CN111485017A - Fluorescent quantitative PCR method for detecting prawn enterogaster hepatica and corresponding kit - Google Patents

Fluorescent quantitative PCR method for detecting prawn enterogaster hepatica and corresponding kit Download PDF

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CN111485017A
CN111485017A CN201910085182.XA CN201910085182A CN111485017A CN 111485017 A CN111485017 A CN 111485017A CN 201910085182 A CN201910085182 A CN 201910085182A CN 111485017 A CN111485017 A CN 111485017A
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hepatica
enterobacter
shrimp
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唐伟
束文圣
杨荣
束浩然
黄斌
蒋华
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Guangdong Magigene Technology Co ltd
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Abstract

The invention discloses a fluorescent quantitative PCR method for detecting prawn enterobacter hepatica and a corresponding kit. The invention skillfully applies specific gene detection to distinguish the shrimp enterobacter hepatica from other genera of the shrimp, and obtains accurate information of the genera through comprehensive judgment. Compared with the existing mainstream detection kit, the kit for detecting the shrimp enterobacter hepatica 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

Fluorescent quantitative PCR method for detecting prawn enterogaster hepatica and corresponding kit
Technical Field
The invention belongs to the technical field of molecular detection, and particularly relates to a method for carrying out fluorescent quantitative PCR detection on prawn enterobacter hepatica through a specific gene and a corresponding detection kit.
Background
Microsporidian (Microsporidium) is a unicellular obligate intracellular parasite with a nucleus, found in a range of hosts from invertebrates to vertebrates, and has microscopic spores, containing a single polar thread and infectious sporogens. After the spores are ingested by the host, the bodies of the insects enter the intestinal epithelium and reach specific tissues via the blood or body cavities.
Prawn hepatopenaei disease (EHP) is a parasitic disease caused by microsporidia. EHP is a new disease which appears in the culture of prawns in China in recent years. A new parasite was discovered in the 2009 culture of Penaeus monodon (Penaeus monodon) and isolated and named, and subsequently found in the culture of Penaeus monodon and water in china, india, indonesia, vietnam, thailand, and other countries. The microsporidian mainly infects the intestinal epidermis of the prawn, further infects the hepatopancreas, parasitizes in epithelial cells of the hepatic duct and the pancreatic duct of the prawn, the diseased prawn is slow in development and occasionally accompanied by white feces, the intestinal absorption function of the newly diseased prawn is reduced, enteritis and hepatopancreas atrophy seriously appear, and huge economic loss is brought to the prawn breeding industry.
At present, the prawns infected with EHP do not show characteristic clinical symptoms and have no effective treatment method, but the prevention is mainly performed. Therefore, the establishment of the quantitative detection and prevention technology of the pathogen strengthens the detection of shrimp seedlings and adult shrimps and the monitoring of baits and the environment of the aquaculture water body, and has important significance for preventing EHP infection, effectively cutting off EHP transmission and promoting the healthy and stable development of the prawn aquaculture industry in China. At present, 5 EHP detection methods mainly comprise a first microscopic observation method, a second immunological detection method, a third nucleic acid probe hybridization method, a fourth common PCR detection method and a fifth loop-mediated isothermal amplification method. The first three methods are complex to operate and long in time consumption, diagnosis needs to be confirmed in a molecular laboratory generally, the detection sensitivity is low, and only prawns with obvious morbidity can be diagnosed; although the common PCR detection method has accurate result, the detection time is long, the operation is complex, and quantification cannot be performed, so that the popularization and application of the common PCR detection method in production are limited to a certain extent; although the loop-mediated isothermal amplification method has the advantages of short detection time and simple operation, the loop-mediated isothermal amplification method has high false positive and is subject to the disadvantages of professionals.
The invention provides a method for quantitatively, quickly and real-timely monitoring prawn enterobacter hepatica, which overcomes the defects of the prior art and ensures that EHP (enterobacter hepatica) is more accurate, sensitive, quick and convenient to detect.
Disclosure of Invention
One of the purposes of the invention is to provide a fluorescent quantitative PCR method for detecting the shrimp enterobacter hepatica.
Specifically, the method comprises the following steps:
s1, collecting a sample;
s2, extracting genome DNA;
s3, detecting the prawn enterobacter hepatica specific gene 18SrRNA gene, β -tubulin gene and SWP1 gene by using fluorescent quantitative PCR;
and S4, reading the Ct value of amplification, wherein when the Ct values of 3 genes in the shrimp enterobacter hepatica specific gene are all less than 35, the detection result of the shrimp enterobacter hepatica is positive, and when the Ct values of the 18SrRNA gene, the β -tubulin gene and the SWP1 gene of the shrimp enterobacter hepatica are all more than 35, the detection result of the shrimp enterobacter hepatica is negative.
As a preferred technical scheme, the detection primer of the prawn shrimp enterobacter hepatica specific gene 18SrRNA is shown as SEQ ID NO: 1-2:
SEQID NO:1(5'-CAGGTCCGTGATGCCCTTA-3')；
SEQ ID NO:2(5'-CATCTCAAATTGCTTTATTCTCTCAAGA-3')。
the sequence of the TaqMan probe is shown in SEQ ID NO. 3;
SEQ ID NO:3(5'-FAM-TATCCTGGGCAGCAAGCGCAATACA-BHQ1-3')。
the detection primer of the prawn shrimp enterobacter hepatica specific gene β -tubulin is shown as SEQ ID NO: 4-5:
SEQ ID NO:4(5'-GATTTGAGAAAATTGGCGGTTAA-3')；
SEQ ID NO:5(5'-CACAGAGGCGCATATCCTACAC-3')。
the sequence of the TaqMan probe is shown as SEQ ID NO. 6;
SEQ ID NO:6(5'-FAMTGATTCCATTTCCACGACTGCACTTTTTC-BHQ1-3')。
the detection primer of the prawn shrimp enterobacter hepatica specific gene SWP1 is shown in SEQ ID NO. 7-8:
SEQ ID NO:7(5'-AAAATGCGTGACGAACTAA-3')；
SEQ ID NO:8(5'-CGCCAAACTCGTAATTACTT-3')。
the sequence of the TaqMan probe is shown as SEQ ID NO. 9;
SEQ ID NO:9(55'-FAM-TGTTTTGCAGAGTGTTGTTAAGGGTT-BHQ1-3')。
the invention also aims to provide a fluorescent quantitative PCR kit for detecting the shrimp enterobacter hepatica, which comprises detection primers and TaqMan probes of the shrimp enterobacter hepatica specific genes 18SrRNA, β -tubulin and SWP 1.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, specific genes capable of distinguishing the shrimp enterobacter hepatica from other bacteria are excavated from a large amount of research data of the early stage shrimp enterobacter hepatica and other bacteria, whether other bacteria samples and environment samples contain the shrimp enterobacter hepatica is accurately judged by detecting the specific genes, the early stage big data mining and the comparison between different species are based, and the selected specific genes have the specificity of species and bacteria.
(2) In the invention, by optimizing the design conditions and the experimental conditions, the primers which are optimized for each gene detection amplification condition are selected as the detection primers, so that the amplification efficiency of the primers is improved, the detection primers 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 judge the enterobacter hepatica and other genera of the prawn, and the result is rigorous and accurate.
(4) The invention can be implemented from 101Diluting to 10 times5The detection range of double dilution is high, and the sensitivity is highThe application range is wide, samples with higher concentration can be detected through dilution, the detection result is stable, and the reproducibility is good.
(5) The detection method can be applied to screening and detecting the source samples in the fields of environmental water body samples and the like, accurately judges whether the samples contain the prawn enterobacter hepatica, and is convenient, rapid and sensitive to operate. The detection kit can be applied to rapid detection of various aspects such as aquaculture, food safety and the like, does not need additional microbial culture, enables gene detection to be applied to daily life, and has the advantages of high sensitivity, strong operability, rapidness, high efficiency and the like.
Drawings
FIG. 1 is a standard curve of 18SrRNA gene detection primers.
FIG. 2 illustrates the β -tubulin gene detection primer standard curve.
FIG. 3 is a SWP1 gene detection primer standard curve.
FIG. 4 is an amplification curve of the 18SrRNA gene in example 1.
FIG. 5 is the β -tubulin gene amplification curve in example 1.
FIG. 6 is the SWP1 gene amplification curve in example 1.
FIG. 7 is an amplification curve of the 18SrRNA gene in example 2.
FIG. 8 is the β -tubulin gene amplification curve in example 2.
FIG. 9 is the SWP1 gene amplification curve in example 2.
FIG. 10 is an amplification curve of three genes specifically tested in example 3.
FIG. 11 is an amplification curve of the 18SrRNA gene at different dilution ratios in example 4.
FIG. 12 is a graph showing the amplification curves of β -tubulin gene at different dilution ratios in example 4.
FIG. 13 is an amplification curve of SWP1 gene 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
Figure BDA0001961516750000061
Figure BDA0001961516750000071
In some embodiments, positive standard plasmids are constructed using the amplification products, and a standard curve of amplification of the primers is detected and plotted to obtain the amplification efficiency of each pair of detection primers, and the amplification efficiency and confidence level of the primer sequences provided by the invention are optimized.
In some embodiments, by detecting specific genes of different positive strains, the prawn enterobacter hepatica and other genera can be correctly distinguished, so that the prawn enterobacter hepatica can be accurately detected and judged.
In some embodiments, by pair 10110 times of210 times of310 times of4Multiple and 105The dilution-multiplied positive bacteria are extracted and detected, and the prawn enterobacter hepatica and other bacterial genera can be accurately distinguished, so that the prawn enterobacter hepatica can be accurately detected and judged. And can ensure positive sample dilution 105The detection can still be accurately carried out, and the microorganism sample with higher titer can be also accurately detected by diluting the microorganism sample to a detection interval.
In some embodiments, a positive sample of the east-China island Zhanjiang, a Zhenyang sample of the Wenchang province and a culture seawater sample are collected, and whether the sample contains the prawn enterobacter hepatica can be quickly and accurately detected through detection of specific genes, so that the method is convenient and quick.
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 method and the kit for detecting the fluorescent quantitative PCR of the enterobacter hepatica of the prawns can be widely applied to a plurality of inspection and quarantine categories such as water body monitoring, drinking water monitoring, food safety monitoring and the like, and can provide a convenient, quick, accurate and efficient monitoring product based on gene detection for prawn culture, breeding and the like.
Example 1 Standard Curve for detection primer amplification
1. Cultivation of microorganisms
The L B culture medium is used as a culture medium of prawn shrimp enterobacter hepatica plasmid, the prawn enterobacter hepatica 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 prawn enterobacter hepatica plasmid is round, smooth and transparent.
2. Genomic DNA extraction
Extraction of genomic DNA was performed according to the QIAamp DNA Mini Kit instructions from Qiagen.
3. Standard quality particle preparation of amplification product
DNA extracted from shrimp enterobacter hepatica is used, primers of an 18SrRNA gene, an β -tubulin gene and an SWP1 gene are used for amplification, an amplification product is added with A, T4 ligase is used for connecting into a T vector, and after competent cells are transduced, a plasmid is obtained to amplify in a large scale, wherein the primer sequences of 3 genes are shown in SEQ ID NO. 1-9, and are specifically shown in Table 2.
TABLE 2
Figure BDA0001961516750000081
Figure BDA0001961516750000091
4. Plasmid extraction and Standard Curve gradient configuration
Respectively plating standard clone bacteria of primer amplification products corresponding to 18SrRNA, β -tubulin gene and SWP1 gene, collecting monoclonal bacterial plaques, culturing and shaking bacteria through L B for overnight amplification to finally obtain Plasmid positive bacterial strains in exponential growth phase of 5ml, 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 method, wherein the concentration gradients are respectively 1 fg/mu L and 1 ×1fg/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
qPCR amplification of concentration gradient standard curves was performed as instructed by QIAamp DNA Mini Kit instructions from Qiagen, and the corresponding amplification curves were plotted to obtain the amplification efficiency for each pair of primers. 3 biological repetitions and 3 technical repetitions are set for each qPCR reaction, the qPCR reaction system and amplification reaction conditions are shown in Table 3, and the drawn standard curves are shown in FIGS. 1-3.
TABLE 3
Figure BDA0001961516750000101
6. Analysis of results
The results of the standard curve for detecting primer amplification show that the primers corresponding to the 18SrRNA, the β -tubulin gene and the SWP1 gene are optimized primer designs and reaction systems, wherein the reliability R of the linear standard curve is2>0.990, proving that the primer design and the reaction system are the optimal state, analyzing the correlation coefficient and the credibility of the amplification curve of 3 genes and the amplification efficiency under 7 concentration gradients, wherein 1 × 101fg/μL～1×106CT values measured under the 6 concentrations of fg/. mu. L are all less than 35 cycles and all reach the requirement of the optimal state, and 3 pairs of primers can meet the detection requirement under the reaction condition, which indicates thatThe method has high plasmid sensitivity, and the coefficient of variation among CT values measured by 3 technical repeated experiments is less than 5%, which proves that the method has high repeatability, the detection results are shown in tables 4-6 and figures 4-6, the figures 4-6 are amplification curves of three genes under different plasmid concentrations, the concentrations of the amplification curves from left to right are 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
Figure BDA0001961516750000111
TABLE 5
Figure BDA0001961516750000112
Figure BDA0001961516750000121
TABLE 6
Figure BDA0001961516750000122
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.
qPCR detection of 2.3 target genes
The qPCR amplification of the samples was performed according to the instructions of TaKaRa Taq PCR Mix, and Ct value readings corresponding to each pair of primers were obtained, the reaction set for qPCR of this experiment was 2 concentrations of 1 × 10 respectively6fg/. mu. L and 1 × 105fg/μ L, 4 biological replicates and 5 technical replicates were set at each concentration, respectively, and the qPCR reaction system and amplification reaction conditions are shown in table 7.
TABLE 7
Figure BDA0001961516750000131
4. Analysis of results
In the embodiment, correlation coefficients and reliability of amplification curves of 3 genes and amplification efficiency under 2 concentrations are analyzed, CT values of measurement are less than 35 cycles and all meet the requirement of an optimal state, 3 pairs of primers can meet the detection requirement under the reaction condition, 4 groups are set for each concentration condition, each group is repeatedly measured for 5 times, the measurement result shows that the variation coefficients are less than 5 percent, the plasmid repeatability of the method is good, the stability is high, the detection results are shown in tables 8-10 and in figures 7-9, figures 7-9 are amplification curves of repeated experiments under different plasmid concentrations of three genes, the concentration of the amplification curves from left to right is 1 × 10 in sequence, and the concentration of the amplification curves from right to left is 1 × 10 in sequence6fg/. mu. L and 1 × 105fg/μL。
TABLE 8
Figure BDA0001961516750000141
TABLE 9
Figure BDA0001961516750000142
Watch 10
Figure BDA0001961516750000143
Figure BDA0001961516750000151
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.
qPCR detection of 3.3 target genes
qPCR amplification of the samples was performed as indicated by TaKaRa Taq PCR Mix instructions and Ct value readings were obtained for each pair of primers. 3 biological replicates and 3 technical replicates were set for each qPCR reaction, and the qPCR reaction system and amplification reaction conditions were the same as in example 2.
4. Analysis of results
As shown in Table 11 and FIG. 10, the results of the 3 primer pairs of qPCR amplification show that the shrimp enterobacter hepatica shows positive results of three of the 18SrRNA, β -tubulin gene and SWP1 gene, while other viruses or genera show negative results, therefore, the detection of the 18SrRNA, β -tubulin gene and SWP1 gene can better distinguish the shrimp enterobacter hepatica from other viruses or genera of shrimp enterobacter.
TABLE 11
Figure BDA0001961516750000161
EXAMPLE 4 sensitivity of Positive samples
1. Sample collection and processing
Respectively collecting healthy litopenaeus vannamei samples and positive samples of the shrimp enterobacter hepatica according to the proportion of 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 QIAampDNA Mini Kit instruction from Qiagen.
2. Genomic DNA extraction
The same as in example 1.
qPCR detection of 3.3 target genes
The same as in example 1.
4. Analysis of results
As shown in Table 12 and FIGS. 11 to 13, the results of qPCR amplification with 3 primers showed a value of 10110 times of2Doubling 10310 times of4Multiple and 105In the range of the double-dilution sample, the shrimp enterobacter hepatica shows positive results of three genes including 18SrRNA, β -tubulin gene and SWP1 gene, so the detection of the 18SrRNA, β -tubulin gene and SWP1 gene can better reach 101Multiple to 105Detecting the enterosporidium hepatolyticum of the prawn in the double dilution range. Simultaneously, diluting 10 from the sample1Multiple to 105Within the range of double, the detection of the prawn enterobacter hepatica can be better completed through the matching detection of 18SrRNA, β -tubulin gene and SWP1 gene, and the detection range can be extended to 10 sample dilution1Multiple to 105And the higher strain concentration in the sample can be diluted to be within the optimal detection range by a dilution method, so that the expected detection requirement can be met.
In fig. 11 to 13, fig. 11 to 13 are amplification curves of three genes at different positive sample concentrations, and the dilution times of the amplification curves from left to right are as follows: 10110 times of210 times of310 times of4Multiple and 105And (4) doubling.
TABLE 12
Figure BDA0001961516750000171
Figure BDA0001961516750000181
EXAMPLE 5 detection of environmental samples
1. Environmental sample collection and extraction of genomic DNA
Different positive samples of Zhanjiang Dongshan, Zhenchang Honda Zhenyang and culture seawater were collected, and genomic DNA was extracted according to the instruction of QIAamp DNAmini Kit of Qiagen.
qPCR detection of 2.3 target genes
The same as in example 2.
3. Analysis of results
As shown in Table 13, the results of the 3 primer pairs of qPCR amplification showed that, in addition to healthy Litopenaeus vannamei, positive sample 2 of Zhanjiang Dongshan island, positive sample 2 of Wenchang Honda and seawater culture samples 1-3, 18SrRNA gene, β -tubulin gene and SWP1 gene were detected in all the samples, and it was confirmed that the corresponding sample sources contained Penaeus vannamei Enteromorpha, and the criterion for finally judging that the Penaeus vannamei Enteromorpha is positive was that Ct value obtained by detection of 18SrRNA gene, β -tubulin gene and SWP1 gene was less than 35 cycles.
Watch 13
Figure BDA0001961516750000182
Figure BDA0001961516750000191
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 PCR method for detecting prawn enterobacter hepatica and corresponding kit
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Claims (8)

1. The fluorescent quantitative PCR method for detecting the shrimp enterobacter hepatica is characterized by comprising the following steps:
s1, collecting a sample;
s2, extracting genome DNA;
s3, detecting the prawn enterobacter hepatica specific gene 18SrRNA gene, β -tubulin gene and SWP1 gene by using fluorescent quantitative PCR;
and S4, reading the Ct value of amplification, wherein when the Ct value of at least one of 3 genes in the shrimp enterobacter hepatica specific gene is 35, the detection result of the shrimp enterobacter hepatica is positive, and when the Ct values of the 18SrRNA gene, the β -tubulin gene and the SWP1 gene of the shrimp enterobacter hepatica are all larger than 35, the detection result of the shrimp enterobacter hepatica is negative.
2. The fluorescent quantitative PCR method for detecting the shrimp enterobacter hepatica according to claim 1, wherein the step S2 further comprises the step of designing detection primers and TaqMan probes for 18SrRNA, β -tubulin and SWP1 genes.
3. The fluorescent quantitative PCR method for detecting the shrimp enterobacter hepatica according to claim 2, characterized in that a detection primer of the shrimp enterobacter hepatica specific gene 18SrRNA is shown as SEQ ID NO 1-2, and a TaqMan probe sequence is shown as SEQ ID NO 3.
4. The fluorescent quantitative PCR method for detecting the shrimp enterobacter hepatica according to claim 2, wherein a detection primer of the shrimp enterobacter hepatica specific gene β -tubulin is shown as SEQ ID NO. 4-5, and a TaqMan probe sequence is shown as SEQ ID NO. 6.
5. The fluorescent quantitative PCR method for detecting the shrimp enterobacter hepatica according to claim 2, wherein a detection primer of a specific gene SWP1 of the shrimp enterobacter hepatica is shown as SEQ ID NO. 7-8, and a TaqMan probe sequence is shown as SEQ ID NO. 9.
6. The fluorescent quantitative PCR method for detecting the shrimp enterobacter hepatica according to claim 1, wherein the reaction system of the fluorescent quantitative PCR detection is 25 μ l, and comprises 2 × Taq PCR Mix 12.5 μ l, total 10uMPrimers Mix1 μ l, 0.5 μ l TaqMan probe, 2 μ l DNA input, H2O 9μl。
7. The fluorescence quantitative PCR method for detecting the shrimp enterobacter hepatica according to claim 1, wherein the reaction conditions of the fluorescence quantitative PCR detection are pre-denaturation at 95 ℃ for 2min30s, denaturation at 94 ℃ for 15s, annealing at 60 ℃ for 30s, fluorescent signal collection and 40 cycles.
8. A fluorescent quantitative PCR kit for detecting prawn enterobacter hepatica is characterized by comprising detection primers and TaqMan probes of specific genes 18SrRNA, β -tubulin and SWP1 of the prawn enterobacter hepatica.
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CN113512607A (en) * 2021-08-12 2021-10-19 杭州市农业技术推广中心 Primer and method for fluorescence quantitative PCR detection of prawn enterobacter hepatica
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CN114250316A (en) * 2021-12-22 2022-03-29 绍兴市水产技术推广中心 Method for detecting shrimp liver enterocytozoon from aquiculture pond water-soil sample

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