Method for visually and rapidly detecting freshwater fish ichthyophthirius multifilis

Abstract

The invention relates to a method for detecting freshwater fish ichthyophthirius multifilis, which is characterized in that a visual LAMP (loop-mediated isothermal amplification) rapid detection method is established by using 18sRNA (ribonucleic acid) gene of ichthyophthirius multifilis as a specific target gene through a specific primer, and a PCR (polymerase chain reaction) solution containing HNB dye and Bst DNA (deoxyribonucleic acid) polymerase are adopted to carry out loop-mediated isothermal amplification reaction, so as to detect the ichthyophthirius multifilis. According to the method, visual detection of ichthyophthirius multifilis can be achieved within 20 min, professional knowledge or special fluorescent dye is not needed, and whether a sample contains the ichthyophthirius multifilis or not can be judged only by observing the color of an amplification product. The method can be used for detecting ichthyophthirius multifilis of which the plasmid DNA concentration of a sample is as low as 0.01 ng / mu L, and is high in sensitivity and good in specificity. According to the method, rapid early warning can be achieved in the early outbreak stage of the ichthyophthirius multifilis, early discovery and early prevention are achieved, economic losses are reduced, and the method has wide application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pathogen detection, and particularly relates to a method for detecting Ichthyophthirius multifiliis of freshwater fish. BACKGROUND

[0002] Ichthyophthirius multifiliis is a specific parasite of freshwater fish, and the white spot disease caused by the parasite is characterized by white cysts on the body surface, which can cause respiratory disorders and movement disorders of fish. Once the Ichthyophthirius multifiliis disease of freshwater fish breaks out, it is difficult to treat and has a very high mortality rate, and is known as the "fish cancer", which causes great economic losses to the aquaculture industry.

[0003] Chemical control drugs (such as formaldehyde and copper sulfate) have general treatment effects, and can cause drug resistance, environmental residues and other problems. In addition, it is difficult to obtain sufficient antigen for vaccine development because the pathogen cannot be cultured in vitro. Therefore, the prevention and early warning of Ichthyophthirius multifiliis disease has been a technical problem to be solved.

[0004] At present, the detection of Ichthyophthirius multifiliis mainly includes traditional microscopy, ordinary PCR detection and fluorescent quantitative PCR detection. However, these methods have obvious limitations. Traditional microscopy depends on mature cysts of the parasite, has low sensitivity and is easy to be confused with other parasites, and can only be detected after the disease occurs. Although PCR detection and fluorescent quantitative PCR detection can detect Ichthyophthirius multifiliis at the molecular level and provide sensitivity, they need to rely on precise instruments, and the existing primers have insufficient specificity and are easy to cause false positives due to cross-reaction with other ciliates, so the accuracy is not high.

[0005] In view of the above problems, it is urgent to develop a technology for specifically detecting Ichthyophthirius multifiliis in aquaculture water and fish at an early stage of Ichthyophthirius multifiliis infection (when the fish has not yet shown obvious symptoms), so as to realize early warning and accurate disinfection, thereby reducing the mortality of fish.

[0006] Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technology, which amplifies target sequences under certain conditions through the strand displacement activity of Bst DNA polymerase and 4-6 specific primers (targeting 6 regions of the target DNA). After the primers are combined with the target DNA, a stem-loop structure is formed, and the amplification is completed in a few tens of minutes without PCR temperature cycling. This technology is particularly suitable for rapid monitoring of pathogens such as Ichthyophthirius multifiliis in aquaculture sites. SUMMARY

[0007] In view of the deficiencies of the prior art, the present application provides a method for rapidly detecting Ichthyophthirius multifiliis of freshwater fish, which has the advantages of high specificity, simple operation, high sensitivity and the like.

[0008] To achieve the above object, the technical scheme adopted by the present application is:

[0009] A primer set for detecting P. multipunga by loop-mediated isothermal amplification, the primer set is as follows:

[0010] FIP: 5'-CCAAGTCGAACCAGTGAAGGGTTCTCTTAGCTAAAATGGGC-3';

[0011] BIP: 5'-TAGAGTGTTTCAGGCAGGTTATAGCACCAATAAAATGGACTCAGTC-3';

[0012] F3: 5'-AGCTCGTAGTTGAACTTCTG-3';

[0013] B3: 5'-CCCTATTAATCATTACCAAATCCA-3';

[0014] LF: 5'-CCGAGTTTGGTTTGCAAACGTAT-3';

[0015] LB: 5'-CCGGATACATCAGCATGGAATAA-3'.

[0016] The primer set for detecting P. multipunga comprises the following steps:

[0017] (1) Collecting samples, obtaining genomic DNA of the samples;

[0018] (2) Using the primer set, mixing with an amplification reaction solution, Bst DNA polymerase, hydroxyl-containing naphthol blue coloring solution and genomic DNA, and performing loop-mediated isothermal amplification;

[0019] (3) After the amplification reaction, judging whether the samples contain P. multipunga by observing the color of the amplification product.

[0020] The amplification system comprises: 2xLAMPMaster Mix 10 μL, primer FIP and BIP each 0.7 μL, primer F3 and B3 each 0.1 μL, primer LF and LB each 0.2 μL, the concentration of each primer is 10 μmol / L, 8U Bst DNA polymerase 1 μL, genomic DNA template 2 μL, glycerol 20 μL, deionized water 5 μL.

[0021] The amplification reaction is at 61-64℃ for 10-20 min.

[0022] The method for judging whether the sample contains Ichthyophthirius multifiliis is: judging by observing the color of the amplification product; if the product color is sky blue, the sample contains Ichthyophthirius multifiliis; if the product color is violet, the sample does not contain Ichthyophthirius multifiliis.

[0023] The application of the loop-mediated isothermal amplification primer set in detecting Ichthyophthirius multifiliis.

[0024] The application of the loop-mediated isothermal amplification primer set in the detection kit of Ichthyophthirius multifiliis.

[0025] The beneficial effects of the present application

[0026] (1) The present application uses loop-mediated isothermal amplification technology to design specific primer sets for Ichthyophthirius multifiliis 18sRNA gene, and establishes a high-efficiency LAMP detection system. The method can complete the detection within 20 minutes, and does not require complex instruments and professional operation skills, but only through the color change of the reaction solution can the detection result be directly judged.

[0027] (2) The present application adopts an optimized reaction system, which contains HNB dye and Bst DNA polymerase and other components, realizes closed tube detection, and can effectively avoid aerosol pollution. Experiments prove that the detection sensitivity of the method to 18S RNA plasmid DNA can reach 0.01 ng / μL, and there is no cross reaction with other common pathogens, the sensitivity is high, and the specificity is good.

[0028] (3) The detection cost of the method of the present application is low, suitable for batch sample screening; without opening the cover, the reaction can be monitored in real time, which greatly improves the accuracy and efficiency of detection; early warning can be realized in the early stage of Ichthyophthirius multifiliis outbreak, early detection and early prevention are achieved, and economic losses are effectively reduced.

[0029] (4) The method of the present application does not require expensive instruments and equipment, and can detect Ichthyophthirius multifiliis in batch samples, especially suitable for primary site detection, only basic experimental equipment is needed to carry out detection, which provides an effective technical means for aquaculture disease prevention and control, and has important popularization value. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is the primer set screening experiment result of example 1.

[0031] Among them, Figure 1 A is the color development result of the LAMP amplification product; Figure 1 B is the gel electrophoresis result of the LAMP amplification product.

[0032] Figure 2 is the primer concentration screening experiment result of example 2.

[0033] Among them, Figure 2A is the color development result of LAMP amplification product; Figure 2 B is the gel electrophoresis result of LAMP amplification product.

[0034] Figure 3 is the screening experiment result of amplification temperature of Example 3.

[0035] wherein, Figure 3 A is the color development result of LAMP amplification product; Figure 3 B is the gel electrophoresis result of LAMP amplification product.

[0036] Figure 4 is the sensitivity detection experiment result of Example 4.

[0037] wherein, Figure 4 A is the color development result of LAMP amplification product; Figure 4 B is the gel electrophoresis result of LAMP amplification product.

[0038] Figure 5 is the specificity experiment result of Example 5.

[0039] wherein, Figure 5 A is the color development result of LAMP amplification product; Figure 5 B is the gel electrophoresis result of LAMP amplification product. DETAILED DESCRIPTION

[0040] The specific embodiments of the present application are further described in detail below in conjunction with the examples.

[0041] The present application utilizes the loop-mediated isothermal amplification technology, takes the 18sRNA gene (Genebank accession number KJ690572) of Phasmarhabditis hermaphrodita as a specific target gene, designs, screens and optimizes specific primers, and further establishes the LAMP detection method of Phasmarhabditis hermaphrodita.

[0042] Example 1: Specific primer screening

[0043] 1. Primer set design

[0044] Taking the 18sRNA gene (Genebank accession number KJ690572) of Phasmarhabditis hermaphrodita as a specific target gene, according to the specific region of the 18sRNA gene of Phasmarhabditis hermaphrodita, according to the loop-mediated isothermal amplification primer design principle, based on the stem-loop structure FIP, BIP, F3, B3 of the sequence, the loop primer LF, LB primer is an optional primer, the introduction of loop primer LF / LB can further shorten the reaction time. Five groups of primers are designed by using software Primer Designer, avoiding hairpin structure and primer dimer, and screening specific primer groups.

[0045] The designed five groups of primers are:

[0046] Primer Set 1

[0047] FIP: 5'-CCAAGTCGAACCAGTGAAGGGTTCTCTTAGCTAAAATGGGC-3' (SEQ ID NO: 1);

[0048] BIP: 5'-TAGAGTGTTTCAGGCAGGTTATAGCACCAATAAAATGGACTC AGTC-3' (SEQ ID NO: 2);

[0049] F3: 5'-AGCTCGTAGTTGAACTTCTG-3' (SEQ ID NO: 3);

[0050] B3: 5'-CCCTATTAATCATTACCAAATCCA-3' (SEQ ID NO: 4);

[0051] LF: 5'-CCGAGTTTGGTTTGCAAACGTAT-3' (SEQ ID NO: 5);

[0052] LB: 5'-CCGGATACATCAGCATGGAATAA-3' (SEQ ID NO: 6);

[0053] Primer Set 2

[0054] FIP: 5'-CATTTCAATGCCGTAGAGAAGAATCCTAATTCAGGGAGGTAGT GAC-3' (SEQ ID NO: 7);

[0055] BIP: 5'-AGAACAGTGTAAATCTCTTAGCGAGTTGGAGCAGGAATTACC G-3' (SEQ ID NO: 8);

[0056] F3: 5'-GGGACGTAAATTACCCAATC-3' (SEQ ID NO: 9);

[0057] B3: 5'-CTGCAACAACTTTAATATACGC-3' (SEQ ID NO: 10);

[0058] LB: 5'-GAACAATTGGAGGGCAAGTCAGGT-3' (SEQ ID NO: 11);

[0059] Primer Set 3

[0060] FIP: 5'-CGTTGTTCACAGGTCGACTAGCTGGTTAATTCCGTTAACGAAC-3' (SEQ ID NO: 12);

[0061] BIP: 5'-TGGAAGTTTAAGGCAATAACAGGTTTTTGAGCCAGCCATTGT-3' (SEQ ID NO: 13);

[0062] F3: 5'-TCTTAGTTGGTGGAGTGATT-3' (SEQ ID NO: 14);

[0063] B3: 5'-ACCTTTCCAGGTCAGGAA-3' (SEQ ID NO: 15);

[0064] LB: 5'-CTGTGATGCCCCTAGACGT-3' (SEQ ID NO: 16);

[0065] Primer Set 4

[0066] FIP: 5'-GCCCGAAGGAGATTGGTTTAATATCATACATGCGAAAAAATCC GT-3' (SEQ ID NO: 17);

[0067] BIP: 5'-GTAACTGATCGAATCGCGGCCCAATACACTACCATCGAGAG-3' (SEQ ID NO: 18);

[0068] F3: 5'-GATAACCGAGCTAATTGTTGG-3' (SEQ ID NO: 19);

[0069] B3: 5'-TTCTCCGTTACCCGTGAG-3' (SEQ ID NO: 20);

[0070] LB: 5'-GCGATAAATCATCTAAGTTTCTGCC-3' (SEQ ID NO: 21);

[0071] Primer Set 5

[0072] FIP: 5'-TCCCCTAACTTTCGTTCTTGATTAAGGACTAACTAATGCGAAA GCA-3' (SEQ ID NO: 22);

[0073] BIP: 5'-TCAGATACCGTCGTAGTCTTAACTATTTCTCATACGGTGCCGA-3' (SEQ ID NO: 23);

[0074] F3: 5'-CAGAGGTGAAATTCTTGGATT-3' (SEQ ID NO: 24);

[0075] B3: 5'-ACCATACTTCCCCCAGAA-3' (SEQ ID NO: 25).

[0076] 2. Nucleic acid extraction

[0077] The genomic DNA of the Ascaris lumbricoides was extracted by using the magnetic bead method genomic DNA extraction kit (Shanghai Biomed, B518749), and the specific method was performed according to the kit instructions. The extracted genomic DNA was diluted to an appropriate concentration and used as a LAMP amplification template.

[0078] 3. Recombinant plasmid construction

[0079] The PUB-T plasmid was used as a vector, and the 18sRNA gene of the Ascaris lumbricoides was used as a target fragment. The 18sRNA gene fragment was inserted into the PUB-T plasmid by T4 ligase catalysis to construct the recombinant plasmid PUB-T-18S. The constructed recombinant plasmid was transformed into E. coli DH5a competent cells, which were cultured in a medium containing 100 mg / mL ampicillin, and positive clones were selected. Then, the recombinant plasmid was extracted by using a plasmid extraction kit, and PCR detection and sequencing were performed to confirm whether the recombinant plasmid was successfully constructed.

[0080] 4. Loop-mediated isothermal amplification

[0081] The primer set for screening was subjected to isothermal amplification, and the amplification experiment was divided into six groups: five primer groups and one control group. The control group had no primers, which were replaced by sterile water.

[0082] The total volume of the amplification system was 40 μL, including LAMP Master Mix (2x, containing HNB color developing solution) 10 μL, 8 U Bst DNA polymerase 1 μL, PUB-T-18S plasmid DNA template 2 μL, primers FIP and BIP (10 μmol / L) each 0.7 μL, primers F3 and B3 (10 μmol / L) each 0.1 μL, primers LF and LB (10 μmol / L) each 0.2 μL, glycerol 20 μL, and deionized water 5 μL.

[0083] If there is no LF or LB primer, the corresponding component is replaced with sterile water; the control group uses sterile water instead of primers.

[0084] The six groups of loop-mediated isothermal amplification systems described above were mixed uniformly and placed in a constant temperature water bath for 20 min at 61°C.

[0085] After the reaction, the color change of the product was observed. If the product was sky blue, it was positive; if the product was violet, it was negative. Gel electrophoresis detection was also performed.

[0086] The results are shown in Table 1. Figure 1 A, B, M, standard molecular weight DL2000; 1, no primer control group; 2, primer group 1; 3, primer group 2; 4, primer group 3; 5, primer group 4; 6, primer group 5.

[0087] Figure 1 A is the color development result of the LAMP amplification product. Label 2 is sky blue, which is positive (+), and labels 1, 3-6 are violet, which are negative (-).

[0088] Figure 1 B is the gel electrophoresis result of the LAMP amplification product. Label 2 has ladder bands, and labels 1, 3-6 have no bands.

[0089] It can be seen that the color development method and gel electrophoresis detection results are consistent. It shows that primer group 1 can specifically amplify the target gene of Phasmarhabditis hermaphrodita. Therefore, primer group 1 is selected for subsequent experiments.

[0090] Example 2: Primer concentration screening

[0091] The amplification system of Example 1 was used to screen the primer concentration based on primer group 1.

[0092] The experiment was divided into 6 groups, and the corresponding primer concentrations of groups 1-6 were 10 μmol / L, 8 μmol / L, 6 μmol / L, 4 μmol / L, 2 μmol / L, and 0 μmol / L (sterile water), respectively.

[0093] Amplification system: including LAMP Master Mix (2x, containing HNB color developing liquid) 10 μL, 8U Bst DNA polymerase 1 μL, PUB-T-18S plasmid DNA 2 μL, primer FIP, BIP each 0.7 μL, primer F3, B3 each 0.1 μL, primer LF, LB each 0.2 μL, glycerol 20 μL, deionized water 5 μL.

[0094] The six groups of amplification systems were mixed uniformly and placed in a constant temperature water bath for 20 min at 61°C. After the reaction, the color of the reaction product was observed, and the product was subjected to gel electrophoresis detection.

[0095] If the product color is sky blue, the sample is positive (+); if the product color is violet, it is negative (-).

[0096] Results as Figure 2 , Figure 2 A is the colorimetric result of LAMP amplification product, Figure 2 B is the gel electrophoresis result of LAMP amplification product. M, standard molecular weight DL2000; the corresponding primer concentrations of 1-6 are 10.0, 8.0, 6.0, 4.0, 2.0, 0 μmol / μL, respectively.

[0097] Figure 2 The product color of 1-4 in A is sky blue, 5 is dark blue, and 6 is violet. 1-4 can be determined as positive by naked eye observation; 5 is dark blue, which cannot be determined by naked eye observation, and is determined as positive by gel electrophoresis; 6 is violet, which is determined as negative.

[0098] Figure 2 B is the gel electrophoresis result of LAMP amplification product, and 1-5 has ladder bands, and 6 has no band.

[0099] The gel electrophoresis result is consistent with the colorimetric method. It can be seen that primer group 1 can effectively detect P. penicillata at a concentration of 2-10 μmol / L, and the preferred primer concentration for on-site detection is 4-10 μmol / L.

[0100] Example 3: Amplification temperature screening

[0101] Using the amplification system of Example 1, the amplification temperature was screened on the basis of primer group 1 and primer concentration of 10 μmol / L.

[0102] The experiment was divided into 6 groups, and the corresponding amplification temperatures of 1-6 were 60℃, 61℃, 62℃, 63℃, 64℃, and 65℃, respectively. Each temperature corresponds to 2 reaction systems, and the genomic component in 0 system is replaced by sterile water, and the genomic component in 1 system is P. penicillata recombinant plasmid.

[0103] Amplification system: LAMP Master Mix (2X, containing HNB color developing liquid) 10 μL, 8U Bst DNA polymerase 1 μL, PUB-T-18S plasmid DNA 2 μL, primer FIP and BIP each 0.7 μL, primer F3 and B3 each 0.1 μL, primer LF and LB each 0.2 μL, and the concentration of each primer is 10 μmol / L; glycerol 20 μL, deionized water 5 μL;

[0104] The above 12 loop-mediated isothermal amplification systems were mixed uniformly and placed in a constant temperature water bath, and photographed every 10 min for 20 min at the temperature of each group.

[0105] The reaction was observed for color change of the product, and the product was detected by gel electrophoresis. If the color of the reaction product was sky blue, it was positive (+); if the color of the product was violet, it was negative (-).

[0106] The results are shown in Figure 3 , Figure 3 A is the color development result of the LAMP amplification product, Figure 3 B is the gel electrophoresis result of the LAMP amplification product.

[0107] In the figure, M, standard molecular weight DL2000; 1-0, 60℃ control; 1-1, 60℃ experiment; 2-0, 61℃ control; 2-1, 61℃ experiment; 3-0, 62℃ control; 3-1, 62℃ experiment; 4-0, 63℃ control; 4-1, 63℃ experiment; 5-0, 64℃ control; 5-1, 64℃ experiment; 6-0, 65℃ control; 6-1, 65℃ experiment.

[0108] Figure 3 A shows that after 10 minutes of reaction, 0 system and 1 system of 60℃ and 65℃ groups are violet, negative; 0 system of 61℃-64℃ four groups are violet, negative; 1 system is sky blue, positive; after 20 minutes of reaction, 0 system of 60℃-65℃ four temperature groups are violet, negative; 1 system is sky blue, positive.

[0109] Figure 3 B is the gel electrophoresis result of the LAMP amplification product, 1-1, 2-1, 3-1, 4-1, 5-1, 6-1 have ladder-like bands, 1-0, 2-0, 3-0, 4-0, 5-0, 6-0 have no ladder-like bands. The gel electrophoresis result is consistent with the color development method.

[0110] It can be seen that primer group 1 can detect whether there is a multiple small cucumber worm in the sample under the condition of 61℃-64℃ for 10 minutes, and under the condition of 60℃ and 65℃ for 20 minutes. Therefore, the recommended reaction temperature for on-site rapid detection is 61℃-64℃.

[0111] Example 4: Sensitivity experiment

[0112] Using the amplification system of Example 1, sensitivity experiments were carried out under different primer concentrations of primer group 1.

[0113] The experiment was divided into 6 groups, and the 18S RNA plasmid DNA concentrations of 1-6 groups were 100.00 ng / μL, 10.00 ng / μL, 1.00 ng / μL, 0.10 ng / μL, 0.01 ng / μL and 0 ng / μL (sterile water) respectively.

[0114] The amplification system comprises: LAMP Master Mix (2X, containing HNB color developing liquid) 10 μL, 8U Bst DNA polymerase 1 μL, PUB-T-18S plasmid DNA 2 μL, primer group 1: primer FIP, BIP each 0.7 μL, primer F3, B3 each 0.1 μL, primer LF, LB each 0.2 μL, the concentration of each primer is 10 μmol / L; glycerol 20 μL, deionized water 5 μL.

[0115] The above 6 groups of loop-mediated isothermal amplification systems are mixed uniformly and placed in a constant temperature water bath, and reacted at 61°C for 20 min.

[0116] After the reaction is completed, whether the color of the product changes is observed, and the product is detected by gel electrophoresis. If the color of the product is sky blue, the sample is positive (+); if the color of the product is violet, the sample is negative (-).

[0117] Figure 4 A is the color developing result of the LAMP amplification product;

[0118] Figure 4 B is the gel electrophoresis result of the LAMP amplification product;

[0119] M, standard molecular weight DL2000; 1, 100.00 ng / μL; 2, 10.00 ng / μL; 3, 1.00 ng / μL; 4, 0.10 ng / μL; 5, 0.01 ng / μL; 6, 0 ng / μL.

[0120] Figure 4 The A result shows that 1-5 are sky blue, and are judged to be positive; 6 is violet, and is judged to be negative. Figure 4 The B result shows that 1-5 have ladder-like bands, and 6 has no band.

[0121] The gel electrophoresis result is consistent with the color developing result. Therefore, the primer group 1 can effectively detect P. peniculata when the concentration is 0.01 ng / μL or more.

[0122] Example 5: Specificity experiment

[0123] Using the amplification system of Example 1, the primer group 1 is used for the specificity experiment on different biological DNA templates.

[0124] The experiment is divided into 7 groups, and the corresponding template DNA of 1-6 groups is the genome of P. peniculata, I. melastoma, Tetrahymena, E. coli, Amur palla, and chironomid larvae, and the volume is 2 μL, and the concentration of the genomic DNA is 0.01 ng / μL; the 7th group is a positive control, and PUB-T-18S plasmid DNA is used.

[0125] The amplification system comprises: 10 μL of LAMP Master Mix (2X, containing HNB color developing liquid), 1 μL of 8U Bst DNA polymerase, 2 μL of template DNA, 0.7 μL of primer group 1 (FIP and BIP), 0.1 μL of primer F3 and B3, 0.2 μL of primer LF and LB, and the concentration of each primer is 10 μmol / L; 20 μL of glycerol and 5 μL of deionized water.

[0126] The extraction method of the genomic DNA of Aphelenchus avenue is shown in Embodiment 1, and the extraction methods of the genomic DNA of Ictalurus furidus, Tetrahymena, Escherichia coli, Acipenser, and Chironomus larvae are the same as that of Aphelenchus avenue.

[0127] The above seven groups of loop-mediated isothermal amplification systems are mixed uniformly and placed in a constant-temperature water bath, and the reaction is carried out at 61℃ for 20 min.

[0128] After the reaction is completed, the color of the product is observed, and the product is detected by gel electrophoresis. If the color of the reaction product is sky blue, it is judged to be positive (+); if the color is violet, it is judged to be negative (-).

[0129] Figure 5 A is the color developing result of the LAMP amplification product,

[0130] Figure 5 B is the gel electrophoresis result of the LAMP amplification product;

[0131] M, standard molecular weight DL2000; 1, PUB-T-18S positive control; 2, Aphelenchus avenue; 3, Ictalurus furidus; 4, Tetrahymena; 5, Escherichia coli; 6, Acipenser; and 7, Chironomus larvae.

[0132] Figure 5 A shows that 1-2 are sky blue, positive; and 3-7 are violet, negative.

[0133] Figure 5 B shows that 1-2 have ladder-shaped bands, and 3-7 have no bands. The gel electrophoresis detection is consistent with the color developing method.

[0134] It can be seen that the primer group 1 can specifically detect Aphelenchus avenue under the condition that the concentration of the genomic DNA is 0.01 ng / μL. The color developing method is consistent with the gel electrophoresis detection, proving that the method of the present application has accurate and reliable detection results, and can batch detect Aphelenchus avenue in samples.

Claims

1. A primer set for detecting Ichthyophthirius multifellows by loop-mediated isothermal amplification, characterized in that: The primer sets are as follows: FIP:5′-CCAAGTCGAACCAGTGAAGGGTTCTCTTAGCTAAAATGGGC-3′; BIP:5′-TAGAGTGTTTCAGGCAGGTTATAGCACCAATAAAATGGACTC AGTC-3′; F3:5′-AGCTCGTAGTTGAACTTCTG-3′; B3:5′-CCCTATTAATCATTACCAAATCCA-3′; LF:5′-CCGAGTTTGGTTTGCAAACGTAT-3′; LB: 5′-CCGGATACATCAGCATGGAATAA-3′.

2. A method for detecting Ichthyophthirius multifellows using the primer set of claim 1, characterized in that: The following steps are involved: (1) Collecting samples and obtaining genomic DNA of the samples; (2) using the primer set, mixing it with an amplification reaction solution, Bst DNA polymerase, a hydroxyl-containing naphthol blue color development solution, and genomic DNA to perform loop-mediated isothermal amplification; (3) After the amplification reaction, the color of the amplified product is observed to determine whether the sample contains Ichthyophthirius multifleus.

3. The method according to claim 2, characterized in that The amplification system includes: 10 μL of 2×LAMP Master Mix, 0.7 μL each of primers FIP and BIP, 0.1 μL each of primers F3 and B3, 0.2 μL each of primers LF and LB, the concentration of each primer is 10 μmol / L, 1 μL of 8U Bst DNA polymerase, 2 μL of genomic DNA template, 20 μL of glycerol, and 5 μL of deionized water.

4. The method according to claim 2, wherein: The amplification reaction was carried out at 61°C to 64°C for 10-20 minutes.

5. The method according to claim 2, wherein: The method for judging whether a sample contains Ichthyophthirius multifellows is to judge by observing the color of the amplified product; if the color of the product is sky blue, the sample contains Ichthyophthirius multifellows; if the color of the product is violet, the sample does not contain Ichthyophthirius multifellows.

6. Use of the loop-mediated isothermal amplification primer set according to claim 1 in detecting Ichthyophthirius multifellows.

7. Use of the loop-mediated isothermal amplification primer set according to claim 1 in a kit for detecting Ichthyophthirius multifellows.

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