A primer-probe combination for detecting the honey bee thermolae mite and a detection method thereof

Abstract

The present invention discloses a primer-probe composition for detecting the heat laelaps of honey bees and a detection method thereof. Specifically disclosed is a primer pair for detecting the heat laelaps of honey bees, the primer pair being a pair of primers for specifically amplifying the COX1 gene or containing the COX1 gene fragment, and the nucleotide sequence of the COX1 gene being SEQ ID No.1. The present invention utilizes multiple strains of heat laelaps of honey bees separated from domestic popular bee colonies for DNA amplification and sequence analysis comparison, screens the conservative nucleic acid sequence as the target, and develops a primer-probe composition for detecting the heat laelaps of honey bees and a real-time fluorescence PCR detection method. The primer-probe composition and the detection method of the present invention have high sensitivity, a minimum detection limit of 2.0 copies / μL, and good specificity, and have no cross reaction with other bee mites, and can be used for the detection of the heat laelaps of honey bees and the identification of the heat laelaps of honey bees from other bee mites, as well as the quarantine of imported and exported bees and bee products.

Description

Technical Field

[0001] The invention relates to the technical field of animal health, and in particular to a primer-probe composition for detecting the honey bee thermolaelaps and a detection method thereof. Background Art

[0002] Tropilelaps mercedesae is a species of bee mite, belonging to the phylum Arthropoda, class Arachnida, subclass Acari, order Parasitoides, order Mesostigmata, superfamily Dermatophoroidea. It belongs to the genus Tropilelaps (Tropilaelaps spp.) together with Tropilelaps koenigerum, Tropilelaps clareae, and Tropilelaps thaii, collectively known as bee mites. Tropilelaps mercedesae is the main parasitic mite of western honey bees. It can be found in western honey bees in its original distribution area, with a parasitic level of up to 90%. Its distribution range is wider than that of Tropilelaps clareae, and it is more harmful. It has developed into a long-term harmful parasite of western honey bees.

[0003] Mei's heat laelaps feed on the hemolymph of bees' caps, often causing a large number of capped larvae and pupae to deform or die. Worker bees that barely leave the hive also have body deformities and physical and physiological defects, causing a serious decline in the productivity of the bee colony, directly affecting the occurrence and development of the bee colony. If the diseased colony is not controlled, the bee colony will soon die. At present, the harmfulness of the small bee mite has attracted widespread attention from the beekeeping industry, researchers, and beekeepers in various countries. At present, the detection of Mei's heat laelaps mainly adopts morphological methods, combined with clinical symptoms, through lid opening inspection and box bottom inspection. Since there is no report of damage caused by Mei's heat laelaps in Europe so far, and the small Varroa mite only causes damage to honey bee colonies in Asia, it did not attract the attention of Western countries in the early stage. There are few studies on this type of mite at home and abroad. There is no morphological description and detailed report on the life history of each stage (egg, larvae, nymph and adult) of Varroa mite, and there is a lack of typical morphological identification characteristics of this mite. In addition, there are more than 100 types of mites related to honey bees in bee colonies, and they are similar in morphology. Therefore, it is impossible to accurately identify them only by morphological examination, and the actual detection effect also depends on the professional level and experience of the detection personnel, which is easy to cause missed detection and wrong detection. At present, there is no report on the real-time fluorescent PCR detection method of Mei's heat laelaps.

[0004] In view of this, researching and developing primer-probe compositions and detection methods for detecting Pyrolae mites in honey bees, which can be used for the detection of Pyrolae mites in honey bees and their identification from other bee mites, as well as the quarantine of imported and exported honey bees and bee products, are of great significance for ensuring the development of my country's beekeeping industry, biosafety and maintaining the natural ecological balance. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to detect the honey bee Tropilaelaps mercedesae simply, quickly, sensitively and / or specifically. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0006] In order to solve the above technical problems, the present invention first provides a primer pair for detecting the honey bee Thermoelaax mites, wherein the primer pair can be a pair of primers for specifically amplifying the mitochondrial cytochrome c oxidase subunit I (cytochrome coxidase subunit I, COX1) gene of the honey bee Thermoelaax mites or a pair of primers containing the COX1 gene fragment, and the nucleotide sequence of the COX1 gene can be SEQ ID No.1.

[0007] Furthermore, the primer pair consists of primer TME-F and primer TME-R, the primer TME-F may be a single-stranded DNA molecule shown in SEQ ID No.2; the primer TME-R may be a single-stranded DNA molecule shown in SEQ ID No.3.

[0008] The primer TME-F is a forward primer, and the primer TME-R is a reverse primer.

[0009] The forward primer TME-F and the reverse primer TME-R are a primer pair for specifically amplifying the COX1 gene of the honey bee Tropilelaps mercedesae, that is, a specific primer pair for detecting the honey bee Tropilelaps mercedesae.

[0010] The present invention also provides a composition (primer-probe composition) for detecting Pseudomonas aeruginosa, which may be composed of the primer pair and a Pseudomonas aeruginosa-specific probe, and the Pseudomonas aeruginosa-specific probe may be a single-stranded DNA molecule that specifically binds to the COX1 gene.

[0011] The probe may be a TaqMan probe named TME-P.

[0012] In the above composition, the nucleotide sequence of the probe TME-P may be SEQ ID No.4.

[0013] In the above composition, the 5' end of the probe TME-P can be labeled with a fluorescent group, and the 3' end can be labeled with a quenching group.

[0014] The fluorescent group is selected from at least one of FAM, 6-FAM, VIC, HEX, TRT, CY3, CY5, ROX, JOE, FITC, TET, NED, TAMRA, LC RED640, LC RED705, Quasar705 and Texas Red.

[0015] The quenching group is selected from at least one of TAMRA, BHQ1, BHQ2, BHQ3, MGB, and Dabcy1.

[0016] In the above composition, the 5' end of the probe may be labeled with ROX, and the 3' end may be labeled with BHQ-2 (BHQ2).

[0017] The present invention also provides a reagent or a kit for detecting Thermoelaea merschmidtii of honey bees, wherein the reagent or the kit comprises the primer pair or any composition described herein.

[0018] Furthermore, the kit also includes Taq DNA polymerase, dNTP, PCR buffer, Mg 2+ One or more of .

[0019] The various reagent components of the kit may be present in separate containers, or may be pre-combined in whole or in part into a reagent mixture.

[0020] Further, the kit may also include a readable carrier recording the method for detecting the honey bee Thermoclase mites described herein. The readable carrier may be a kit instruction sheet (e.g., a printed instruction sheet) for practicing the method of the present invention or a computer-readable medium (e.g., a floppy disk, a CD, etc.) on which information has been recorded.

[0021] The present invention also provides any of the following uses of the primer pair, and / or any of the compositions described herein:

[0022] A1) Use in detecting the heat lacerta mites of honey bees or in preparing a product for detecting the heat lacerta mites of honey bees;

[0023] A2) Use in detecting COX1 gene or preparing a product for detecting COX1 gene;

[0024] A3) Application in identifying or assisting in identifying the Pyrolae mites of honey bees;

[0025] A4) Use of the invention in the preparation of a product for identifying or assisting in identifying the Pyrolae mites of honey bees;

[0026] A5) Use of the method for identifying and distinguishing the honey bee's Pyrolae mites from other honey bee mites or for preparing a product for identifying and distinguishing the honey bee's Pyrolae mites from other honey bee mites;

[0027] A6) Application in the control of honey bee pyrolyticus mites.

[0028] The product described herein may be a reagent, a kit, a chip or a test paper.

[0029] The present invention also provides a method for detecting the honey bee's Pyrolae mites for non-disease diagnosis purposes, which may include using the primer pair or any of the compositions described herein to perform a PCR amplification reaction on a sample to be tested, and determining whether the sample to be tested contains the honey bee's Pyrolae mites or whether the sample to be tested is the honey bee's Pyrolae mites based on the PCR amplification product.

[0030] Furthermore, in the above method, the PCR amplification reaction may be a real-time fluorescence quantitative PCR reaction.

[0031] Furthermore, in the above method, the real-time fluorescence quantitative PCR reaction system includes: PCR buffer, MgCl2, dNTPs, primer TME-F, primer TME-R, probe TME-P, DNA polymerase, sample DNA to be tested, and ddH2O.

[0032] Furthermore, in the above reaction system, the molar ratio of primer TME-F to primer TME-R is 1:1.

[0033] Furthermore, in the above reaction system, the molar ratio of primer TME-F, primer TME-R and probe TME-P is 1:1:3.

[0034] Furthermore, in the above reaction system, the final concentration of primer TME-F and primer TME-R is 0.1 μmol / L.

[0035] Furthermore, in the above reaction system, the final concentration of the probe TME-P was 0.3 μmol / L.

[0036] Furthermore, the real-time fluorescence quantitative PCR reaction system can be: 2×PCR buffer 10μL, 25mmol / LMgCl2 2.0μL, 2.5mmol / L dNTPs 1.5μL, 10μmol / L primer TME-F 0.2μL, 10μmol / L primer TME-R0.2μL, 10μmol / L probe TME-P 0.6μL, 5U / μL DNA polymerase 0.5μL, test sample DNA 2.0μL, ddH2O3.0μL.

[0037] The real-time fluorescence PCR reaction procedure may be: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing at 59°C for 30 seconds, a total of 40 cycles, and single-point fluorescence detection at 59°C.

[0038] Furthermore, the sample to be tested may be bee mites, bees, or bee products such as honey and pollen.

[0039] In the above method, the method for determining whether the sample to be tested contains the honey bee's Thermoelaeae mites or whether the sample to be tested is the honey bee's Thermoelaeae mites according to the PCR amplification product can be any of the following:

[0040] B1) determining whether the sample to be tested contains the honeybee thermolaelaps or is the honeybee thermolaelaps according to whether the PCR amplification product contains a DNA fragment of 171 bp in size: if the PCR amplification product contains a DNA fragment of 171 bp in size, the sample to be tested contains the honeybee thermolaelaps or is the honeybee thermolaelaps; if the PCR amplification product does not contain a DNA fragment of 171 bp in size, the sample to be tested does not contain the honeybee thermolaelaps or is not the honeybee thermolaelaps;

[0041] B2) determining whether the sample to be tested contains the honey bee Meissler mite or is the honey bee Meissler mite according to the amplification curve and / or the melting curve of the PCR amplification product: if the amplification curve is an S-shaped curve, it is determined that the sample to be tested contains the honey bee Meissler mite or is the honey bee Meissler mite; if the amplification curve is not an S-shaped curve, it is determined that the sample to be tested does not contain the honey bee Meissler mite or is not the honey bee Meissler mite; if the melting curve is a single peak curve, it is determined that the sample to be tested contains the honey bee Meissler mite or is the honey bee Meissler mite; if the melting curve is not a single peak curve or has no peak, it is determined that the sample to be tested does not contain the honey bee Meissler mite or is not the honey bee Meissler mite;

[0042] B3) determining whether the sample to be tested contains the honey bee Thermoelaax mites or is the honey bee Thermoelaax mites according to the Ct value of the PCR amplification product: if the Ct value is ≤35, it is determined that the sample to be tested contains the honey bee Thermoelaax mites or is the honey bee Thermoelaax mites; if the Ct value is >35, it is determined that the sample to be tested does not contain the honey bee Thermoelaax mites or is not the honey bee Thermoelaax mites.

[0043] The purposes of the above-mentioned applications and methods may be disease diagnosis, disease prognosis and / or disease treatment, and their purposes may also be non-disease diagnosis, non-disease prognosis and non-disease treatment purposes; their direct purpose may be to obtain information on intermediate results of disease diagnosis results, disease prognosis results and / or disease treatment results, and their direct purpose may be non-disease diagnosis, non-disease prognosis and / or non-disease treatment purposes.

[0044] The sample to be tested in the above method may be a sample from a non-living human or animal body, such as a beehive environment sample, an insect sample or a bee product sample.

[0045] The above method can be used for the classification and identification of Thermoelaea merschmidtii.

[0046] The present invention utilizes multiple strains of Melson's heat laelaps isolated from domestic popular bee colonies to perform DNA amplification and sequence analysis comparison, screens conservative nucleic acid sequences as targets, designs and screens optimal primers and probes, optimizes reaction systems and reaction conditions, and obtains a primer-probe combination and a detection method for detecting Melson's heat laelaps of honey bees through a series of creative work. The combination is used for the detection of Melson's heat laelaps of honey bees for non-diagnostic purposes and their identification from other bee mites, as well as the quarantine of imported and exported honey bees and bee products, and is of great significance for ensuring the development of my country's beekeeping industry, biosafety, and maintaining the balance of natural ecology.

[0047] The technical solution of the present invention has the following advantages:

[0048] (1) Good specificity and stability: The present invention screens the conserved sequence fragment of the honey bee Thermoelaeae merantii as the target through a large number of DNA sequencing and sequence comparison analysis; the best primer is selected after comparing multiple sets of primers, and a specific fluorescent probe is designed and screened, which can perform dual control on the target during the real-time fluorescent PCR reaction process, and has a very strong detection specificity for the honey bee Thermoelaeae merantii, and has no cross reaction with other bee mites; compared with the recombinase polymerase isothermal nucleic acid amplification technology, the real-time fluorescent PCR primer has a short length requirement, is not easy to produce primer dimers, has a better amplification effect, and has good repeatability;

[0049] (2) High sensitivity: The present invention uses TaqMan-BHQ-2 probe, which has higher sensitivity because BHQ-2 itself does not produce fluorescence and has a low fluorescence background. The sensitivity is further improved by optimizing the reaction system and reaction conditions multiple times, reaching about 2.0 copies / μL.

[0050] (3) Simple and fast operation: Real-time fluorescence data collection, no need for electrophoresis to detect nucleic acid amplification, the entire reaction can be completed within 1 hour. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is a diagram showing the optimization results of the real-time fluorescence PCR annealing temperature (where only the annealing temperature of 59° C. has the smallest Ct value and the highest fluorescence intensity).

[0052] Figure 2 This is a diagram of the optimization results of the real-time fluorescence PCR reaction system (in which, the Ct value, fluorescence intensity and amplification curve morphology are most ideal only when the final concentration of TME-F / R in the reaction system is 0.1 μmol / L and the final concentration of TME-P is 0.3 μmol / L).

[0053] Figure 3This is a sensitivity detection diagram (wherein, when the positive clone plasmid is 2.0 copies / μL, it can still be amplified to obtain the corresponding curve). Figure 3 1 to 9 represent the concentration of plasmid DNA standard, which is 2×10 8 Copies / μL~2×10 0 copies / μL.

[0054] Figure 4 is a standard curve (wherein the standard curve shows that the amplification efficiency E is 99.1%, the determination coefficient R2 is 0.999, and the standard curve Y=-3.344x+33.751).

[0055] Figure 5 This is a graph for determining the Ct value (except for the positive control, the 4 replicates at 2.0 copies / μL all showed amplification curves, the average amplification Ct value was 35.0225, and the 4 replicates at 1.0 copies / μL and 0.5 copies / μL did not show amplification curves).

[0056] Figure 6 The specificity test result diagram is shown in Figure 2. Figure 6 The amplification curve in the figure is the amplification curve of Thermoelaeae mellifera of honey bees (only Thermoelaeae mellifera of honey bees has an amplification curve, while other honey bee mites do not have an amplification curve).

[0057] Figure 7 This is a graph of clinical sample testing results. DETAILED DESCRIPTION

[0058] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0059] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0060] Example 1. Design of primers and probes

[0061] In this embodiment, multiple strains of Thermo laelaps mei'sii isolated from popular bee colonies in China were used for DNA amplification and sequence analysis and comparison, and conservative nucleic acid sequences were screened as targets. The highly conserved region screened was the COX1 gene, and the nucleotide sequence of the COX1 gene was SEQ ID No.1. The present invention uses a recombinant plasmid (plasmid pUC57-TME) carrying the COX1 gene as a test sample (i.e., a plasmid standard). The recombinant plasmid pUC57-TME is a recombinant vector obtained by replacing the fragment (small fragment) between the NruI and ApaI restriction endonuclease recognition sites of the pUC57 vector with the DNA fragment shown in SEQ ID No.1 in the sequence table, while keeping the other nucleotide sequences of the pUC57 vector unchanged.

[0062] A specific primer pair and probe for detecting the Thermo laelaps meisenii were designed for the highly conserved region COX1 gene (SEQ ID No.1) of the screened Thermo laelaps meisenii. The designed primer pair (TME-F / R) consists of primer TME-F and primer TME-R, wherein the primer TME-F can be a single-stranded DNA molecule as shown in SEQ ID No.2; the primer TME-R can be a single-stranded DNA molecule as shown in SEQ ID No.3, and the primer pair can be used to specifically amplify the COX1 gene (SEQ ID No.1). The designed probe for specifically recognizing the COX1 gene is a TaqMan probe named TME-P, the nucleotide sequence of the probe TME-P is SEQ ID No.4, the 5' end of the probe TME-P is labeled with ROX, and the 3' end is labeled with BHQ-2 (BHQ2).

[0063] Example 2: Optimization of real-time fluorescence PCR annealing temperature

[0064] The real-time fluorescence PCR reaction program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing temperature set to 55-65°C (8 gradient temperatures, i.e., annealing temperatures of 55°C, 55.7°C, 57°C, 59°C, 61.4°C, 63.3°C, 64.5°C, and 65°C), annealing extension for 30 s, 3 repetitions for each gradient temperature setting, for a total of 40 cycles;

[0065] The real-time fluorescence PCR reaction system was: 2×PCR buffer 10μL, 25mmol / L MgCl2 2.0μL, 2.5mmol / L dNTPs 1.5μL, 10μmol / L upstream and downstream primers 0.8μL each, 10μmol / L probe 0.8μL, 5U / μL DNA polymerase 0.5μL, and 2.0μL of the sample DNA to be tested. Then, ddH2O 1.6μL was added to make the total reaction volume 20.0μL.

[0066] The annealing temperature of real-time fluorescence PCR detection of honey bee thermolaelaps was optimized according to the above method. Figure 1 described.

[0067] The results showed that when the annealing temperature was 59°C, the Ct value was the smallest and the fluorescence intensity was high. On the basis of maintaining the amplification efficiency, a higher annealing temperature was selected to improve the specificity, so the optimal annealing temperature was selected as 59°C.

[0068] Example 3: Optimization of real-time fluorescence PCR reaction system

[0069] According to the lowest CT value, higher fluorescence intensity increase value and amplification curve morphology and the principle of economy, the real-time fluorescence PCR reaction system was optimized according to the reaction procedure optimized in Example 2. The amounts of other reagents in the reaction system remained unchanged, and the final concentrations of upstream and downstream primers (0.1 μmol / L to 1.0 μmol / L) and the final concentration of the probe (0.1 μmol / L to 0.5 μmol / L) were optimized and selected by using a square array ratio test.

[0070] The reaction system of real-time fluorescence PCR detection of honey bee Thermoelaeae merantii was optimized according to the above method. Figure 2 described.

[0071] The results showed that when the content of TME-F / R (10 μmol / L) in the reaction system was 0.2 μL (final concentration was 0.1 μmol / L) and the content of TME-P (10 μmol / L) was 0.6 μL (final concentration was 0.3 μmol / L), the Ct value, fluorescence intensity and amplification curve morphology could all reach an ideal state. Therefore, the real-time fluorescence PCR reaction system was selected as follows: 2×PCR buffer 10 μL, 25 mmol / LMgCl2 2.0 μL, 2.5 mmol / L dNTPs 1.5 μL, 10 μmol / L upstream and downstream primers 0.2 μL each, 10 μmol / L probe 0.6 μL, 5 U / μL DNA polymerase 0.5 μL, 2.0 μL of the sample DNA to be tested, and then 3.0 μL of ddH2O was added.

[0072] Example 4: Establishment of real-time fluorescence PCR detection method

[0073] 1. Preparation of plasmid standards

[0074] A plasmid standard was prepared according to the highly conserved region COX1 gene (SEQ ID No. 1) screened in Example 1. The construction method was as follows: DNA of Thermoelaea mertensii was used as a template, and PCR amplification was performed according to the self-designed primers TME-F / R, the reaction system: 2×Taq PCR mix 12.5 μL, upstream and downstream primers TME-F / R 1.0 μL each, template DNA 2.0 μL, ddH2O supplemented to 25 μL; reaction conditions: 94°C pre-denaturation for 5 min; 94°C denaturation for 45 s, 55°C annealing for 30 s, 72°C extension for 45 s, a total of 35 cycles; 72°C for 10 min. After the reaction, the product was identified by 2.0% agarose gel electrophoresis, and the target fragment was recovered by a DNA gel recovery kit, cloned into the pUC57 vector, transformed into DH5α, cloned into LB liquid culture medium containing ampicillin, cultured at 37°C for 8h, and the recombinant plasmid was extracted by a plasmid extraction kit and sequenced. The recombinant plasmid with the correct sequencing result was used as the positive standard.

[0075] The constructed plasmid standard is named pUC57-TME. The recombinant plasmid pUC57-TME is a recombinant vector obtained by replacing the fragment (small fragment) between the NruI and ApaI restriction endonuclease recognition sites of the pUC57 vector (purchased from Fuzhou Boshang Biotechnology Co., Ltd.) with the DNA fragment shown in SEQ ID No. 1 in the sequence table, while keeping the other nucleotide sequences of the pUC57 vector unchanged.

[0076] 2. Extract DNA from samples to be tested

[0077] The genomic DNA of the sample to be tested was extracted using a genomic DNA extraction kit (blood / cell / tissue genomic DNA extraction kit, Tiangen Biochemical Technology Co., Ltd.), and the extraction method was based on the kit instructions.

[0078] 3. Real-time fluorescence PCR detection method

[0079] Using the DNA extracted in step 2 as a template, the primers TME-F, TME-R and probe TME-P designed in Example 1 were used to perform real-time fluorescent PCR amplification reaction on the sample to be tested, wherein:

[0080] The real-time fluorescence PCR reaction system was: 2×PCR buffer 10μL, 25mmol / L MgCl2 2.0μL, 2.5mmol / L dNTPs 1.5μL, 10μmol / L upstream and downstream primers 0.2μL each, 10μmol / L probe 0.6μL, 5U / μL DNA polymerase 0.5μL, sample DNA 2.0μL, and then ddH2O 3.0μL was added.

[0081] The real-time fluorescence PCR reaction program was: 95°C pre-denaturation for 5 min; 95°C denaturation for 15 s, 59°C annealing for 30 s, for a total of 40 cycles. Single-point fluorescence detection was performed at 59°C.

[0082] The negative control was sterile water.

[0083] The positive control was plasmid pUC57-TME.

[0084] 4. Result determination

[0085] Determine whether the sample to be tested contains or is a honey bee thermolaelaps mite based on the PCR amplification product in step 3. The specific criteria for determining the test results are:

[0086] Under the condition that the experiment is valid, if the sample to be tested has no Ct value and no amplification curve, it means that the sample does not contain the honey bee Meissori Thermolae mite; if the sample to be tested has a Ct value ≤35 and a typical amplification curve appears, it means that the sample contains the honey bee Meissori Thermolae mite; if the sample to be tested has a Ct value >35, the sample is tested repeatedly, and if the repeated test results have no Ct value or the Ct value is >35, the sample does not contain the honey bee Meissori Thermolae mite; if the repeated test results have a Ct value and the Ct value is ≤35, the sample contains the honey bee Meissori Thermolae mite;

[0087] The experiment is effective when: the negative control has no Ct value and no amplification curve, while the positive control Ct value is ≤35 and a typical amplification curve appears, indicating that the experiment is an effective experiment.

[0088] Example 5: Sensitivity experiment

[0089] The positive clone plasmid containing the COX1 gene of the honey bee thermolaelaps (i.e., the plasmid standard prepared in step 1 of Example 4) was diluted 10 times in increments to 2×10 8 Copies / μL~2×10 0 Plasmid DNA standards at copies / μL concentration.

[0090] The diluted plasmid DNA standard was used as a template, and each standard sample was treated 3 times, and healthy bee tissue DNA was used as a control, and sterile water was used as a blank control. Real-time fluorescence PCR detection was performed according to the detection method in step 3 of Example 4. Figure 3 The amplification curves shown were used to test the sensitivity and Figure 4 The standard curve of the honey bee Thermoelaea mellifera.

[0091] from Figure 3 It can be seen that the method can detect a minimum of about 2.0 copies / μL of the honey bee Thermoelaea mertensii DNA template. Figure 4 The regression equation of the constructed standard curve is Y = -3.344x + 33.751; R represents the correlation coefficient, and the determination coefficient R2 The sensitivity of the real-time fluorescence PCR detection method is 0.999 and the amplification efficiency is 99.1%.

[0092] Example 6. Determination of Ct value

[0093] Using 2.0 copies / μL standard plasmid (i.e., the plasmid standard prepared in step 1 of Example 4) as the starting concentration, serial 2-fold dilutions were performed to obtain 3 dilutions of 2.0 copies / μL, 1.0 copies / μL, and 0.5 copies / μL. Using the plasmid standard with these 3 dilutions as templates, real-time fluorescence PCR detection was performed according to the detection method of step 3 of Example 4. The detection results are shown in FIG. Figure 5 shown.

[0094] Figure 5 The results showed that when the template concentration was 2.0 copies / μL, amplification occurred in all four replicate tubes, with an average Ct value of 35.0225; when the template concentrations were 1.0 copies / μL and 0.5 copies / μL, no amplification curve appeared, so the positive judgment Ct value of this method was determined to be 35.

[0095] Example 7, specificity experiment

[0096] Seven common bee mites, namely, Pyrolae meridienne, Pyrolae leucodermi, Pyrolae taenii, Pyrolae koroi, Varroa destructor, Shield mite of bees, and Buchner locust mite, were used as samples (these seven samples were collected and preserved for this experiment. The public can obtain the above-mentioned bee mite biological materials from the applicant. The biological materials are only used to repeat the experiment of the present invention and cannot be used for other purposes). After DNA was extracted according to the method of step 2 of Example 4, real-time fluorescence PCR detection was performed using the detection method of step 3 of Example 4, and the result determination method of step 4 of Example 4 was used for determination. The detection results are as follows. Figure 6 shown.

[0097] Depend on Figure 6 It can be seen that only the honey bee Thermoelaeae mesenterica produced a typical amplification curve (Ct value was 18.87), and the other samples had no amplification curve, indicating that the real-time fluorescent PCR detection method of the present invention has good specificity.

[0098] Example 8, Clinical Sample Experiment

[0099] The method of the present invention was used to detect 10 clinically isolated bee mite samples. After extracting DNA from each sample according to the method in step 2 of Example 4, real-time fluorescence PCR detection was performed according to the detection method in step 3 of Example 4. The result was determined by the determination method in step 4 of Example 4. The test results are shown in FIG. Figure 7 shown.

[0100] Depend on Figure 7 It can be seen that 5 out of 10 samples were positive, with Ct values ​​ranging from 22 to 34, the positive control was 17.52, and the other 5 samples and the negative control had no Ct value, which was consistent with the expected results.

[0101] As can be seen from the above examples, the present invention provides a primer-probe combination and a detection method thereof for detecting the honey bee Thermoelaeae mertensii. It has been verified that the primer-probe combination and the detection method thereof of the present invention have high sensitivity, a minimum detection limit of 2.0 copies / μL, good specificity, and no cross reaction with other bee mites.

[0102] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A composition for detecting Pyrolacrida mites, characterized in that: The composition consists of a primer pair and a specific probe for detecting Thermoelaea merschmidtii; The primer pair consists of primer TME-F and primer TME-R, wherein the primer TME-F is a single-stranded DNA molecule shown in SEQ ID No.2; and the primer TME-R is a single-stranded DNA molecule shown in SEQ ID No.3; The nucleotide sequence of the probe is SEQ ID No.

4.

2. The composition according to claim 1, characterized in that The 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

3. The composition according to claim 2, characterized in that The 5' end of the probe is labeled with ROX, and the 3' end is labeled with BHQ-2.

4. A reagent for detecting Pyrolaea mertensii, characterized in that: The reagent comprises the composition according to any one of claims 1 to 3.

5. Use of the composition of any one of claims 1 to 3 for any of the following purposes other than disease diagnosis and treatment: A1) Use in detecting the Pyrolae mites or in preparing a product for detecting the Pyrolae mites; A2) Use of the method for distinguishing the Pyrolae meroni from other bee mites or for preparing a product for distinguishing the Pyrolae meroni from other bee mites; A3) Application in the control of Pyrocheteus fuscata.

6. A method for detecting Pyrolae mertensii for non-disease diagnosis purposes, characterized in that: The method comprises using the composition described in any one of claims 1 to 3 to perform a PCR amplification reaction on a sample to be tested, and determining whether the sample to be tested contains Thermoelaea merschmidt according to the PCR amplification product.

7. The method according to claim 6, characterized in that The method for determining whether the sample to be tested contains the Pyrolae mertensii mite according to the PCR amplification product is as follows: When the experiment is valid, if the sample to be tested has no Ct value and no amplification curve, it means that the sample to be tested does not contain the Thermolae meroni; if the Ct value of the sample to be tested is ≤35 and a typical amplification curve appears, it means that the sample to be tested contains the Thermolae meroni; if the Ct value of the sample to be tested is >35, the sample to be tested is tested repeatedly, and if the repeated test result has no Ct value or the Ct value is >35, the sample to be tested does not contain the Thermolae meroni; if the repeated test result has a Ct value and the Ct value is ≤35, the sample to be tested contains the Thermolae meroni.

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