CN115725742A

CN115725742A - PCR kit for diagnosing psoroptes and detection method

Info

Publication number: CN115725742A
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: 2023-03-03
Anticipated expiration: 2042-07-20
Also published as: CN115725742B

Abstract

The invention discloses an application of ITS2, 16S, 12S and COX1 gene sequences of prurus mite in preparing a kit for diagnosing pruritic acariasis, and in addition, the invention discloses a PCR kit for diagnosing pruritic acariasis, wherein specific primers in the kit can be specifically combined with ITS2, and/or 12S, and/or 16S, and/or COX1 gene sequences of pruritic acarid. The qualitative PCR kit for diagnosing the psoriatic acariasis and the PCR detection method for the psoriatic acariasis by using the kit have higher detection sensitivity than 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 infected cases with low-load parasitic infection, and can distinguish the existing infection from the emergent infection.

Description

PCR kit for diagnosing psoroptes and detection method

Technical Field

The invention belongs to the technical field of in-vitro diagnosis, and particularly relates to a PCR kit for diagnosing psoroptes and a detection method.

Background

The Psoroptes ovis is a common ectoparasite disease which is popular in the world and is parasitized on the skin surfaces of herbivorous animals and wild animals by Psoroptes ovis, clinically takes pruritus, scabbing, skin thickening and depilation as main clinical symptoms, causes great economic loss to the breeding industry and threatens the survival of the wild animals. The clinical symptoms of visible scab can appear 2 weeks and later after animals are artificially infected with P.ovis once, while the time for the clinical symptoms of visible scab to appear is longer when healthy animals in actual production are infected with P.ovis by contacting affected animals or vectors polluted by mites, and the period can be prolonged to 1 month or even several months.

In the existing production, the diagnostic means of the psoriatic mite is to confirm the diagnosis by observing the mites or mite eggs from scabs through a microscope, the detection rate of severe infection cases is high, but subclinical infection and slight infection cases cannot be detected because scabs are not visible to the naked eye, and the undetected cases become invisible infection sources of the psoriatic mite infection in animal groups, and finally cause the severe epidemic of the psoriatic mite in the animal groups. In addition, the microscopic diagnosis method requires a diagnostician to have a certain professional knowledge of parasites to identify the bodies or eggs, and thus, the microscopic etiology diagnosis method is time-consuming, requires a certain professional knowledge for the operator, and is missing for cases infected with low mite numbers.

In recent years, indirect ELISA diagnostic methods of a plurality of recombinant antigens have been proved to be applicable to the diagnosis of the cases of subclinical infection and stealth infection of the psoriatic mite, and the indirect ELISA method (rPsoSP 3-iELISA) using the recombinant serine protease inhibitor 3 has been reported to have the best diagnostic effect in serological diagnostic methods. Although the rpsop 3-iiisa can be used for early diagnosis of subclinical cases and cases of stealth infection by detecting specific IgG, the detection method cannot distinguish between an existing infection and a present infection due to the long period of IgG antibody regression.

Therefore, there is a need for a method for diagnosing pruritic mite, which has high sensitivity, high accuracy, and low operator requirements, and can discriminate the past infection from the present infection of pruritic mite.

Disclosure of Invention

Recently, molecular diagnostic methods based on PCR technology are used for detecting infection of parasites, viruses and bacteria, and research reports about the molecular marker gene of the itch mite in the prior art are mainly used for evaluating genetic studies of itch mite populations with different geographical sources and host sources, and the research reports about the molecular diagnostic method of the itch mite disease are not seen at present. In order to more efficiently diagnose the itch mite disease, the invention provides a PCR kit for diagnosing the itch mite disease and a PCR detection method for the itch mite disease of non-diagnosis purposes.

In a first aspect, the invention provides an application of ITS2, 16S, 12S and COX1 gene sequences of prunychus urticae in preparing a kit for diagnosing prunychus urticae.

Preferably, the gene sequence is ITS2, 12S.

Most preferably, the gene sequence is ITS2.

In a second aspect, the present invention provides a PCR kit for diagnosing psoroptes, wherein the kit comprises a specific primer, wherein the specific primer can be specifically combined with the ITS2, and/or 12S, and/or 16S, and/or COX1 gene sequence of psoroptes;

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

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

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

the sequence of the specific primer aiming at the COX1 gene is shown as SEQ ID NO. 7-SEQ ID NO. 8.

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

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

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

Preferably, the kit further comprises a negative control substance, and the negative control substance is normal saline.

In a third aspect, the invention provides a PCR detection method for non-diagnostic itch mite, 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 (A)	Volume of
		2×Taq PCR Master Mix	5μL
Specific primer	0.25-1μL
		Template DNA	0.5μL
Ultrapure water	3.5-4.25μL
		In total	10μL

(3) PCR amplification was performed, the procedure was as follows: performing 25-35 cycles of pre-denaturation at 95 deg.C for 3min, denaturation at 95 deg.C for 30s, annealing at 50-64 deg.C for 45s, and extension at 72 deg.C for 30s, maintaining at 72 deg.C for 5min, and storing at 4 deg.C;

(4) The PCR product was subjected to gel electrophoresis.

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

Preferably, the PCR amplification procedure in step (3) is: pre-denaturing at 95 deg.C for 3min, denaturing at 95 deg.C for 30s, annealing at 50 deg.C, 60 deg.C, and 64 deg.C, performing 25 cycles at 45s and extension at 72 deg.C for 30s, maintaining at 72 deg.C for 5min, and storing at 4 deg.C.

The sample in the step (1) is a dry swab taken from an animal to be detected, wherein the animal to be detected comprises domestic animals and wild animals. Method for collection of dry swabs: 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 centrifugal tube, and storing at-20 ℃.

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

1. the invention provides a thought for designing specific primers aiming at ITS2, 16S, 12S and COX1 gene sequences of prurus armeniaca to prepare a qualitative PCR kit or a fluorescent quantitative PCR kit, and the inventor designs and screens experiments to prove that the specific primers designed aiming at ITS2 and 12S genes have good specificity, and further, the sensitivity of the specific primers aiming at ITS2 is higher and can reach 40.3 pg/mu L (20 times of DNA dilution of 1 mite).

2. The qualitative PCR kit for diagnosing the psoriatic acariasis and the method for carrying out PCR detection on the psoriatic acariasis by using the kit have higher detection sensitivity than 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 infected cases with low-load parasitic infection, provide better guarantee for disease prevention and control, and can distinguish the existing infection from the existing infection.

Drawings

FIG. 1 12S, 16S, ITS2, COX1 gene PCR amplification, from left to right lane D2000DNA marker;12S;16S; ITS2; COX1; and (5) negative control.

FIG. 2 12S and ITS2 and 16S genes, D2000DNA marker from left to right; itch mite DNA; scabies mite DNA; rabbit skin tissue DNA; and (5) negative control.

FIG. 3 shows the sensitivity test of ITS2 and 12S genes, D2000DNA marker is shown in the left and right lanes; DNA of 10 itch mites; DNA of 5 itch mites; 1 itch mite DNA;1, diluting the DNA of the itch mite 1: 10; 1 itch mite DNA1:20 dilution; 1 itch mite DNA1:40 dilution; 1, diluting the DNA of the itch mite 1: 80; 1 itch mite DNA1:100 dilution; and (5) negative control.

FIG. 4 shows the optimization of PCR amplification procedure for ITS2 gene, D2000DNA marker in the left and right lanes; 25 circulation; 30 cycles; 35, circulating; negative control; d2000DNA marker;0.25 μ L;0.5 mu L;1 mu L of the solution; negative control; d2000DNA marker;50 ℃;55 ℃;60 ℃;65 ℃; and (5) negative control.

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

Detailed Description

The technical solutions in 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, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Primer design

ITS2, 12S, 16S, COX gene sequences of p.ovis were downloaded from GeneBank, primers were designed using Primer Premier 5.0 software, specificity was confirmed using Primer blast for the designed primers, and primers were designed for ITS2, 12S, 16S, COX gene sequences as follows:

PCR detection method for psoroptes

S1: taking off the crust in the external auditory canal of the itch mite disease breeding rabbit by using tweezers, putting the scurf into a culture dish, putting the culture dish in a 37 ℃ incubator for 30min, collecting the worm bodies which climb out from the scurf, putting the collected mites in a 1.5mL EP tube for preservation at the temperature of minus 20 ℃ after morphological identification and confirmation, and extracting the DNA of the itch mites according to the instruction of an OMEGA micro-genome DNA extraction kit (Chengdu Baisbobo biotechnology Co., ltd.);

s2: adding 0.5 mu L of each specific primer, 0.5 mu L of template DNA, 5 mu L of 2 XTaq PCR Master Mix and 3.5 mu L of ultrapure water into a PCR reaction tube, wherein the total amount is 10 mu L, and oscillating by using a vortex instrument to fully Mix the primers; here, 4 PCR reaction systems are set, and specific primers are (1) 0.5. Mu.L of SEQ ID NO:1+ 0.5. Mu.L of SEQ ID NO:2, (2) 0.5. Mu.L of SEQ ID NO:3+ 0.5. Mu.L of SEQ ID NO:4, (3) 0.5. Mu.L of SEQ ID NO:5+ 0.5. Mu.L of SEQ ID NO:6, (4) 0.5. Mu.L of SEQ ID NO:7+ 0.5. Mu.L of SEQ ID NO:8;

s3: PCR amplification is carried out by taking prurus DNA as a template, and the program is as follows:

ITS2 amplification conditions: performing 25 cycles of pre-denaturation at 95 deg.C for 3min, denaturation at 95 deg.C for 30s, annealing at 50 deg.C for 45s, and extension at 72 deg.C for 30s, maintaining at 72 deg.C for 5min, and storing at 4 deg.C;

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

16S amplification conditions: performing 25 cycles of pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 64 ℃ for 45s and extension at 72 ℃ for 30s, finally keeping at 72 ℃ for 5min, and storing at 4 ℃;

COX1 amplification conditions: performing 25 cycles of pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 64 ℃ for 45s and extension at 72 ℃ for 30s, finally keeping at 72 ℃ for 5min, and storing at 4 ℃; (ii) a

S4: an appropriate amount of the PCR amplification product was added to a gel block loading well containing a nucleic acid dye at an agarose concentration of 1.5% for electrophoresis, and observed using a gel imaging system, and the results are shown in FIG. 1.

The specific primers designed aiming at ITS2, 12S and 16S successfully amplify DNA extracted from the itch mite body through PCR, and the bands of PCR amplification products in gel are consistent with the expected sizes, so that the successful design of the ITS2, 12S and 16S primers is proved, but two bands are amplified by the COX1 specific binding primers and are not suitable for serving 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 an OMEGA minigenome DNA extraction kit (Chengdabangsbuba biotechnology limited), wherein the scabies mite and rabbit ear skin tissue are provided by the research center of parasitic diseases of Sichuan agriculture university;

s2: adding 0.5 mu L of each specific primer, 0.5 mu L of template DNA, 5 mu L of 2 XTaq PCR Master Mix and 3.5 mu L of ultrapure water into a PCR reaction tube, wherein the total amount is 10 mu L, and oscillating by using a vortex instrument to fully Mix the primers; a total of 4 PCR reaction systems are set, and the specific primers are (1) ' 0.5 μ L SEQ ID NO:1+0.5 μ L SEQ ID NO:2, (2) ' 0.5 μ L SEQ ID NO:3+0.5 μ L SEQ ID NO:4, (3) ' 0.5 μ L SEQ ID NO:5+0.5 μ L SEQ ID NO:6;

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

s4: an appropriate amount of the PCR amplification product was added to a gel block loading well containing a nucleic acid dye at an agarose concentration of 1.5% for electrophoresis, and observed using a gel imaging system, and the results are shown in FIG. 2.

No amplification products of ITS2 and 12S genes are detected in DNA of the sarcoptidosis and rabbit skin tissue samples, but 16S amplification products appear after the DNA of the rabbit skin tissue samples is amplified. PCR result analysis shows that the specificity of the primers of ITS2 and 12S is better, and the cross reaction (false positive) of the itch mite with the DNA of scabies mite and rabbit skin tissue samples can not occur.

Sensitivity testing of primers

S1: extracting prurus DNA according to the instruction of the OMEGA minigenome DNA extraction kit (venter baisbane biotechnology limited), setting the DNA concentration gradient: extracting DNA of 10 itch mites, 5 itch mites and 1 itch mite, and diluting the DNA of 1 itch mite into a sequence of 1;

s2: adding 0.5 mul of each specific upstream primer and downstream primer, 0.5 mul of template DNA, 5 mul of 2 XTaq PCR Master Mix and 3.5 mul of ultrapure water into a PCR reaction tube, wherein the total amount is 10 mul, and fully and uniformly mixing the primers by using a vortex instrument; 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 DNA of the prurus armeniaca with different concentrations set in the S1 as templates respectively, and the program is as follows:

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

s4: an appropriate amount of the PCR amplification product was added to a gel loading well containing a nucleic acid dye at an agarose concentration of 1.5% for electrophoresis, and the gel imaging system was used for observation, and the results are shown in FIG. 3.

Comparing the band brightness under 8 different DNA concentrations after electrophoresis, the result shows that the 12S specific primer can reach the sensitivity (0.403 ng/. Mu.L) of diluting the DNA of 1 itch mite by 10 times, while the ITS2 specific primer has high sensitivity to diluting the DNA of 1 itch mite by 20 times (40.3 pg/. Mu.L), so the ITS2 is more suitable for being used as a molecular target to carry out PCR diagnosis, and then the ITS2 purpose band gel is sent to sequence to be compared with the itch mite ITS2 sequence reported in a Genebank database, and the similarity between the sequence obtained by amplification and a reference sequence is 98.9%.

Optimization of PCR amplification procedure

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

The PCR detection method comprises the following steps:

s1: extracting prurus DNA according to the instruction of the OMEGA minigenome DNA extraction kit (chengdabangsbuba biotechnology limited);

s2: adding specific primers ITS2-F + ITS2-R, template DNA 0.5 muL, 2 XTaq PCR Master Mix 5 muL and ultrapure water 3.5-4.25 muL into a PCR reaction tube, wherein the total volume is 10 muL, and oscillating by using a vortex instrument to fully and uniformly Mix the primers;

s3: carrying out PCR amplification by using prurus armeniaca DNA as a template, wherein the program comprises the following steps: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 45s, extension at 72 ℃ for 30s, performing a certain number of cycles, keeping at 72 ℃ for 5min, and storing at 4 ℃;

s4: an appropriate amount of the PCR amplification product was added to a gel block loading well containing a nucleic acid dye at an agarose concentration of 1.5% for electrophoresis, and observed using a gel imaging system, and the results are shown in FIG. 4.

The variables are set as follows:

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

2. cycling from denaturation to extension, wherein the number of cycles is set to 25 times, 30 times and 35 times respectively;

3. the annealing temperatures were 50 ℃, 55 ℃ and 60 ℃.

The PCR program was optimized by the above variables, 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, microscopic examination and ELISA comparison

1. Establishment of New Zealand rabbit itch mite disease artificial infection model and sample collection

Taking off the scab skin in the auditory meatus of the sick rabbit by using tweezers, putting the skin scraps into a culture dish, placing the culture dish in a 37 ℃ incubator for 2-3h, collecting the itch mite in the skin scraps, subpackaging 100 itch mite bodies into a 1.5mL centrifuge tube, and collecting enough itch mite bodies according to the amount of 100 infected itch mite bodies in one rabbit ear.

Rabbits to be infected (n = 9) were kept and their ear canals were plugged with cotton. And directly pouring the collected prurus armeniaca into ear pits of rabbit ears, sealing the ears with 5 x 7cm of medical dressing, and leaving a small part of gaps. The lower part of the rabbit ear is wound by using an adhesive tape (ensuring the blood circulation of the rabbit ear to be normal), and then an Elizabeth ring is worn on the rabbit to prevent the rabbit from scratching. Observing the rabbit after artificial infection, releasing the adhesive plaster after 24h, taking out the dressing and the plug after 2 days, taking off the Elizabeth ring after 3 days, and waiting for the rabbit to continuously infect the itch mite. Rabbits were treated with the drug 6 weeks post-infection and weighed to be injected subcutaneously with ivermectin in an amount of 0.1mL/kg.w, only once.

And (4) extracting a sample to be tested, including collection of a dry swab and serum.

And (3) collecting dry swabs, namely repeatedly wiping the infected 9 rabbits at the ear pits of the rabbits for 3-5 times by using a cotton swab after the infected 9 rabbits are well preserved, putting the cotton swab into a centrifugal tube, collecting 2 samples in total, marking the tube wall, storing 1 sample at normal temperature for traditional microscope detection, and storing the other 1 sample at-20 ℃ for PCR detection.

Collecting serum, collecting 3mL blood from ear vein of 9 infected rabbits, placing in 37 deg.C incubator for 30min, placing in 4 deg.C refrigerator for 3h, centrifuging supernatant and centrifuge tube at 3000g for 10min, and storing at-20 deg.C for ELISA detection.

The sampling time was 1, 2, 4, 6 weeks after infection and 3, 7, 11 days after treatment.

2. PCR detection

Extracting DNA of the collected dry swab by using an OMEGA micro-genome DNA extraction kit, detecting the stored dry swab DNA by using an optimized and established PCR method, and judging that a strip with an expected size is positive after gel electrophoresis.

3. Conventional microscopy

Placing the dry swab sample preserved at normal temperature into a culture dish, placing the culture dish in a 37 ℃ incubator for 30min, taking out the dry swab sample, placing the dry swab sample into a large glass plate, and cushioning the large glass plate on white paper, picking the live insect body on a glass slide, observing the live insect body by using a microscope, and judging the shape of the obvious itch mite body to be positive.

4. Detection of rPsoSP3-iELISA

The collected serum is detected by an rPsoSP3-iELISA method screened at the earlier stage of a subject group, and the basic steps are to take a 96-well enzyme label plate for antigen coating, dilute the rPsoSP3 to 35.5 mu g/mL by using a coating buffer solution, add 100 mu L of sample to each well of the 96-well plate, and coat overnight at 4 ℃. After overnight, the wells were rinsed by draining the liquid, washing each well with 200. Mu.L PBST, and washing with shaking. After completion, 300. Mu.L of blocking solution was added to each well and incubated at 37 ℃ for 1-2h. After washing, 100. Mu.L of the serum to be tested diluted with PBS to 1:40 was added to each well and incubated at 37 ℃ for 1 hour. 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. And finally washing for color development, adding 100 mu L of TMB color development liquid into each concave hole, and standing for 20min in a dark place. Adding 100 μ L of stop solution, and measuring OD450 with enzyme-labeled color developing instrument, wherein the result is positive when cut-off value exceeds 0.401, and the result is negative otherwise.

All rabbits in this trial were successfully infected with prurus mites, and samples taken at 1, 2, 4, 6 weeks of infection and 3, 7, 11 days after treatment were examined by conventional microscopy, PCR and iELISA, with results shown in FIG. 5.

At week 1, the traditional microscopy revealed only 33.3% (3/9) of positive rates, reaching 100% (9/9) at week 2. The positive rates of the microscopic examination were reduced to 66.6% (6/9) and 11.1% (1/9) at 3 days after the treatment and 7 days after the treatment, respectively, and the positivity at 7 days after the treatment was due to the microscopic examination of the dead and dead bodies in the ear of rabbit No. 9.

The positivity of the iELISA at week 1 was 77.7% (7/9), and the OD450 values were greater than cut-off value 0.401 from week 2 to day 11 after treatment, and were generally stable after rising.

As a result of the PCR diagnosis, the positive rate reached 88.9% (8/9) at

week

1 and 100% (9/9) at week 2. The positive rates of 3 days after treatment and 7 days after treatment are 66.6% and 0% respectively, but the condition of yang recovery of No. 8 rabbit is found on 11 days after treatment, but the condition of yang recovery is not detected by microscopic examination and iELISA, and a small amount of dead insect bodies or debris may remain in No. 8 rabbit after treatment, so that the residual itch mite bodies or debris can not be collected every time when the dry swab is sampled, and subsequent yang recovery is caused, therefore, the sampling of the dry swab can be carried out for multiple times, and then the dry swab is mixed for PCR, so that the detection sensitivity is improved.

By combining the experimental result, the positive detection rate of the 1 st week PCR detection method is 88.9 percent and is obviously higher than the detection rate of 33.3 percent of microscopic examination, which shows that the PCR detection sensitivity is higher than that of microscopic examination in the diagnosis of the psoriatic acariasis, so that early infected rabbits can be discovered more early and more comprehensively, and a better guarantee is provided for the prevention and control of the psoriatic acariasis.

Compared with ELISA, PCR has similar sensitivity, but does not have complicated steps for preparing antigen protein, and has good specificity, thereby avoiding the occurrence of false positive, most importantly, due to the long-term existence of the antibody in the host body, the ELISA can not distinguish the existing infection from the existing infection, and the PCR can make up for the defect.

Practical application of PCR in rabbit farm

36 dry swab samples collected from a rabbit field with a history of the scrapie epidemic disease are subjected to traditional microscope and PCR detection, wherein 4 positive samples are detected by a microscope, the detection rate is 11.1%, the PCR detection of the 4 positive samples is positive, but 2 positive samples are detected by 2 negative samples, 6 positive samples are detected in the 36 samples, the detection rate is 16.7%, and the results are shown in the following table.

TABLE 1

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of ITS2, 16S, 12S and COX1 gene sequences of the itch mite in preparing a kit for diagnosing the itch mite disease.

2. The use according to claim 1, wherein the gene sequence is ITS2, 12S.

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

4. A PCR kit for diagnosing itch mite disease, wherein said kit comprises a specific primer, said specific primer can be specifically combined with ITS2, and/or 12S, and/or 16S, and/or COX1 gene sequence of itch mite;

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

5. The kit according to claim 4, wherein the specific primer in the kit is a specific primer sequence for ITS2 gene and/or a specific primer sequence for 12S gene.

6. The kit according to claim 5, wherein the specific primer in the kit is a specific primer sequence for ITS2 gene.

7. The kit according to claim 4, wherein the kit further comprises conventional reagents used in DNA extraction process, and/or conventional reagents used in PCR amplification reaction, and/or conventional reagents used in detection of PCR amplification product.

8. A PCR detection method for non-diagnostic itch mite is characterized by comprising the following steps:

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

(4) The PCR product was subjected to gel electrophoresis.

9. The detection method according to claim 8, wherein the specific primer is added in a volume of 1. Mu.L in the step (2), and the upstream primer and the downstream primer are each added in a volume of 0.5. Mu.L.

10. The detection method according to claim 8, wherein the PCR amplification procedure in step (3) is as follows: pre-denaturing at 95 deg.C for 3min, denaturing at 95 deg.C for 30s, annealing at 50 deg.C, 60 deg.C, and 64 deg.C, performing 25 cycles at 45s and extension at 72 deg.C for 30s, maintaining at 72 deg.C for 5min, and storing at 4 deg.C.