CN111518928A - TaqMan probe quantitative detection method for detecting vibrio harveyi and corresponding kit - Google Patents
TaqMan probe quantitative detection method for detecting vibrio harveyi and corresponding kit Download PDFInfo
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Abstract
The invention discloses a TaqMan probe quantitative detection method for detecting Vibrio harveyi and a corresponding kit. The invention skillfully applies specific gene detection to distinguish the vibrio harveyi and other species of strains, and obtains accurate genus information through comprehensive judgment. Compared with the existing mainstream detection kit, the kit for detecting the vibrio harveyi 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 the technical field of molecular biology, in particular to a method for carrying out TaqMan probe quantitative detection on Vibrio harveyi through specific genes and a corresponding detection kit.
Background
Vibrio harveyi (Vibrio harveyi) belongs to the family Vibrionaceae (Vibrionaceae) and Vibrio (Vibrio). Widely distributed in marine environments, it is an important pathogenic bacteria of aquaculture animals that has been recognized for over the last 10 years. Vibrio harveyi is an important pathogenic bacterium for culturing prawns in Indonesia, Thailand, India, Australia, Ecuador, China and other countries and regions, and is also a pathogenic bacterium for culturing fishes.
Vibrio harveyi is gram-negative, luminous marine bacteria, is short rod-shaped, has straight or slightly bent thalli, two blunt ends, is not capsulated, does not form spores, can move in an extreme single flagellum, exists singly, rarely has two or chain-shaped arrangements, and has the size of 0.5-0.9 Mum multiplied by 1.1-1.9 Mum. The strain does not grow in a salt-free culture medium, well grows on a salt-containing nutrient agar plate, is circular and smooth, has neat edges, slightly bulges and flashes after being cultured for 24 hours at the temperature of 28 ℃, is yellow on a TCBS plate, and cannot grow below 4 ℃ and above 40 ℃.
Extracellular products (ECP) of vibrio harveyi have pathogenicity, and extracellular proteases, lipopolysaccharides, phospholipases or hemolysins secreted therefrom play an important role in the pathogenicity of shrimps and fishes and damage host tissue organs and the like. The shrimp with strong susceptibility includes Penaeus chinensis, Penaeus vannamei Boone larva, Penaeus elongatus and Penaeus japonicus adult shrimp, and the fish includes Perch, grouper, mullet, rainbow trout, Pseudosciaena crocea, and turbot.
After Vibrio harveyi infection, no effective method is available for treatment at present, and the possibility of preventing and controlling outbreak of the disease can be achieved only by strengthening epidemic disease monitoring and quarantine. Through a large amount of research, scholars at home and abroad establish a series of detection methods for vibrio harveyi, such as a histopathological anatomy method, an electron microscope observation method, a Leeb's liquid thioglycolate culture method, a PCR detection method and a LAMP method. However, the first three methods are complex in operation, long in time consumption and low in detection sensitivity, and only can diagnose the prawns with obvious morbidity; although the PCR detection method has accurate result, the detection time is long, the operation is complex, quantification cannot be realized, and the method is difficult to popularize and apply in production; although the LAMP method has short detection time, the false positive is high, and the requirement of accurate detection cannot be met.
The invention provides a method for quantitatively, quickly and real-timely monitoring vibrio harveyi, which aims to overcome the defect that vibrio harveyi is monitored at present
Disclosure of Invention
One of the purposes of the invention is to provide a TaqMan probe quantitative detection method for detecting Vibrio harveyi, which can quantitatively, rapidly and real-timely detect Vibrio harveyi and make the detection of Vibrio harveyi more accurate, sensitive, rapid and safe.
Specifically, the method comprises the following steps:
s1, collecting a sample;
s2, extracting genome DNA;
s3, quantitatively detecting the specific gene ToxR gene of the vibrio harveyi by using a TaqMan probe;
s4, reading the Ct value of the amplification; when the Ct value of the specific gene ToxR of the vibrio harveyi is less than 35, the detection result of the vibrio harveyi is positive; and when the Ct value of the specific gene ToxR of the Vibrio harveyi is more than 35, the detection result of the Vibrio harveyi 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 ToxR gene.
As a preferred technical scheme, a detection primer of the vibrio harveyi specific gene ToxR gene is shown as SEQID NO: 1-2;
SEQ ID NO:1(5'-AGAGCCCACTGCTGAGACAAA-3')；
SEQ ID NO:2(5'-CAGGTGCAGATTTTAATGGTTGAG-3')。
the probe sequence is shown as SEQ ID NO. 3;
SEQ ID NO:3(5'-FAM-AGAAACAGCCGTCGAACAAGCACCG-BHQ1-3)。
as a preferred technical scheme, the reaction system of the fluorescent quantitative PCR detection is 25 ul, and comprises 12.5 ul of 2 × TaqPCR Mix, 1 ul of total 10uM Primers Mix, 0.5 ul of TaqMan probe,DNA input 2μl，H2O 9μl。
as a preferred technical scheme, the reaction conditions of the fluorescent quantitative PCR detection are denaturation at 95 ℃ for 2min30s, annealing at 94 ℃ for 15s, annealing at 60 ℃ for 30s, collecting fluorescent signals and performing 40 cycles.
The invention also aims to provide a TaqMan probe quantitative detection kit for detecting the Vibrio harveyi, which comprises a detection primer of the specific gene ToxR gene of the Vibrio harveyi and a TaqMan primer probe.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, the orphan gene capable of distinguishing the vibrio harveyi and the specificity of other species of strains is excavated from a large amount of early-stage research data on the vibrio harveyi and other species of strains, whether other species of positive samples and environmental samples contain the vibrio harveyi is accurately judged by detecting the specificity gene, the early-stage big data mining and the comparison between different species are provided, and the selected specificity gene has 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 the toxR 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 trace sample can be correctly detected.
(3) Compared with other detection methods in the market, the detection strategy of the invention can intuitively judge the infection of the vibrio harveyi and the infection of other strains, and the result is more rigorous and accurate.
(4) The detection method can be applied to screening and detecting the culture water body sample or litopenaeus vannamei and other multi-aspect source samples, accurately judges whether the culture water body sample contains the vibrio harveyi, and is convenient, rapid and sensitive to operate. The detection kit can be applied to the rapid detection of various culture samples without additional 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. 1 illustrates a standard curve of primers for detecting ToxR gene.
FIG. 2 is a graph showing the amplification curve of the ToxR gene in example 1.
FIG. 3 is a curve of amplification of the ToxR gene in example 2.
FIG. 4 is an amplification curve of the specific experimental toxR gene in example 3.
FIG. 5 is an amplification curve of the toxR gene of sample 1 at the breeding ground in example 4.
Detailed Description
In order to clearly understand the technical contents of the present invention, the following embodiments are described in detail with reference to the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
The following reagents used in the present invention can be purchased from conventional sources.
TABLE 1
| Name (R) | Brand | Model number | Specification of |
| Healthy litopenaeus vannamei | Zhanjiang certain farm | 100g | |
| Vibrio harveyi | ATCC | Freezing | |
| Cryptocaryon irritans | Certain aquatic product promotion station | Freezing | |
| Vibrio vulnificus | ATCC | Freezing | |
| Vibrio parahaemolyticus | ATCC | Freezing | |
| Streptococcus agalactiae | ATCC | 244793 | Freezing |
| Edwardsiella tarda | ATCC | 8117 | Freezing |
| QIAamp DNA Mini Kit | Qiagen | 51304 | 50 reactions |
| QIA group GE extraction of mi test d M dose id box i Kit from Pl group N | QIAGEN | 12145 | 100 reactions |
| Quantitative test P agent C box R premixed reaction liquid for Taq PCR Mix fluorescent particle light extraction | TaKaRa | RR391 | 200 reactions |
| Peptone yeast extract agar | Sigma | 7A 7196- | 500 g |
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 for different strains, samples infected by Vibrio harveyi and samples of other species can be correctly distinguished, thereby accurately detecting and judging Vibrio harveyi.
In some embodiments, pure water, Vibrio harveyi samples, culture base samples and culture water samples are collected, and whether the samples contain Vibrio harveyi can be quickly and accurately detected through the detection of specific genes, so that the method is convenient and quick.
It will be appreciated 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 by designing other sections of the same detection object.
The method and the kit for detecting the Vibrio harveyi TaqMan probe quantitative detection can be widely applied to a plurality of inspection and quarantine categories such as the detection of a Litopenaeus vannamei sample and the detection of a culture base, and can provide a convenient, quick, accurate and efficient gene detection-based monitoring product for the culture, propagation and the like of the Litopenaeus vannamei.
Example 1 Standard Curve for primer amplification detection and plasmid sensitivity test
1. Cultivation of microorganisms
LB medium was used as the medium for Vibrio harveyi plasmid, and the growth was good in the plate with pH7.0-7.4. The colony diameter on the plate is 2mm, round, smooth and transparent. And selecting a microorganism sample with vigorous growth for subsequent experiments.
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 Vibrio harveyi is used, amplification is carried out with primers for the ToxR gene, the amplification product is treated with A, T4 ligase is used for ligation into T vector, and plasmid is amplified on a large scale after transduction of competent cells. The primer sequence and TaqMan probe sequence of the ToxR 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
Amplification of coated plates separatelyCloning bacteria as standard substance of primer amplification product corresponding to ToxR gene, collecting monoclonal bacterial plaque, culturing and shaking bacteria via LB to amplify overnight, finally obtaining plasmid positive bacterial strain in exponential growth phase of 5ml, extracting plasmid DNA according to QIAGENPismid Midi Kit instruction, detecting plasmid DNA concentration after extraction, and configuring 7 standard substances with concentration gradient of 1 fg/mu L and 1 × 10 by 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 performing q-PCR amplification of a concentration gradient standard curve according to the instruction of TaKaRa Taq PCR Mix instruction, and drawing a corresponding amplification curve to obtain the amplification efficiency of the primer. 3 biological replicates and 3 technical replicates were set for each q-PCR reaction, the q-PCR reaction system and amplification reaction conditions are shown in Table 3, and the standard curve is plotted as 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 toxR gene are optimized primer design and reaction systems. Linear standard curve confidence R2>0.990, the primer design and the reaction system were proved to be the optimal state, the standard deviation and the coefficient of variation of the amplification curve of the toxR gene and the amplification efficiency under 7 concentration gradients were both satisfied with the requirements of the optimal state, the primers could satisfy the detection requirements under the reaction conditions, which indicated that the method had high plasmid sensitivity, and the coefficient of variation between CT values measured by 3 technical repeat experiments was less than 5.0%, which proved that the method had high reproducibility, the detection results were shown in Table 4 and FIG. 2, FIG. 2 is the amplification curve of the toxR gene at different plasmid concentrations, the concentration of the amplification curve from left to right was 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. q-PCR detection of target genes
The q-PCR amplification of the samples was performed according to the instructions of TaKaRa Taq PCR Mix, and Ct values corresponding to the primers were read.2 concentrations were set for the q-PCR reaction of this experiment, 1 × 106fg/. mu.L and 1 × 105fg/. mu.L, 4 biological replicates and 5 technical replicates per concentration, respectively, and the q-PCR reaction system and amplification reaction conditions are shown in Table 5.
TABLE 5
4. Analysis of results
In this example, it is analyzed that the correlation coefficient and reliability of the amplification curve of the ToxR gene and the amplification efficiency under 2 concentrations all meet the requirements of the optimal state, and the primer can satisfy 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 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. q-PCR detection of target genes
q-PCR amplification of the samples was performed as indicated in TaKaRa Taq PCR Mix instructions and Ct value readings were obtained for the primers. The q-PCR reaction set 3 biological and 3 technical repeats, and the q-PCR reaction system and amplification reaction conditions were the same as in example 2.
4. Analysis of results
As shown in Table 7 and FIG. 4, the results of q-PCR amplification of the primers revealed that Vibrio harveyi exhibited a positive result for the ToxR gene, while all other genera exhibited negative results. Therefore, the detection of the ToxR gene can be carried out on the Vibrio harveyi and other genera, so that the Vibrio harveyi and other genera can be better distinguished. In summary, the corresponding primers and probes of the toxR gene can meet the requirements of detection and judgment of Vibrio harveyi.
TABLE 7
Example 4 detection of environmental samples
1. Environmental sample collection and extraction of genomic DNA
And respectively collecting a pure water sample, a Vibrio harveyi sample, a culture base sample and multiple culture water samples, and extracting the genomic DNA according to the instruction of the QIAamp DNA Mini Kit of Qiagen.
2. q-PCR detection of target genes
The same as in example 2.
3. Analysis of results
As shown in Table 8 and FIG. 5, the results of q-PCR amplification of the primers showed that the ToxR gene was detected in the Vibrio harveyi sample and one of the culture plots, which confirmed that the corresponding sample source contained Vibrio harveyi. The final criterion for judging that the Vibrio harveyi is positive is that the Ct value obtained by the toxR gene detection is less than 35 cycles. If the Ct value obtained by the ToxR gene detection is more than 35 cycles, the Vibrio harveyi negative is judged to be undetected.
TABLE 8
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> TaqMan probe quantitative detection method for detecting Vibrio harveyi and corresponding kit
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Claims (6)
1. The TaqMan probe quantitative detection method for detecting the vibrio harveyi is characterized by comprising the following steps of:
s1, collecting a sample;
s2, extracting genome DNA;
s3, quantitatively detecting and detecting the specific gene ToxR gene of the vibrio harveyi by using a TaqMan probe;
s4, reading the Ct value of the amplification; when the Ct value of the specific gene ToxR of the vibrio harveyi is less than 35, the detection result of the vibrio harveyi is positive; and when the Ct value of the specific gene ToxR of the Vibrio harveyi is more than 35, the detection result of the Vibrio harveyi is negative.
2. The TaqMan probe quantitative detection method for detecting Vibrio harveyi according to claim 1, wherein the step S2 further comprises a step of designing a detection primer of the ToxR gene and a TaqMan probe.
3. The TaqMan probe quantitative detection method for detecting Vibrio harveyi according to claim 1, wherein a detection primer of the specific gene ToxR gene of Vibrio harveyi is shown as SEQ ID NO 1-2, and a sequence of the TaqMan probe is shown as SEQ ID NO 3.
4. The method of claim 1, wherein the reaction system of the quantitative PCR assay is 25 μ l, and comprises 2 × Taq PCR Mix 12.5 μ l, total 10uMPrimers Mix 1 μ l, TaqMan probe 0.5 μ l, DNA input 2 μ l, H2O 9μl。
5. The TaqMan probe quantitative detection method for Vibrio harveyi according to claim 1, wherein the reaction conditions of the fluorescent quantitative PCR detection are denaturation at 95 ℃ for 2min30s, annealing at 94 ℃ for 15s, annealing at 60 ℃ for 30s, collecting a fluorescent signal, and 40 cycles.
6. A TaqMan probe quantitative detection kit for detecting Vibrio harveyi is characterized by comprising a detection primer of the specific gene ToxR gene of Vibrio harveyi and a TaqMan probe.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296617A (en) * | 2015-10-10 | 2016-02-03 | 宁波大学 | Gene chip for detecting vibrio harveyi colony and using method of gene chip |
| CN105602945A (en) * | 2015-11-04 | 2016-05-25 | 中国水产科学研究院南海水产研究所 | Detection primer group, detection kit, and detection method used for simultaneous detection of three pathogenic bacteria of marine fishes |
| CN108546743A (en) * | 2018-05-09 | 2018-09-18 | 宁波海洋研究院 | A kind of Vibrio harveyi rapid detection method |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296617A (en) * | 2015-10-10 | 2016-02-03 | 宁波大学 | Gene chip for detecting vibrio harveyi colony and using method of gene chip |
| CN105602945A (en) * | 2015-11-04 | 2016-05-25 | 中国水产科学研究院南海水产研究所 | Detection primer group, detection kit, and detection method used for simultaneous detection of three pathogenic bacteria of marine fishes |
| CN108546743A (en) * | 2018-05-09 | 2018-09-18 | 宁波海洋研究院 | A kind of Vibrio harveyi rapid detection method |
Non-Patent Citations (6)
| Title |
|---|
| D. SCHIKORSKI等: "Development of TaqMan real-time PCR assays for monitoring Vibrio harveyi infection and a plasmid harbored by virulent strains in European abalone Haliotis tuberculata aquaculture", 《AQUACULTUR》 * |
| JIANHUPANG等: "A real-time recombinase polymerase amplification assay for the rapid detection of Vibrio harveyi", 《MOLECULAR AND CELLULAR PROBES》 * |
| YOUHEI FUKUI等: "Rapid Detection of Vibrio harveyi in Seawater by Real-Time PCR", 《MICROBES ENVIRON》 * |
| 姚东瑞等: "基于toxR基因病原哈氏弧菌PCR检测方法的建立", 《水产科学》 * |
| 尚琨等: "患腹水病牙鲆病原菌分离、鉴定及病原菌的特性", 《水产学报》 * |
| 林佳琪等: "四重PCR检测副溶血性弧菌及其毒力基因方法的建立", 《微生物学通报》 * |
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Application publication date: 20200811 |