CN114250317B - Primer group and kit for detecting Erjiangmeiqi yeast - Google Patents
Primer group and kit for detecting Erjiangmeiqi yeast Download PDFInfo
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Abstract
The invention belongs to the technical field of pathogenic bacteria detection, and provides a primer group and a kit for detecting Ericaria acuminata. The primer group comprises an upstream detection primer and a downstream detection primer; the nucleotide sequence of the upstream detection primer is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream detection primer is shown as SEQ ID NO. 2. The kit containing the detection primer has the advantages of high sensitivity, strong specificity and high detection accuracy, can effectively detect the bicinchondric yeast, has simple and convenient method, does not have cross reaction with adjacent species of the bicinchondric yeast, also has no cross reaction with other common aquatic animal pathogenic bacteria, and has important significance for disease prevention and control of aquatic animals.
Description
Technical Field
The invention relates to the technical field of pathogenic bacteria detection, in particular to a primer group and a kit for detecting Ericaria acuminata.
Background
The Erjiangqi yeast is a pathogenic yeast, and can cause diseases of various aquatic animals such as Eriocheir sinensis, Macrobrachium rosenbergii, and Oncorhynchus marmoreus. At present, the disease of the eriocheir sinensis is particularly serious. The main symptoms of the sick crabs are slow reaction and a large amount of white milky liquid can be seen in the heart surrounding cavity, and the crabs are called 'milk disease' by farmers. The disease causes a great deal of death of the eriocheir sinensis, the morbidity can reach 90 percent in serious cases, the mortality rate exceeds 50 percent, and great economic loss is caused. At present, no effective treatment measures are available for the disease.
Early detection is carried out, and the cultivation of eriocheir sinensis without carrying Ericaria bicolor is an effective method for preventing the disease. However, the current detection method is still imperfect, and mainly depends on a microbial culture method for detection, which takes a long time, and usually needs more than 3 days. In the prior art, Chinese patent 202011134597.0 discloses a PCR detection method of Ericaceae yeast, which is used for detecting the Ericaceae yeast with the minimum limit of 8.62 multiplied by 10 per mu L in terms of sensitivity; in the aspect of specificity, detection shows that the bacillus subtilis preparation has no specific reaction with lactobacillus reuteri, lactobacillus bulgaricus, streptococcus thermophilus, lactobacillus plantarum, lactobacillus casei, bacillus natto, staphylococcus aureus, vibrio cholerae, pseudomonas putida, saccharomyces cerevisiae, candida parapsilosis, pichia guilliermondii, pichia stipitis, lodoria elongata, cladosporium sporum and fusarium equiseti.
The quality of the PCR detection method is evaluated, and the sensitivity and the specificity are main indexes. The higher the sensitivity and the stronger the specificity, the better the effect is. In the PCR detection method established in the prior art, the lowest detection limit (sensitivity) has a space for improvement in the sensitivity. In terms of specificity, although no cross reaction with more neighboring species of Ericaria biciper was found, according to reports, identification of other pathogens, such as Vibrio alginolyticus, Vibrio harveyi (Portunus trituberculatus) "milk disease" pathogen and histopathological studies thereof [ D ]. university of agriculture and forestry, northwest 2006 ], can also cause crab "milk disease", and methods established in the prior art do not determine whether there is a cross reaction with these pathogens. In addition, river crabs mainly live in water and are often attacked by pathogens of aquaculture animals, and the methods established in the prior art do not determine whether cross reaction exists with common pathogens of aquaculture animals (such as flavobacterium columnare, aeromonas hydrophila, aeromonas sobria, aeromonas veronii, streptococcus agalactiae, streptococcus dysgalactiae and pseudomonas fluorescens). Thus, the method can be further improved and improved.
Disclosure of Invention
The invention aims to provide a primer group and a kit for detecting bicinchondric yeast, wherein the kit has the advantages of high sensitivity, strong specificity and high detection accuracy, can effectively detect the bicinchondric yeast, is simple and convenient in method, has no cross reaction with adjacent species of the bicinchondric yeast, also has no cross reaction with other common aquatic animal pathogenic bacteria, and has important significance for disease prevention and control of aquatic animals.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a primer group for detecting Ericaria bicolor, which comprises an upstream detection primer and a downstream detection primer; the nucleotide sequence of the upstream detection primer is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream detection primer is shown as SEQ ID NO. 2.
Upstream detection primer (SEQ ID NO: 1): 5'-ATGAACCCTCGTCCCAACT-3', respectively;
downstream detection primer (SEQ ID NO: 2): 5'-GATAGCCTTGCCATTACTTCC-3' are provided.
The invention also provides a kit for detecting the Ericaria acuminata, which comprises upstream and downstream detection primers and a detection reagent.
Further, the detection reagent includes 2 × taq mastermix and sterile water.
Further, the upstream detection primer and the downstream detection primer in the primer group are independently used at a concentration of 8-12 mM.
Further, the amplification system for detection by using the kit comprises the following components in 50 μ L: 2 XTaq Master Mix 25. mu.L, upstream detection primer 0.5. mu.L, downstream detection primer 0.5. mu.L, template 1. mu.L, sterile water 23. mu.L.
Further, the amplification procedure in the detection using the kit is as follows: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 1min, annealing at 62 ℃ for 1min, extension at 72 ℃ for 30s, and 35 cycles of denaturation, annealing and extension; extension at 72 ℃ for 10 min.
The invention has the beneficial effects that:
1. the kit of the invention improves the sensitivity of the existing detection method, and the detection limit of the sensitivity of the bicinchondric yeast is 2.41 multiplied by 10 per mu L.
2. The kit has strong specificity and high detection accuracy, not only ensures that the kit has no cross reaction with adjacent species of the bicinchondric yeast, but also ensures that the kit has no cross reaction with other pathogens (vibrio alginolyticus and vibrio harveyi) of crab type 'milk disease' and common aquatic animal pathogenic bacteria (flavobacterium columnare, aeromonas hydrophila, aeromonas sobria, aeromonas veronii, streptococcus agalactiae, streptococcus dysgalactiae, vibrio parahaemolyticus, vibrio vulnificus, photobacterium mermairei, nocardia seriolae, pseudomonas fluorescens and the like), has simple and convenient method, and has important significance for preventing and controlling diseases of aquatic animals.
Drawings
FIG. 1 is a PCR electrophoretogram using genomic DNA of Ericaceae Saccharomyces cerevisiae as a template; in the figure, lane M: DNAMarkerDL 2000; lane 1: genomic DNA of Erjiangmei yeast; lane 2: negative control (sterile water);
FIG. 2 is a PCR electrophoretogram showing the sensitivity of primers for detection of Ericaria bicolor; in the figure, lanes 1 to 10 correspond to copy numbers of 2.41X 10 9 、2.41×10 8 ，2.41×10 7 ，2.41×10 6 ，2.41×10 5 ，2.41×10 4 ，2.41×10 3 ，2.41×10 2 ，2.41×10 1 ，2.41×10 0 (ii) a Lane "+" is a positive control (bicinchondric yeast genomic DNA); lane "-" is a negative control (sterile water); lane M is DNA markerDL 2000;
FIG. 3 is a PCR electrophoretogram of specificity of detection primers for Saccharomycotina bicolor; in the figure, lane 1 is a positive control; lane 2 is a sterile water negative control; lane M is DNAmarker, Standard molecular weight, DL 2000; lanes 3-24 are Metronickel, Saccharomycotina, Saccharomyces cerevisiae, Candida humus, Candida tropicalis, Pichia kudriavzevii, Rhodotorula palustris, Rhodotorula glutinis, Cryptococcus aurea, Vibrio alginolyticus, Vibrio harveyi, Flavobacterium columnare, Aeromonas hydrophila, Aeromonas sobria, Aeromonas veronii, Streptococcus agalactiae, Streptococcus dysgalactiae, Vibrio parahaemolyticus, Vibrio vulnificus, Photobacterium merus, Nocardia seriolae, Pseudomonas fluorescens, respectively;
FIG. 4 is a PCR electrophoretogram of 24 river crab samples injected with sterile water; in the figure, lane M is DNA markerDL 2000; lanes 1-24 are river crab samples;
FIG. 5 is a PCR electrophoretogram of 24 river crab samples injected with Ericaria bicolor; in the figure, lane M is DNAmarkerDL 2000; lanes 1-24 are river crab samples;
FIG. 6 is a diagram showing the death status of the healthy river crabs after artificial infection of bicinchondric yeast by three ways of intramuscular injection, dipping bath and mixing and feeding.
Detailed Description
The invention provides a primer group for detecting Ericaria bicolor, which comprises an upstream detection primer and a downstream detection primer; the nucleotide sequence of the upstream detection primer is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream detection primer is shown as SEQ ID NO. 2; the method comprises the following specific steps:
upstream detection primer (SEQ ID NO: 1): 5'-ATGAACCCTCGTCCCAACT-3', respectively;
downstream detection primer (SEQ ID NO: 2): 5'-GATAGCCTTGCCATTACTTCC-3' is added.
The invention also provides a kit for detecting the Ericaria acuminata, which comprises upstream and downstream detection primers and a detection reagent.
In the present invention, the detection reagent includes 2 × Taq MasterMix and sterile water.
In the present invention, 2 × TaqMasterMix was purchased from Bao bioengineering (Dalian) Co.
In the present invention, the upstream detection primer and the downstream detection primer in the primer set are preferably used at a concentration of 8 to 12mM independently, and more preferably at a concentration of 9 to 11mM independently.
In the invention, the amplification system used in the detection of the kit comprises the following components in 50 μ L: 2 XTaq MasterMix 25. mu.L, upstream detection primer 0.5. mu.L, downstream detection primer 0.5. mu.L, template 1. mu.L, sterile water 23. mu.L.
In the present invention, the amplification procedure in the detection using the kit is as follows: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 1min, annealing at 62 ℃ for 1min, extension at 72 ℃ for 30s, and 35 cycles of denaturation, annealing and extension; extension at 72 ℃ for 10 min.
After the amplification is finished, judging whether the sample contains bicinchonium metschnikowii yeast or not according to the electrophoresis result of the amplification product, wherein if the amplification product has a 339bp band, the bicinchonium metschnikowii yeast exists in the sample; if the amplified product does not show a 339bp band, the Ericaria does not exist in the sample.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The embodiment provides a method for verifying the effectiveness of a primer for detecting bicinchondric yeast, which adopts a yeast DNA extraction kit (purchased from Takara Bio-engineering (Dalian) Co., Ltd.) to extract DNA of a bicinchondric yeast strain JMB-1 (from aquatic research institute in Tianjin) as a template to verify the effectiveness of the primer, and uses sterile water as negative control.
The PCR amplification is carried out by using the detection primer of the invention, wherein the sequence of the upstream detection primer is (5'-ATGAACCCTCGTCCCAACT-3'), the sequence of the downstream detection primer is (5'-GATAGCCTTGCCATTACTTCC-3'), and the detection primer is designed according to the hypothetical protein gene of Ericacillus lanceolatus. PCR (50. mu.L reaction) amplification conditions were as follows: add 2 XTaq Master Mix 25. mu.L, 10mM upstream detection primer 0.5. mu.L, 10mM downstream detection primer 0.5. mu.L, template 1. mu.L, sterile water 23. mu.L. The reaction procedure is as follows: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 1min, annealing at 62 ℃ for 1min, extension at 72 ℃ for 30s, and 35 cycles of denaturation, annealing and extension; extension at 72 ℃ for 10 min.
As shown in FIG. 1, the amplification result showed that about 339bp of the desired band was amplified using the DNA of Saccharomyces dicentri as a template, and that no band was observed in the negative control.
Example 2
This example provides a method for testing the sensitivity of a primer for detecting Ericaria Bijiangensis by using a yeast DNA extraction kit (purchased from Saccharomyces cerevisiae)From Takara Bio Inc.) the PCR product of example 1 was recovered by cutting a gel, ligated with a pMD18-T vector (purchased from Takara Bio Inc.), transferred into E.coli TOP10 competent cells (purchased from Takara Bio Inc.) and the positive clone was selected and named Top10 (18T-0815). The strain Top10(18T-0815) was inoculated into LB medium containing ampicillin, cultured at 150rpm at 37 ℃ for 36 hours, and then the plasmid was extracted using a plasmid extraction kit (purchased from Takara Bio Inc.), and the plasmid concentration was measured to be 80 ng/. mu.L according to the general method. Calculating the copy number according to the formula, wherein the copy number (copies/. mu.L) [ < 80X 10> -9 ×(6.02×10 23 )]Div (660 × 3031), calculated to be 2.41 × 10 copy number 10 copies/μL。
The plasmid was serially diluted 10-fold with sterile water, and 1. mu.L of each concentration of plasmid was used as a template for PCR amplification, and the genomic DNA of Ericaria distachya was used as a positive control and sterile water was used as a negative control. PCR (50. mu.L reaction) amplification conditions were as follows: mu.L of 2 XTaq MasterMix25, 0.5. mu.L of 10mM upstream detection primer, 0.5. mu.L of 10mM downstream detection primer, 1. mu.L of template, and 23. mu.L of sterile water were added. The reaction procedure is as follows: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 1min, annealing at 62 ℃ for 1min, extension at 72 ℃ for 30s, and 35 cycles of denaturation, annealing and extension; extension at 72 ℃ for 10 min. The results are shown in FIG. 2, where the lowest detection limit was 2.41X 10 1 mu.L/L.
Example 3
This example provides a specific test method for detecting primers of Metschnikowianulerrima, Lactobacillus cerevisiae, Candida humilis, Candida tropicalis, Pichia pastoris, Rhodotorula palustricorne, Rhodotorula palusteruvia, Rhodotorula glutinis, Gluconobacter glutinosa, Cryptococcus aurea, Vibrio alginolyticus, Vibrio harveyi, Vibrio Flavobacterium columniferus, Aeromonas hydrophila, Pseudomonas hydrophylla, Pseudomonas aeruginosa, Streptococcus faecalis, Streptococcus lactis, Streptococcus faecalis, Streptococcus strain, Streptococcus faecalis, Streptococcus strain, Streptococcus faecalis strain, Streptococcus lactiae, Streptococcus strain, Streptococcus lactibacillus, Streptococcus strain, Lactobacillus strain, Streptococcus strain, Lactobacillus strain, Streptococcus strain, Lactobacillus strain, and strain, Streptococcus strain, Lactobacillus strain, Streptococcus strain, Lactobacillus strain, Streptococcus strain, Lactobacillus strain, Streptococcus strain, Lactobacillus strain, Streptococcus strain, Lactobacillus strain, Lactobacillus strain, etc. strain, etc, the specificity of the detection primer of the Ericaria distachys is verified. PCR (50. mu.L reaction) amplification conditions were as follows: add 2 XTaqMasterMix 25. mu.L, 10mM upstream detection primer 0.5. mu.L, 10mM downstream detection primer 0.5. mu.L, template 1. mu.L, sterile water 23. mu.L. The reaction procedure is as follows: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 1min, annealing at 62 ℃ for 1min, extension at 72 ℃ for 30s, and 35 cycles of denaturation, annealing and extension; extension at 72 ℃ for 10 min. As shown in FIG. 3, it can be seen that the detection primers are highly specific and do not cross-react with the pathogenic fungi (Metronickel, Lamivy, Saccharomyces cerevisiae, Candida humus, Candida tropicalis, Pichia kudriavzevii, Rhodotorula palustris, Rhodotorula glutinis, Cryptococcus aureofaciens) and common aquatic animals (Vibrio alginolyticus, Vibrio harveyi, Flavobacterium columnare, Aeromonas hydrophila, Aeromonas sobria, Aeromonas veronii, Streptococcus agalactiae, Streptococcus dysgalactiae, Vibrio parahaemolyticus, Vibrio vulnificus, Photobacterium mermairei, Nocardia seriolae, Pseudomonas fluorescens).
Experimental example 1 detection of clinical samples by primer
Taking 48 healthy river crabs, wherein 24 of the healthy river crabs are injected from the plantar ganglion joint membrane of the 4 th step to have the concentration of 2 multiplied by 10 7 CFU/mL of Ericaria dubia 0.2mL, and 24 additional injections of sterile water of equal volume. After culturing in water at 25 ℃ for 7 days, respectively extracting DNA samples of liver and pancreas of the river crabs as templates, and carrying out PCR detection. PCR (50. mu.L reaction) amplification conditions were as follows: add 2 XTaq Master Mix 25. mu.L, 10mM upstream detection primer 0.5. mu.L, 10mM downstream detection primer 0.5. mu.L, template 1. mu.L, sterile water 23. mu.L. The reaction procedure is as follows: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 deg.C for 1min, annealing at 62 deg.C for 1min, and annealing at 72 deg.CExtending for 30s, and performing 35 cycles of denaturation, annealing and extension; extension at 72 ℃ for 10 min. As shown in FIGS. 4 and 5, the samples of the non-injected and infected river crabs were all negative, and the samples of the injected and infected river crabs were all positive. The established PCR detection method is suitable for detecting river crab samples.
Experimental example 2 Artificial infection experiment
The method comprises the steps of adopting three ways of intramuscular injection, dipping bath and mixing and feeding to carry out artificial infection on healthy river crabs, carrying out an infection experiment in a plastic box with the volume of 64cm multiplied by 46cm multiplied by 39cm and the water depth of 30cm, dividing into 4 groups (3 experimental groups and 1 control group), continuously inflating 25 healthy river crabs in each group, and carrying out the infection experiment after temporary culture for 7 days. During the experiment, the water temperature is 21 ℃, the water is changed 1/3 every day, and the compound feed for the river crabs is fed according to 2 percent of the weight of the river crabs every day. Observing the activity and ingestion of the river crabs every day, recording the death number of the river crabs, and taking the moribund crabs for pathogenic bacteria separation.
The density of the bacterial suspension is adjusted to be 2 multiplied by 10 by the injection group 7 CFU/mL, injected at step 4 plantar ganglion articular membrane (50. mu.L/mouse);
the bathing group removes 1-2 feet of healthy river crabs with no damage to body surfaces by using sterile scissors, and places the crabs in a bacterial suspension with the density of 2 multiplied by 10 7 Continuously soaking in CFU/mL water for 3h, and then transferring into clear water for culture;
the density of the bacterial suspension of the feeding group is adjusted to be 2 multiplied by 10 8 CFU/mL, at 2X 10 7 Mixing the CFU/g with river crab compound feed uniformly, adding biological phycocolloid, stirring, drying in the shade for 1h at room temperature, storing in a refrigerator at 4 deg.C, and feeding daily instead of river crab basic feed;
the control group was injected with an equal dose of 0.85% sterile saline.
The artificial infection experiment is carried out by adopting the purified strain JMB-1, the result is shown in figure 6 and table 1, the death of the river crabs occurs in each injection group, bathing group and feeding group at 3 days after the river crabs are infected, the river crabs in the injection groups start not to eat any more at 2 days, obvious action reaction is observed at 7 days, the death amount is maximal at 9 days and 10 days, all the river crabs die at 14 days, the cumulative death rate is 100 percent, the death river crabs die at 6 days have obvious 'milk disease' symptoms, and a large amount of thalli with the same form as the strain JMB-1 can be observed by taking white emulsion smears of the artificially infected crabs. Dead river crabs begin to appear in the dipping group at the 2 nd day, the cumulative death rate is 28 percent by 14 days after the test is finished, the river crabs dying in the initial stage do not show obvious milk disease symptoms, but bacterial colony and thallus forms of bacterial strains separated from the hepatopancreas of the river crabs are consistent with JMB-1, and more obvious white emulsion is observed in the pericardial cavity of the river crabs dying on the 9 th day. Dead river crabs appear on the feeding group from 3 days, the food intake is reduced, the cumulative death rate is 12% till the infection experiment is finished, but the dead river crabs do not show obvious symptoms of milk diseases. None of the control groups died and the feeding activity was good by the end of the test.
TABLE 1 Artificial infection test results for JMB-1 Strain
After the infection test is finished, pathogeny is separated from the hepatopancreas of the rest river crabs (22) in the feeding group, wherein the bacterial strain separated from 1 river crab has the same colony and thallus form as JMB-1, and the proportion is 4.5%. Pathogen was isolated from the hepatopancreas of the remaining river crabs (18) in the bathing group, wherein the strains isolated from 3 river crabs were identical in colony and thallus morphology to JMB-1, accounting for 16.7%.
EXAMPLE 3 half lethal experiment
Adjusting the density of the Erjiangmei yeast suspension to 1.2 × 10 8 cfu/mL, 10-fold diluted to concentrations of 1.2X 10, respectively 7 cfu/mL，1.2×10 6 cfu/mL，1.2×10 5 cfu/mL, assay was divided into 5 groups, each group with the following bacteria concentration, group 1: 1.2X 10 8 cfu/mL; group 2: 1.2X 10 7 cfu/mL; group 3: 1.2X 10 6 cfu/mL; group 4: 1.2X 10 5 cfu/mL; group 5: control group, 30 healthy river crabs per group. The test groups were injected with the plantar ganglion joint membranes of step 4 of river crab, 50. mu.L each. The control group was injected with an equal dose of 0.85% sterile saline. During the experiment, the water temperature is kept at 15 ℃, the water is changed 1/3 every day, and the basic compound feed for the river crabs is fed according to 2 percent of the weight of the river crabs every day. Observing river crab activity every day and recording death situation, and collecting dying crab as pathogenSeparating bacteria, and calculating half lethal dose.
The results of half lethal dose detection (Table 2) show that, with the increase of infection concentration, the proliferation speed of yeast in the river crab is accelerated, the death of the river crab is accelerated, the river crab of group 1 dies completely at 13d after infection, the river crab of group 2 dies completely at 14d, the death rates of group 3 and group 4 at 14d respectively reach 43.3 percent and 33.3 percent, the time node is used, and the half lethal concentration of bicinchondric yeast to the river crab is 6.6 multiplied by 10 when the water temperature is 15 ℃ and the infection period is 14 days according to the improved Korotkoff method 5 cfu/mL. However, at 17d of the infection experiment, all of the river crabs in the groups 3 and 4 died, and each group had a concentrated death phase.
TABLE 2 death result records of river crabs with different infection concentrations
Experimental example 4 pathogenicity test
Adjusting the density of the Erjiangmei yeast suspension to 4.6 × 10 3 cfu/mL，4.6×10 2 cfu/mL, the test was divided into 3 groups of 30 healthy river crabs, of which 1 group was a control group. Injecting test river crab into the plantar node joint membrane of step 4, 50 μ L each, and injecting 0.85% sterile saline into the control group. During the experiment, the water temperature is kept at 20 ℃, the water is changed 1/3 every day, and the basic compound feed for the river crabs is fed according to 2 percent of the weight of the river crabs every day. Observing river crab activities and recording death situation every day, and taking dying crabs to observe symptoms and separate pathogenic bacteria.
The results show (Table 3), 4.6X 10 3 All the cfu/mL river crabs died, 4.6X 10 2 The 21-day mortality rate of the cfu/mL group of river crabs is 90 percent, and the rest 3 river crabs are in the state of morbidity.
TABLE 3 death result record of river crab with different infection concentration
The infection tests show that the two-tip meiqi yeast has strong pathogenicity on the river crabs, can be spread by oral administration and body surface wounds, once enters the bodies of the river crabs, and greatly proliferate in the bodies of the river crabs under proper conditions to cause milk-like pathological changes of the river crabs, and finally die. Therefore, the research and development of a rapid and sensitive detection technology for early and timely detection of pathogens in the environment have great significance for prevention and control of the pathogens.
The above examples and experimental examples show that the invention provides a primer group and a kit for detecting bicinchondric yeast, the kit has the advantages of high sensitivity, strong specificity and high detection accuracy, can effectively detect the bicinchondric yeast, is simple and convenient in method, has no cross reaction with adjacent species of the bicinchondric yeast, also has no cross reaction with other common aquatic animal pathogenic bacteria, and has important significance for disease prevention and control of aquatic animals.
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 principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> aquatic research institute in Tianjin
<120> primer group and kit for detecting Erjiangqi yeast
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<213> Artificial Sequence (Artificial Sequence)
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Claims (6)
1. A primer group for detecting Erjiangqiyeyu is characterized by comprising an upstream detection primer and a downstream detection primer; the nucleotide sequence of the upstream detection primer is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream detection primer is shown as SEQ ID NO. 2.
2. A kit for detecting Ericaria distachya, comprising the primer set of claim 1 and a detection reagent.
3. The kit of claim 2, wherein the detection reagents comprise 2 x Taq MasterMix and sterile water.
4. The kit according to claim 2, wherein the upstream detection primer and the downstream detection primer in the primer set are independently used at a concentration of 8-12 mM.
5. The kit according to claim 2, wherein the amplification system used in the kit detection comprises the following components in 50 μ L: 2 XTaq MasterMix 25. mu.L, upstream detection primer 0.5. mu.L, downstream detection primer 0.5. mu.L, template 1. mu.L, sterile water 23. mu.L.
6. The kit according to claim 2, wherein the amplification procedure in the detection using the kit is as follows: pre-denaturation at 94 ℃ for 10 min; denaturation at 94 ℃ for 1min, annealing at 62 ℃ for 1min, extension at 72 ℃ for 30s, and 35 cycles of denaturation, annealing and extension; final extension at 72 ℃ for 10 min.
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| Metschnikowia bicuspidata isolated from milky diseased Eriocheir sinensis: Phenotypic and genetic characterization,antifungal susceptibility and challenge models;Hongli Ma等;《J Fish Dis》;20210915;第45卷(第1期);第41-49页 * |
| 中华绒螯蟹"牛奶病"病原及其致病性研究;徐晓丽等;《中国海洋大学学报》;20211110;第51卷(第12期);第23-32页 * |
| 辽宁地区中华绒螯蟹"牛奶病"的病原分离与鉴定;马红丽等;《大连海洋大学学报》;20200921;第35卷(第5期);第714-718页 * |
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