CN115725742B

CN115725742B - PCR (polymerase chain reaction) kit for diagnosing itch mite disease and detection method

Info

Publication number: CN115725742B
Application number: CN202210856989.0A
Authority: CN
Inventors: 古小彬; 杨光友; 谢跃; 何冉; 徐璟
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2022-07-20
Filing date: 2022-07-20
Publication date: 2024-03-26
Anticipated expiration: 2042-07-20
Also published as: CN115725742A

Abstract

The invention discloses application of ITS2, 16S, 12S and COX1 gene sequences of itch mites in preparing a kit for diagnosing itch mites, and further discloses a PCR kit for diagnosing itch mites, wherein specific primers in the kit can be specifically combined with ITS2, and/or 12S, and/or 16S, and/or COX1 gene sequences of itch mites. The qualitative PCR kit for diagnosing the itch mite and the PCR detection method for detecting the itch mite by using the kit have higher detection sensitivity than that of microscopic examination, can discover early infected animals more comprehensively and earlier, have higher sensitivity and specificity, can be used for diagnosing subclinical cases and invisible infection cases of low-load parasitic infection, and can distinguish existing infection from existing infection.

Description

PCR (polymerase chain reaction) kit for diagnosing itch mite disease and detection method

Technical Field

The invention belongs to the technical field of in-vitro diagnosis, and particularly relates to a PCR (polymerase chain reaction) kit for diagnosing itch mite disease and a detection method.

Background

The itch mite disease is a common ectoparasitic disease which is parasitic on the skin surface of herbivores and wild animals by sheep itch mites (Psoropates ovis), clinically takes itch, crusting, skin thickening and dehairing as main clinical symptoms, causes great economic loss for the breeding industry and threatens the survival of the wild animals. Macroscopic clinical signs of crusting can appear 2 weeks and later after the animals are once infected with P.ovis artificially, but when healthy animals are infected with P.ovis by contacting with diseased animals or vectors polluted by mites in actual production, the time for the macroscopic clinical signs of crusting can be longer, and the period can be prolonged to 1 month or even several months.

In the existing production, the diagnosis means of the itch mite disease is confirmed by observing mites or mite eggs from scab through a microscope, the diagnosis method has high detection rate of severe infection cases, but subclinical infection and mild infection cases cannot be detected because scab is not visible, and the missed detection cases become invisible infection sources of the itch mite disease infection in animal groups, and finally cause severe epidemic of the itch mite disease in animal groups. In addition, microscopic diagnostic methods require that the diagnostic personnel have a certain expertise of the parasite to identify the parasite or eggs, and thus it can be seen that microscopic etiology diagnostic methods are time consuming, that the operator requires a certain expertise, and that they miss cases of low mite number infection.

In recent years, the indirect ELISA diagnosis method of a plurality of recombinant antigens has been confirmed to be useful for diagnosis of subclinical infection and invisible infection cases of itch mite, and the diagnosis effect of the indirect ELISA method (rPsoSP 3-iELISA) using recombinant serine protease inhibitor 3 has been reported to be optimal among serological diagnosis methods. Although the rPsoSP3-iELISA can be used for early diagnosis of subclinical cases and invisible infection cases by detecting specific IgG, the detection method cannot distinguish between past infection and present infection due to too long a period of IgG antibody resolution.

Therefore, there is a need to provide a method for diagnosing itch mite which has high sensitivity, good accuracy, low operator requirements, and can identify itch mite infestations from present infections.

Disclosure of Invention

Recently, molecular diagnostic methods based on PCR technology are used for detecting parasites, viruses and bacteria infection, and research reports on the molecular marker genes of the itch mites in the prior art mainly aim at evaluating genetic research of itch mite populations of different geographic sources and host sources, and research reports on the molecular diagnostic methods of itch mite diseases are not seen at present. In order to more efficiently diagnose the itch mite, the invention provides a PCR kit for diagnosing the itch mite and a PCR detection method for the non-diagnostic purpose of the itch mite.

In a first aspect, the invention provides the use of ITS2, 16S, 12S and COX1 gene sequences of itch mites in the preparation of a kit for diagnosing itch mites.

Preferably, the gene sequences are ITS2, 12S.

Most preferably, the gene sequence is ITS2.

In a second aspect, the invention provides a PCR kit for diagnosing itch mite disease, characterised in that the kit comprises specific primers which can specifically bind to ITS2, and/or 12S, and/or 16S, and/or COX1 gene sequences of itch mites;

wherein, the specific primer sequence for ITS2 gene is shown in SEQ ID NO. 1-SEQ ID NO. 2;

the specific primer sequence for the 12S gene is shown as SEQ ID NO. 3-SEQ ID NO. 4;

the specific primer sequence for the 16S gene is shown as SEQ ID NO. 5-SEQ ID NO. 6;

the specific primer sequence for COX1 gene is shown in SEQ ID NO. 7-SEQ ID NO. 8.

Preferably, the specific primer in the kit is a specific primer sequence for ITS2 gene and/or a specific primer sequence for 12S gene.

Most preferably, the specific primer in the kit is a specific primer sequence for ITS2 gene.

The kit also comprises a conventional reagent in the DNA extraction process, and/or a conventional reagent in the PCR amplification reaction, and/or a conventional reagent for detecting a PCR amplification product.

Preferably, the kit further comprises a negative control product, wherein the negative control product is physiological saline.

In a third aspect, the invention provides a PCR detection method for non-diagnostic purpose itch mite disease, which is characterized by comprising the following steps:

(1) Extracting DNA of a sample to be detected;

(2) Preparing a PCR reaction system, which specifically comprises the following steps:

composition of the components	Volume of
		2×Taq PCR Master Mix	5μL
Specific primers	0.25-1μL
		Template DNA	0.5μL
Ultrapure water	3.5-4.25μL
		Sum up	10μL

(3) PCR amplification was performed as follows: pre-denaturing at 95deg.C for 3min, denaturing at 95deg.C for 30s, annealing at 50-64deg.C for 45s, extending at 72deg.C for 30s for 25-35 cycles, and preserving at 72deg.C for 5min at 4deg.C;

(4) The PCR products were subjected to gel electrophoresis.

Preferably, the specific primer in step (2) is added in a volume of 1. Mu.L, and the upstream primer and the downstream primer are each 0.5. Mu.L.

Preferably, the PCR amplification procedure in the step (3) is: pre-denaturation at 95℃for 3min, denaturation at 95℃for 30s, one of annealing at 50℃and 60℃and 64℃for 45s and extension at 72℃for 30s for 25 cycles, and finally holding at 72℃for 5min and preserving at 4 ℃.

The sample in the step (1) is a dry swab obtained from an animal to be tested, wherein the animal to be tested comprises domestic and wild animals. Method for collecting dry swab: repeatedly wiping the ear pit of the animal to be detected for 3-5 times by using a cotton swab, putting the cotton swab into a centrifuge tube, and storing the cotton swab at-20 ℃.

The technical scheme provided by the invention has the following advantages:

1. the invention provides a thought that specific primers are designed for ITS2, 16S, 12S and COX1 gene sequences of itch mites and are used for preparing a qualitative PCR kit or a fluorescent quantitative PCR kit, and the inventor designs and tests prove that the specific primers designed for ITS2 and 12S genes have good specificity, and further, the sensitivity of the specific primers for ITS2 is higher and can reach 40.3 pg/mu L (1 mite DNA is diluted by 20 times).

2. The qualitative PCR kit for diagnosing the itch mite and the PCR detection method for detecting the itch mite by using the kit are higher in detection sensitivity than that of microscopic examination, can discover early infected animals more comprehensively and early, have higher sensitivity and specificity, can be used for diagnosing subclinical cases and invisible infection cases of low-load parasitic infection, provide better guarantee for disease prevention and control, and can distinguish existing infection from present infection.

Drawings

FIG. 1, lanes from left to right, respectively, shows the PCR amplification of the 12S, 16S, ITS, COX1 genes, D2000DNA markers; 12S;16S; ITS2; COX1; negative control.

FIG. 2, lanes from left to right, respectively, are D2000DNA markers for the specificity tests of the 12S and ITS2 and 16S genes; itch mite DNA; scabies DNA; rabbit skin tissue DNA; negative control.

FIG. 3 sensitivity test of ITS2 and 12S genes, D2000DNA markers from left to right lanes, respectively; 10 itch mite DNA;5 itch mite DNA;1 itch mite DNA;1, 10 dilution of 1 itch mite DNA;1, diluting 1 to 20 of itch mite DNA;1 itch mite DNA1:40 dilution; 1 itch mite DNA1:80 dilution; 1, 1 part of itch mite DNA is diluted by 100 parts; negative control.

The PCR amplification procedure of the ITS2 gene of FIG. 4 was optimized, with D2000DNA markers from left to right lanes, respectively; 25 cycles; 30 cycles; 35 cycles; a negative control; d2000 A DNA marker; 0.25. Mu.L; 0.5. Mu.L; 1 μl; a negative control; d2000 A DNA marker;50 ℃;55 ℃;60 ℃;65 ℃; negative control.

FIG. 5 comparison of microscopic, ELISA, PCR diagnostic methods.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Primer design

Downloading the ITS2, 12S and 16S, COX1 gene sequences of P.ovis from GeneBank, designing primers by using Primer Premier 5.0 software, confirming the specificity of the designed primers by using Primer blast, and designing the primers for the ITS2, 12S and 16S, COX gene sequences respectively as follows:

PCR detection method for itch mite disease

S1: taking down scab skin in external auditory canal of the itch mite seed-retaining rabbit with forceps, placing dander into a culture dish, placing into a 37 ℃ incubator for 30min, collecting insect bodies climbing out of the dander, placing the collected mites into a 1.5mL EP tube for preservation at-20 ℃ after morphological identification and confirmation, and extracting itch mite DNA according to the instruction of OMEGA micro genome DNA extraction kit (Chengdu Baisi Biotechnology Co.);

s2: adding 0.5 μl of each specific primer, 0.5 μl of template DNA, 2× Taq PCR Master Mix 5 μl, and 3.5 μl of ultrapure water into a PCR reaction tube, adding 10 μl, and mixing thoroughly by vortex oscillation; here, 4 PCR reaction systems were set up, and specific primers were (1) 0.5. Mu.L SEQ ID NO: 1+0.5. Mu.L SEQ ID NO:2, (2) 0.5. Mu.L SEQ ID NO: 3+0.5. Mu.L SEQ ID NO:4, (3) 0.5. Mu.L SEQ ID NO: 5+0.5. Mu.L SEQ ID NO:6, (4) 0.5. Mu.L SEQ ID NO: 7+0.5. Mu.L SEQ ID NO:8, respectively;

s3: PCR amplification was performed using the itch mite DNA as a template, and the procedure was as follows:

ITS2 amplification conditions: pre-denaturing at 95deg.C for 3min, denaturing at 95deg.C for 30s, annealing at 50deg.C for 45s, extending at 72deg.C for 30s for 25 cycles, and maintaining at 72deg.C for 5min, and preserving at 4deg.C;

12S amplification conditions: pre-denaturing at 95deg.C for 3min, denaturing at 95deg.C for 30s, annealing at 60deg.C for 45s, extending at 72deg.C for 30s for 25 cycles, and maintaining at 72deg.C for 5min and preserving at 4deg.C;

16S amplification conditions: pre-denaturing at 95deg.C for 3min, denaturing at 95deg.C for 30s, annealing at 65deg.C for 45s, and extending at 72deg.C for 30s for 25 cycles, maintaining at 72deg.C for 5min, and preserving at 4deg.C;

COX1 amplification conditions: pre-denaturing at 95deg.C for 3min, denaturing at 95deg.C for 30s, annealing at 65deg.C for 45s, and extending at 72deg.C for 30s for 25 cycles, maintaining at 72deg.C for 5min, and preserving at 4deg.C; the method comprises the steps of carrying out a first treatment on the surface of the

S4: the appropriate amount of PCR amplified product was added to agarose 1.5% and the gel cake containing nucleic acid dye was subjected to electrophoresis, and the result was observed by using a gel imaging system, as shown in FIG. 1.

Specific primers designed for ITS2, 12S and 16S successfully amplify DNA extracted from itch mite bodies by PCR, and the bands of PCR amplification products in gel are consistent with expected sizes, so that the ITS2, 12S and 16S primers are proved to be successfully designed, but COX1 specific binding primers amplify two bands and are not suitable for being used as diagnostic molecular targets.

Specificity test of primers

S1: extracting itch mite DNA, scabies mite DNA and rabbit skin tissue DNA according to the instruction of OMEGA micro genome DNA extraction kit (Chengdu Baisi Bo Biotechnology Co., ltd.), wherein scabies mite and rabbit ear skin tissue are provided by Sichuan agricultural university parasitosis research center;

s2: adding 0.5 μl of each specific primer, 0.5 μl of template DNA, 2× Taq PCR Master Mix 5 μl, and 3.5 μl of ultrapure water into a PCR reaction tube, adding 10 μl, and mixing thoroughly by vortex oscillation; 4 PCR reaction systems were provided in total, and specific primers were (1) 0.5. Mu.L SEQ ID NO: 1+0.5. Mu.L SEQ ID NO:2, (2) 0.5. Mu.L SEQ ID NO: 3+0.5. Mu.L SEQ ID NO:4, (3) 0.5. Mu.L SEQ ID NO: 5+0.5. Mu.L SEQ ID NO:6, respectively;

s3: PCR amplification is carried out by taking itch mite DNA, scabies mite DNA and rabbit skin tissue DNA as templates, and the procedure is as follows:

s4: the appropriate amount of PCR amplified product was added to agarose 1.5% and the gel cake containing nucleic acid dye was subjected to electrophoresis, and the result was observed by using a gel imaging system, as shown in FIG. 2.

No amplification products of ITS2 and 12S genes were detected in the DNA of the scabies and rabbit skin tissue samples, but 16S amplification products appeared after the DNA of the rabbit skin tissue samples was amplified. The PCR result analysis shows that the specificity of the specific primers of ITS2 and 12S is better, and the cross reaction (false positive) of itch mites and scabies and rabbit skin tissue sample DNA can not occur.

Sensitivity test of primers

S1: the itch mite DNA was extracted according to the instructions of OMEGA minigenome DNA extraction kit (Chengdu Bessel Biotechnology Co., ltd.) and the DNA concentration gradient was set: extracting 10 itch mites, 5 itch mites and 1 itch mite DNA, and diluting 1 itch mite DNA into 1:10, 1:20, 1:40, 1:80 and 1:100;

s2: adding 0.5 μl of each of the specific upstream and downstream primers, 0.5 μl of template DNA, 2× Taq PCR Master Mix 5 μl, and 3.5 μl of ultrapure water into a PCR reaction tube, adding 10 μl, and mixing thoroughly by vortex oscillation; 2 PCR reaction systems are arranged in total, and specific primers are respectively (1) 0.5 mu L SEQ ID NO:1+0.5 mu L SEQ ID NO:2, (2) 0.5 mu L SEQ ID NO:3+0.5 mu L SEQ ID NO:4;

s3: PCR amplification is carried out by taking the S1 set itch mite DNA with different concentrations as a template, and the procedure is as follows:

12S: amplification conditions: pre-denaturing at 95deg.C for 3min, denaturing at 95deg.C for 30s, annealing at 60deg.C for 45s, extending at 72deg.C for 30s for 25 cycles, and maintaining at 72deg.C for 5min and preserving at 4deg.C;

s4: the appropriate amount of PCR amplified product was added to agarose 1.5% and the gel cake containing nucleic acid dye was subjected to electrophoresis, and the result was observed by using a gel imaging system, as shown in FIG. 3.

The results of comparing the strip brightness under 8 different DNA concentrations after electrophoresis show that the 12S specific primer can achieve 10 times of sensitivity (0.403 ng/. Mu.L) of 1 itch mite DNA dilution, while the ITS2 specific primer has very high sensitivity to 20 times (40.3 pg/. Mu.L) of 1 itch mite DNA dilution, so that ITS2 is more suitable for being used as a molecular target for PCR diagnosis, and then ITS2 target strip gel is sent to be compared with the itch mite ITS2 sequence reported in a Genebank database after sequencing, and the similarity between the amplified sequence and a reference sequence is 98.9%.

Optimization of PCR amplification procedure

Several factors that generally have a relatively large impact on the PCR results are primer concentration, cycle number, and annealing temperature, respectively, and the above parameters are optimized for this experiment.

The PCR detection method comprises the following steps:

s1: extracting itch mite DNA according to the instruction of OMEGA micro genome DNA extraction kit (Chengdu Baibo biotechnology Co., ltd.);

s2: adding specific primers ITS2-F+ITS2-R, template DNA 0.5 μL, 2X Taq PCR Master Mix 5 μL and ultrapure water 3.5-4.25 μL with different volumes into a PCR reaction tube, adding 10 μL, and shaking by a vortex instrument to fully mix;

s3: PCR amplification is carried out by taking the itch mite DNA as a template, and the procedures are as follows: pre-denaturing at 95deg.C for 3min, denaturing at 95deg.C for 30s, annealing at 45deg.C, extending at 72deg.C for 30s, performing a certain number of cycles, and preserving at 72deg.C for 5min at 4deg.C;

s4: the appropriate amount of PCR amplified product was added to agarose 1.5% and the gel cake containing nucleic acid dye was subjected to electrophoresis, and the result was observed by using a gel imaging system, as shown in FIG. 4.

The variables were set as follows:

1. the primers in the PCR reaction system are respectively 0.25 mu L, 0.5 mu L and 1 mu L;

2. cycling is carried out from denaturation to extension, and the cycle numbers are respectively set to 25 times, 30 times and 35 times;

3. the annealing temperatures were set at 50℃and 55℃and 60℃respectively.

The PCR procedure was optimized by the variables described above, and it was determined that the optimal cycle number was 25, the optimal annealing temperature was 50℃and the optimal primer volume was 1. Mu.L.

PCR detection method, microscopy and ELISA comparison

1. Establishing an artificial infection model and collecting samples of New Zealand rabbit itch mite patients

Taking away scab skin in the ear canal of a diseased rabbit with forceps, placing the dander into a culture dish, placing the culture dish in a temperature box at 37 ℃ for 2-3 hours, collecting itch mites in the dander, subpackaging each 100 itch mite bodies into a 1.5mL centrifuge tube, and collecting enough multiple itch mite bodies according to the amount of 100 itch mites infected by one rabbit ear.

Rabbits to be infected (n=9) were kept well, and the ear canal of the rabbit was plugged with cotton. And pouring the collected itch mites directly into the position of the ear pit of the rabbit, and sealing the ears by using a medical dressing of 5 x 7cm, so as to leave a small part of gaps. The lower part of the rabbit ear is entangled by using the adhesive tape (ensuring the normal blood circulation of the rabbit ear), and then the illisha white ring is worn on the rabbit to prevent the rabbit from being scratched. After artificial infection, the rabbits are observed, the adhesive tape can be detached after 24 hours, the dressing and the plug are taken out after 2 days, the Elizabeth circle is taken off after 3 days, and the rabbits are continuously infected with itch mites. Rabbits were treated with the drug at week 6 post infection, and ivermectin was subcutaneously injected in an amount of 0.1mL/kg.w, with only one injection.

Samples to be tested are extracted, including collection of dry swabs and serum.

The method for collecting the dry swab comprises the steps of repeatedly wiping the infected 9 rabbits for 3-5 times at the rabbit ear fossa by using a cotton swab after the infected rabbits are well preserved, placing the cotton swab into a centrifuge tube, collecting 2 samples in total, labeling on the tube wall, storing 1 sample at normal temperature for detection by a traditional microscope, and storing the other 1 sample at-20 ℃ for detection by PCR.

Serum is collected, after 3mL of blood is collected from the ear vein of 9 infected rabbits, the rabbits are placed in a constant temperature box at 37 ℃ for 30min, placed in a refrigerator at 4 ℃ for 3h, and the supernatant and a centrifuge tube are centrifuged for 10min at 3000g and stored at-20 ℃ for ELISA detection.

Sampling times were 1, 2, 4, 6 weeks post-infection and 3, 7, 11 days post-treatment.

2. PCR detection

And extracting the collected dry swab DNA by using an OMEGA micro genome DNA extraction kit, detecting the preserved dry swab DNA by using an optimized and established PCR method, and judging that the band with the expected size appears to be positive after gel electrophoresis.

3. Conventional microscopy

Placing a dry swab sample stored at normal temperature into a culture dish, placing the dry swab sample into a 37 ℃ incubator for 30min, taking out the dry swab sample, placing the dry swab sample into a large glass plate, placing the dry swab sample on white paper, picking a viable insect body on a glass slide, observing the insect body by using a microscope, and judging that the insect body has obvious itch mite body morphology is positive.

4. Detection of rPsoSP3-iELISA

The collected serum is detected by rPsoSP3-iELISA method of subject group pre-screening, the basic steps are that 96-well ELISA plate is taken for antigen coating, rPsoSP3 is diluted to 35.5 mug/mL by coating buffer, 100 mug/well of 96-well plate is added, and the coating is carried out at 4 ℃ overnight. After overnight washing was performed by beating the liquid in the wells to dry, adding 200. Mu.L of PBST to each well for washing, and shaking for washing. After the completion, 300. Mu.L of blocking solution was added to each well, and incubated at 37℃for 1-2 hours. After washing, 100. Mu.L of serum to be tested diluted 1:40 with PBS was added to each well and incubated at 37℃for 1h. After washing, 100. Mu.L of HRP-labeled sheep anti-rabbit IgG antibody (1:10000) diluted in PBS was added to each well and incubated at 37℃for 1h. Finally, washing is carried out for color development, 100 mu L of TMB color development liquid is added into each concave hole, and the mixture is placed for 20min in a dark place. And adding 100 mu L of stop solution, measuring an OD450 value by using an enzyme-labeled color developing instrument, and judging positive if the OD450 value exceeds a cut-off value by 0.401, and otherwise, judging negative.

All rabbits were successfully infected with the itch mites, and samples taken 1, 2, 4, 6 weeks of infection and 3, 7, 11 days after treatment were tested by conventional microscopy, PCR and iielisa, and the results are shown in fig. 5.

The positive rate detected by the traditional microscopic examination is only 33.3% (3/9) at week 1, and reaches 100% (9/9) at week 2. The positive rates of the 3-day and 7-day post-treatment microscopic examination decreased to 66.6% (6/9) and 11.1% (1/9), respectively, with positives of 7-day post-treatment due to microscopic examination of body debris and dead bodies in rabbit ear # 9.

The positive rate of iELISA was 77.7% (7/9) at week 1, the OD450 value was greater than the cut-off value of 0.401 from week 2 to 11 days after treatment, and the period was stable after the overall upward trend.

According to the PCR diagnosis result, the positive rate reaches 88.9% (8/9) at week 1, and the positive rate reaches 100% (9/9) at week 2. The positive rate of the rabbit at the day 3 after treatment and the positive rate of the rabbit at the day 7 after treatment are 66.6% and 0% respectively, but the condition of re-yang appears in the rabbit at the day 8 after treatment, but the condition of re-yang is not detected by microscopic examination and iELISA, and a small amount of dead bodies or remains possibly remained in the rabbit at the day 8 after treatment, so that the dry swab sampling can not always collect the remained itch mite bodies or remains every time, thereby leading to the appearance of subsequent re-yang, the dry swab sampling can be carried out for a plurality of times later, and the PCR is carried out after mixing, thereby improving the detection sensitivity.

By combining the experimental result, the positive detection rate of the PCR detection method at week 1 is 88.9 percent, which is obviously higher than the detection rate of 33.3 percent of microscopic examination, so that the detection sensitivity of PCR on the diagnosis of the itch mite disease is higher than that of microscopic examination, early infected rabbits can be found earlier and more comprehensively, and better guarantee is provided for the prevention and control of the itch mite disease.

PCR has similar sensitivity compared with ELISA, but does not have complicated steps for preparing antigen protein, has good specificity, avoids false positive, and most importantly, ELISA can not distinguish between existing infection and current infection due to long-term existence of antibodies in a host body, so that PCR can compensate the defect.

Practical application of PCR in rabbit field

The conventional microscope and PCR test was performed on 36 dry swab samples taken from a rabbit farm with a history of itch mite epidemics, wherein 4 samples were positive for microscopic examination, the detection rate was 11.1%, the PCR test was positive for the 4 positive samples for microscopic examination, but 2 samples were positive for microscopic examination, so that 6 positive samples were detected in total in 36 samples, the detection rate was 16.7%, and the results are shown in the following table.

TABLE 1

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. Application of specific primers for detecting ITS2 and/or 12S genes of itch mites in preparing a kit for diagnosing itch mites; wherein, the specific primer sequence for ITS2 gene is shown in SEQ ID NO. 1-SEQ ID NO. 2; the specific primer sequence for the 12S gene is shown as SEQ ID NO. 3-SEQ ID NO. 4.

2. The use according to claim 1, wherein the gene is ITS2.

3. A PCR kit for diagnosing itch mite disease, characterized in that the kit comprises a specific primer that can specifically bind to ITS2 and/or 12S genes of itch mites;

wherein, the specific primer sequence for ITS2 gene is shown in SEQ ID NO. 1-SEQ ID NO. 2; the specific primer sequence for the 12S gene is shown as SEQ ID NO. 3-SEQ ID NO. 4.

4. A kit according to claim 3, wherein the specific primer in the kit is a specific primer sequence for ITS2 gene.

5. A kit according to claim 3, wherein the kit further comprises conventional reagents for DNA extraction and/or conventional reagents for PCR amplification reactions and/or conventional reagents for detection of PCR amplification products.

6. The PCR detection method for the non-diagnostic purpose itch mite disease is characterized by comprising the following steps of:

(1) Extracting DNA of a sample to be detected;

composition of the components Volume of 2×Taq PCR Master Mix 5μL A specific primer according to any one of claims 3 to 4 0.25-1μL Template DNA 0.5μL Ultrapure water 3.5-4.25μL Sum up 10μL

(4) The PCR products were subjected to gel electrophoresis.

7. The method according to claim 6, wherein the specific primer in the step (2) is added in a volume of 1. Mu.L, and the upstream primer and the downstream primer are each 0.5. Mu.L.

8. The method according to claim 6, wherein the PCR amplification procedure in the step (3) is: pre-denaturation at 95℃for 3min, denaturation at 95℃for 30s, one of annealing at 50℃and 60℃and 64℃for 45s and extension at 72℃for 30s for 25 cycles, and finally holding at 72℃for 5min and preserving at 4 ℃.