. The nervous necrosis disease is popular in seawater fishes in almost all regions except south America and Africa, the regions of Guangdong, Fujian, Hainan and Taiwan are main high-incidence regions of nervous necrosis disease of fishes in China, and the larva fishes and juvenile fishes mainly infected with grouper, abalone in red fin east and sparus fasciatus have the death rate of 40-100 percent generally. The death rate of serious patients in one week can reach 100%. The fish with viral nervous necrosis disease has black body, prominent eyeball, reduced appetite, emaciation, neurological dysfunction, floating on water surface, circling in water or spiral swimming. Pathological examination shows that the diseased fish maw is swollen, nervous tissue is necrotic and vacuole appears.
The early stage of the nervous necrosis disease of the infected fishes can be controlled by vaccines, but the epidemiological research on the nervous necrosis virus of the fishes is limited, so the disease control effect is poor. In China, intensive aquaculture is prevalent, the variety is single, the aquaculture density is high, once infection occurs, the disease propagation speed is very high, and therefore detection and prevention of the disease are extremely important.
At present, the fish nervous necrosis virus detection is mainly based on various diagnosis methods such as a pathological section method, immunology, RT-PCR and L AMP technologies, but the existing methods have the defects that the detection methods based on the pathological section method and the immunology are complex in operation, long in time consumption and low in sensitivity, the RT-PCR method is sensitive and accurate, but the process is complex, needs to be operated by professionals and is not suitable for popularization and application, the L AMP technology is rapid and simple, but amplification products are easy to cross-contaminate, so that false positive is high, the amplification products are generally only used as disease primary screens, and certain limitations exist in detection application.
Disclosure of Invention
One of the purposes of the invention is to provide a fluorescent quantitative RT-PCR method for detecting fish viral nervous necrosis, which can detect fish nervous necrosis quantitatively, rapidly and in real time, so that VNNV detection is more accurate, sensitive, rapid and safe.
Specifically, the method comprises the following steps:
s1, collecting a sample;
s2, extracting genome DNA;
s3, detecting the specific gene CP gene of the fish viral nervous necrosis disease by using fluorescent quantitative RT-PCR;
s4, reading the Ct value of the amplification; when the Ct value of the specific gene CP gene of the fish viral nervous necrosis disease is less than 35, the detection result of the fish viral nervous necrosis disease is positive; and when the Ct value of the specific gene CP gene of the fish viral nervous necrosis disease is more than 35, the detection result of the fish viral nervous necrosis disease is negative.
As a preferred technical scheme, the step S2 further comprises the step of designing a detection primer and a TaqMan probe of the CP gene.
As a preferred technical scheme, the detection primer of the specific gene CP gene of the fish viral nervous necrosis disease is shown as SEQ ID NO. 1-2;
SEQ ID NO:1(5'-GGACCTCGTCGGGAAAGGAG-3')；
SEQ ID NO:2(5'-GACACAGCACTGACACGTTGA-3')。
the probe sequence is shown as SEQ ID NO. 3;
SEQ ID NO:3(5'-FAM-CGTCACCTGGTCGGCTGATACTCCTGTA-BHQ1-3)。
as a preferred technical scheme, the reaction system for the fluorescent quantitative PCR detection is 25 mu l, and comprises 12.5 mu l of 2 × TaqPCR Mix, 1 mu l of total10uM Primers Mix, 0.5 mu l of TaqMan probe, 2 mu l of DNA input, H2O 9μl。
As a preferred technical scheme, the reaction conditions of the fluorescent quantitative PCR detection are 50 ℃ pre-denaturation for 15min, 95 ℃ denaturation for 10min, 94 ℃ annealing for 15s, 58 ℃ annealing for 40s, fluorescent signals are collected, and 40 cycles are carried out.
The invention also aims to provide a fluorescent quantitative RT-PCR kit for detecting the fish viral nervous necrosis disease, which comprises a detection primer and a TaqMan primer probe of the specific gene CP gene of the fish viral nervous necrosis disease.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, orphan genes capable of distinguishing the fish viral nervous necrosis disease and the bacterial species and virus specificity of other species are excavated from a large amount of research data on the fish viral nervous necrosis disease and other species and viruses in the early stage, whether other bacterial species or virus positive samples and environmental samples contain the fish viral nervous necrosis disease or not is accurately judged by detecting the specific genes, the early stage big data mining and the comparison basis between different species are provided, and the selected specific genes have the specificity of species and genus.
(2) In the invention, by optimizing the design conditions and the experimental conditions, the primer which is optimized for CP gene detection amplification conditions is selected as the detection primer, so that the amplification efficiency of the primer is improved, the detection primer with stability, high efficiency, high sensitivity and reproducibility can be achieved, and the correct detection of a trace sample is realized.
(3) Compared with other detection methods in the market, the detection method provided by the invention has the advantages that the detection strategy can intuitively judge the infection of the fish viral nervous necrosis disease and the infection of other viruses or strains, and the result is more rigorous and accurate.
(4) The invention can be implemented from 101Diluting to 10 times5The detection range of the dilution is doubled, the sensitivity is high, the application range is wide, a sample with higher concentration can be detected through dilution conversion, the detection result is stable, and the reproducibility is good.
(5) The detection method can be applied to screening and detecting various source samples such as culture water body samples or grouper samples and the like, accurately judges whether the culture water body samples contain the fish viral nervous necrosis diseases or not, and is convenient, rapid and sensitive to operate. The detection kit can be applied to the rapid detection of various culture samples without additionally carrying out microbial culture, so that the gene detection is applied to the detection of culture water and daily life, and has the advantages of high sensitivity, strong operability, rapidness, high efficiency and the like.
Drawings
FIG. 1CP Gene detection primer Standard Curve.
FIG. 2 is a CP gene amplification curve in example 1.
FIG. 3 is a CP gene amplification curve in example 2.
FIG. 4 is an amplification curve of CP gene in the specific experiment in example 3.
FIG. 5 is a graph showing the amplification curves of CP gene of sample 1 at different dilution ratios in example 4.
FIG. 6 is a graph showing the amplification curves of CP gene of sample 2 at different dilution ratios in example 4.
FIG. 7 is a graph showing the amplification curves of CP gene of sample 3 at different dilution ratios in example 4.
Detailed Description
In order to make the technical contents of the present invention more clearly understandable, the following examples are described in detail with reference to the accompanying drawings, it being understood that these examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.
The following reagents used in the present invention can be purchased from conventional sources.
TABLE 1
In some embodiments, positive standard plasmids are constructed using the amplification products, and the amplification standard curve of the primers is detected and plotted to obtain the amplification efficiency of the detection primers, and the amplification efficiency and the confidence degree of the primer sequences provided by the invention are optimized.
In some embodiments, by detecting specific genes of different positive strains, samples infected by the fish viral nervous necrosis disease and samples infected by other species of fungi and viruses can be correctly distinguished, so that the fish viral nervous necrosis disease can be accurately detected and judged.
In some embodiments, the number of pairs is 10, 100, 1000, 10000, and 105The positive bacteria are extracted and detected, and the fish viral nervous necrosis disease and microorganisms of other species can be accurately distinguished, so that the fish viral nervous necrosis disease can be accurately detected and judged. And can guarantee that as low as 10 positive bacteria can still be accurately detected, and a microbial sample with higher titer can be also accurately detected by diluting to a detection interval.
In some embodiments, the method comprises the steps of collecting pure water, samples of healthy grouper, samples of the pike of the pearl sea and culture water samples of the grouper, and rapidly and conveniently detecting whether the samples contain the fish viral nervous necrosis disease or not by detecting specific genes.
It will be understood by those skilled in the art that the genes to be detected and the specific primers to be designed in the method can be designed to achieve the corresponding detection purpose through the design of other sections of the same detection object.
The fluorescent quantitative RT-PCR method and the kit for detecting the fish viral nervous necrosis disease can be widely applied to a plurality of inspection and quarantine categories such as grouper sample detection, culture water body detection and the like, and can provide convenient, quick, accurate and efficient gene detection-based monitoring products for grouper culture, propagation and the like.
Example 1 Standard Curve for primer amplification detection and plasmid sensitivity test
1. Cultivation of microorganisms
The L B culture medium is used as a culture medium of the fish viral nervous necrosis disease plasmid, the fish viral nervous necrosis disease plasmid grows well in a plate with the pH value of 7.0-7.4, the diameter of a bacterial colony on the plate is 2mm, and the plate is round, smooth and transparent.
2. Genomic DNA extraction
Extraction of genomic DNA was performed as indicated in the QIAamp DNAMini Kit instruction by Qiagen.
3. Standard quality particle preparation of amplification product
DNA extracted from fish viral nervous necrosis disease is used, amplification is carried out with a primer of CP gene, the amplification product is treated by adding A, T4 ligase is used for connecting into T vector, and plasmid is amplified in large scale after competent cell is transduced. The primer sequence and TaqMan probe sequence of the CP gene are shown in SEQ ID NO 1-3, and are specifically shown in Table 2.
TABLE 2
4. Plasmid extraction and Standard Curve gradient configuration
Respectively plating standard clone bacteria of primer amplification products corresponding to the amplified CP gene, collecting monoclonal bacterial plaques, culturing and shaking the bacteria by L B for overnight amplification, finally obtaining plasmid positive strains in 5ml exponential growth phase, extracting plasmid DNA according to the instruction of QIAGEN plasmid Midi Kit, detecting the concentration of the plasmid DNA after extraction, and configuring 7 standard products with concentration gradients of 1 fg/mu L and 1 × 10 by a 10-fold dilution method1fg/μL，1×102fg/μL，1×103fg/μL，1×104fg/μL，1×105fg/μL，1×106fg/μL。
5. Drawing a standard curve of the detection primer
And (3) performing qRT-PCR amplification of a concentration gradient standard curve according to the instruction of TaKaRa Taq RT-PCR Mix instruction, and drawing a corresponding amplification curve to obtain the amplification efficiency of the primer. 3 biological repeats and 3 technical repeats are set for each qRT-PCR reaction, the qRT-PCR reaction system and the amplification reaction conditions are shown in Table 3, and the drawn standard curve is shown in FIG. 1.
TABLE 3
6. Analysis of results
The result of the standard curve for detecting primer amplification shows that the primers corresponding to the CP gene are optimized primer design and a reaction system. Linear standard curve confidence R2>0.980, the primer design and the reaction system are proved to be in the optimal state, the standard deviation and the variation coefficient of the amplification curve of the CP gene and the amplification efficiency under 7 concentration gradients are both satisfied with the requirement of the optimal state, the primer can meet the detection requirement under the reaction condition, which shows that the plasmid sensitivity of the method is high, the variation coefficient between CT values measured by 3 technical repeated experiments is less than 5.0%, the method is proved to have high repeatability, the detection result is shown in table 4 and fig. 2, fig. 2 is the amplification curve of the CP gene under different plasmid concentrations, the concentration of the amplification curve from left to right is 1 × 10 in sequence6fg/μL，1×105fg/μL，1×104fg/μL，1×103fg/μL，1×102fg/μL，1×101fg/μL，1×100fg/μL。
TABLE 4
Example 2 plasmid reproducibility test
1. Cultivation of microorganisms
The same as in example 1.
2. Genomic DNA extraction
The same as in example 1.
3. qRT-PCR detection of target genes
According to the instruction of TaKaRa Taq RT-PCR MixqRT-PCR amplification of samples and Ct value readings corresponding to the primers 2 concentrations were set for the qRT-PCR reaction of this experiment, 1 × 10 respectively6fg/. mu. L and 1 × 105fg/. mu. L, 4 biological replicates and 5 technical replicates were set at each concentration, respectively, and the qRT-PCR reaction system and amplification reaction conditions are shown in Table 5.
TABLE 5
4. Analysis of results
In the embodiment, the correlation coefficient and the reliability of the amplification curve of the CP gene and the amplification efficiency under 2 concentrations are analyzed, the requirements of the optimal state are met, and the primers can meet the detection requirements under the reaction conditions. The detection results are shown in table 6 and fig. 3, 4 groups are set for each concentration condition, each group is repeatedly measured for 5 times, and the measurement results show that the coefficient of variation is less than 5.0 percent, which proves that the method has good repeatability and high stability.
TABLE 6
EXAMPLE 3 Positive and negative sample detection
1. Cultivation of microorganisms
The same as in example 1.
2. Genomic DNA extraction
The same as in example 1.
3. qRT-PCR detection of target genes
qRT-PCR amplification of samples was performed according to TaKaRa Taq RT-PCR Mix instructions and Ct value readings corresponding to the primers were obtained. The qRT-PCR reaction set 3 biological repeats and 3 technical repeats, the qRT-PCR reaction system and amplification reaction conditions were the same as example 2.
4. Analysis of results
As shown in Table 7 and FIG. 4, the qRT-PCR amplification results of the primers showed that the fish viral nervous necrosis disease showed a positive result of the CP gene, while other viruses or genera all showed negative results. Therefore, the CP gene is detected for the fish viral nervous necrosis disease and other viruses or genera, so that the fish viral nervous necrosis disease and other viruses or genera can be better distinguished. In conclusion, the corresponding primers and probes of the CP gene can meet the detection and judgment of the viral nervous necrosis of the fish.
TABLE 7
EXAMPLE 4 sensitivity of Positive samples
1. Sample collection and processing
Respectively collecting healthy rockfish samples and positive samples of viral nervous necrosis disease according to 10110 times of210 times of310 times of4Multiple and 105The respective dilutions were performed in a gradient and the genomic DNA was extracted as indicated in the QIAamp DNAMini Kit instruction from Qiagen.
2. Genomic DNA extraction
The same as in example 1.
3. qRT-PCR detection of target genes
The same as in example 2.
4. Analysis of results
As shown in Table 8 and FIGS. 5 to 7, the qRT-PCR amplification results of CP gene primers for 3 samples showed a value from 10110 times of210 times of310 times of4Multiple and 105In the range of the dilution sample, the fish viral nervous necrosis disease shows the positive result of the CP gene. Therefore, the detection of CP gene can be preferably carried out to a level of from 101Multiple to 105Detecting the viral nervous necrosis of the fish in the dilution range. Simultaneously, diluting 10 from the sample1Multiple to 105In a double range, the detection of CP gene can be performedThe detection of the fish viral nervous necrosis disease is well finished, and the detection range can span to 10 sample dilution1Multiple to 105And the higher strain concentration can be diluted to be within the optimal detection range by a dilution method, so that the expected detection requirement can be met.
TABLE 8
EXAMPLE 5 detection of environmental samples
1. Environmental sample collection and extraction of genomic DNA
And respectively collecting a pure water sample, a healthy grouper sample, a positive specimen of Zhuhai fighter, a positive specimen of Zhanjiang Chikan and a culture seawater sample, and extracting the genome DNA according to the instruction of QIAamp DNAmii Kit of Qiagen.
2. qRT-PCR detection of target genes
The same as in example 2.
3. Analysis of results
As shown in Table 9, the qRT-PCR amplification results of the primers show that CP genes are detected from all samples except for a pure water sample, a healthy grouper sample, a positive sample of Binhancmen, a positive sample of Zhanjiang Chikan and a sample of aquaculture seawater, and the corresponding sample sources are proved to contain the fish viral nervous necrosis disease. The final criterion for judging that the fish viral nervous necrosis disease is positive is that the Ct value obtained by CP gene detection is less than 35 cycles. If the Ct value obtained by the CP gene detection is more than 35 cycles, the negative fish viral nervous necrosis disease is judged to be undetected.
TABLE 9
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, but should be considered as the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above examples only show some embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
<110> Guangdong Meige Gene science and technology Co., Ltd
<120> fluorescent quantitative RT-PCR method for detecting fish viral nervous necrosis and corresponding kit
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>20
<212>DNA
<213> CP Gene Forward primer (Artificial Sequence)
<400>1
<210>2
<211>21
<212>DNA
<213> CP Gene reverse primer (Artificial Sequence)
<400>2
gacacagcac tgacacgttg a 21
<210>3
<211>28
<212>DNA
<213> CP Gene Probe (Artificial Sequence)
<400>3
cgtcacctgg tcggctgata ctcctgta 28
Claims (6)
1. The fluorescent quantitative RT-PCR method for detecting the fish viral nervous necrosis is characterized by comprising the following steps:
s1, collecting a sample;
s2, extracting genome DNA;
s3, detecting the specific gene CP gene of the fish viral nervous necrosis disease by using fluorescent quantitative RT-PCR;
s4, reading the Ct value of the amplification; when the Ct value of the specific gene CP gene of the fish viral nervous necrosis disease is less than 35, the detection result of the fish viral nervous necrosis disease is positive; and when the Ct value of the specific gene CP gene of the fish viral nervous necrosis disease is more than 35, the detection result of the fish viral nervous necrosis disease is negative.
2. The fluorescent quantitative RT-PCR method for detecting fish viral nervous necrosis disease according to claim 1, wherein the step S2 further comprises a step of designing a detection primer of CP gene and TaqMan probe.
3. The fluorescent quantitative RT-PCR method for detecting the fish viral nervous necrosis disease according to claim 1, wherein the detection primer of the specific gene CP gene of the fish viral nervous necrosis disease is shown as SEQ ID NO. 1-2, and the TaqMan probe sequence is shown as SEQ ID NO. 3.
4. The fluorescent quantitative RT-PCR method for detecting fish viral nervous necrosis as claimed in claim 1, wherein the reaction system of the fluorescent quantitative PCR detection is 25 μ l, comprising 2 × Taq PCR Mix 12.5 μ l, total10uM Primers Mix 1 μ l, TaqMan probe 0.5 μ l, DNA input 2 μ l, H2O 9μl。
5. The fluorescence quantitative RT-PCR method for detecting fish viral nervous necrosis disease as claimed in claim 1, wherein the reaction conditions of the fluorescence quantitative PCR detection are pre-denaturation at 50 ℃ for 15min, denaturation at 95 ℃ for 10min, annealing at 94 ℃ for 15s, annealing at 58 ℃ for 40s, and collection of fluorescence signals for 40 cycles.
6. A fluorescent quantitative RT-PCR kit for detecting fish viral nervous necrosis is characterized by comprising a detection primer of a specific gene CP gene of the fish viral nervous necrosis and a TaqMan probe.
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| CN114231664A (en) * | 2021-11-30 | 2022-03-25 | 华南农业大学 | Epinephelus akaara nervous necrosis virus RGNNV detection kit and detection method |
| CN114657283A (en) * | 2021-10-21 | 2022-06-24 | 沈阳海关技术中心 | Primer and probe combination for simultaneously detecting EHNV, VNNV and RSIV |
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| CN114657283A (en) * | 2021-10-21 | 2022-06-24 | 沈阳海关技术中心 | Primer and probe combination for simultaneously detecting EHNV, VNNV and RSIV |
| CN114231664A (en) * | 2021-11-30 | 2022-03-25 | 华南农业大学 | Epinephelus akaara nervous necrosis virus RGNNV detection kit and detection method |
| CN114231664B (en) * | 2021-11-30 | 2023-07-28 | 华南农业大学 | Epinephelus akaara nerve necrosis virus RGNNV detection kit and detection method |
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