Application of Cat PKD1 Gene Mutation Loci, Reagents and Diagnostic Kits

Abstract

This application relates to a reagent and a diagnostic kit for detecting cat PKD1 gene mutation sites, and the mutation site is PKD1:NM_058383.1:exon29:c.9864C>A:p.Cys3288X. For the first time, this application detects cat polycystic kidney-related gene mutation sites by multiplex Taqman-MGB real-time fluorescence PCR technology, which has the advantages of simple operation, strong specificity, high sensitivity, strong reliability and low cost.

Description

Technical Field

[0001] The present application relates to the application of a cat PKD1 gene mutation site, a reagent and a diagnostic kit, and belongs to the technical field of gene detection. Background Art

[0002] Feline polycystic kidney disease (PKD) is a hereditary disease in Persian cats and cats related to Persian cats, which is characterized by cysts in the kidneys, liver and pancreas and has an autosomal dominant inheritance pattern. Currently, the disease is highly prevalent in the cat population, making it the most prominent hereditary feline disease. However, there are limited methods for identifying cats carrying PKD before the disease develops. Currently, ultrasonic examination is commonly used clinically to detect feline polycystic kidney. However, ultrasonic examination is rarely performed before clinical symptoms appear (usually when about 66% of normal renal function is lost), and in the early stage of the disease, due to the small size of the cysts, it is not easy to detect by ultrasonic examination.

[0003] In the prior art, the only method for detecting cat PKD1 gene mutation is the ARMS-PCR method combined with capillary electrophoresis detection technology. This method has the following disadvantages:

[0004] (1) Complicated operation: First, a common upstream fluorescent primer and ARMS primers for the wild-type template and ARMS primers for the mutant template are used together to amplify a sequence containing the gene mutation site to be detected, generating amplification products with specific lengths and fluorescent labels. Subsequently, capillary electrophoresis is also required to detect the amplification products, and the genotype of the sample is determined by the fragment size and the product peak height ratio of the gene locus.

[0005] (2) Time-consuming: Due to the complicated operation, this detection takes a long time.

[0006] (3) Expensive detection cost: The detection relies on a PCR amplifier and a gene detector, and the instrument cost is relatively high.

[0007] (4) Low detection sensitivity: According to the literature report, its detection sensitivity is only 0.5 ng / μL.

[0008] (5) Low accuracy: Capillary electrophoresis of the amplification products is prone to product contamination, resulting in false positives.

[0009] Based on this, it is necessary to provide a more simple and efficient detection method. Summary of the Invention

[0010] The purpose of the present application is to provide the application of a cat PKD1 gene mutation site, a reagent and a diagnostic kit, which can accurately, effectively, simply and efficiently detect cats carrying PKD.

[0011] To achieve the above object, the present invention provides the following technical solutions:

[0012] In a first aspect, the present application provides an application of a feline PKD1 gene mutation site in the preparation of a reagent for diagnosing or treating feline polycystic kidney. The feline PKD1 gene mutation site is

[0013] PKD1:NM_058383.1:exon29:c.9864C>A:p.Cys3288X.

[0014] In a second aspect, the present application provides a reagent for detecting a feline PKD1 gene mutation site that causes feline polycystic kidney. The reagent includes primers for detecting the feline PKD1 gene mutation site c.9864C>A;

[0015] The primers for detecting the feline PKD1 gene mutation site c.9864C>A include an upstream primer with a nucleotide sequence as shown in

[0016] SEQ ID NO:1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:2.

[0017] In one or more feasible embodiments, the reagent further includes a wild-type probe with a nucleotide sequence as shown in SEQ ID NO:3; and / or,

[0018] the reagent further includes a mutant probe with a nucleotide sequence as shown in SEQ ID NO:4.

[0019] In one or more feasible embodiments, different fluorescent groups are provided on the wild-type probe and the mutant probe, and each of the fluorescent groups is independently selected from any one of FAM, VIC, HEX, Cy5 or ROX.

[0020] In one or more feasible embodiments, a quenching group is further provided on the wild-type probe and the mutant probe, and the quenching group is MGB.

[0021] In a third aspect, the present application provides a diagnostic kit for feline polycystic kidney, including the reagent described in the second aspect and a PCR amplification reaction solution.

[0022] In one or more feasible embodiments, the PCR amplification reaction solution includes dNTP, a PCR amplification buffer, magnesium ions and Taq polymerase.

[0023] In one or more feasible embodiments, the diagnostic kit further includes a sample preservation solution, a positive control product and a negative control product.

[0024] Fourthly, the present application provides an application of primers for detecting feline PKD1 gene mutation sites in the preparation of a feline polycystic kidney disease auxiliary diagnosis kit, and the feline PKD1 gene mutation site is PKD1:NM_058383.1:exon29:c.9864C>A:p.Cys3288X.

[0025] In one or more feasible embodiments, the primers include an upstream primer with a nucleotide sequence as shown in SEQ ID NO:1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:2.

[0026] In one or more feasible embodiments,

[0027] A method for using the kit to assist in diagnosing feline polycystic kidney gene mutation includes the following steps:

[0028] Use the kit to detect whether the feline PKD1 gene in the sample carries a mutation site to diagnose whether an individual has feline polycystic kidney disease or carries PKD;

[0029] If a c.9864C>A heterozygous mutation is detected in the genotype, the individual has feline polycystic kidney disease or carries PKD;

[0030] If the detected genotype is wild type, the tested individual is diagnosed as a normal cat.

[0031] According to one or more embodiments of the present application, the beneficial effects of the present application are as follows:

[0032] The present application provides an application of a feline PKD1 gene mutation site in the preparation of a reagent for diagnosing or treating feline polycystic kidney disease. The feline gene mutation site is PKD1:NM_058383.1:exon29:c.9864C>A:p.Cys3288X. The present application detects polycystic kidney disease-related mutation sites by multiplex Taqman-MGB real-time fluorescence PCR technology, which has the advantages of simple operation, strong specificity, high sensitivity, strong reliability and low cost.

[0033] On the one hand, by detecting whether a feline individual carries the above mutation, the present application is used for screening or diagnosing feline polycystic kidney disease, and cats can be selectively bred and PKD can be removed from the population to improve the overall health of domestic and wild cat breeds; on the other hand, the present application is conducive to carrying out gene mutation screening work for feline polycystic kidney disease. Cats identified as PKD carriers can be used as models for disease research and PKD treatment development; thirdly, the present application can provide possible drug targets for treating feline polycystic kidney disease.

[0034] The above description is only an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and implement it according to the content of the specification, the following describes in detail with reference to the preferred embodiments of the present invention and the accompanying drawings. Description of the Drawings

[0035] Figure 1 It is the amplification map obtained by using the kit of the present application to detect a cat carrying PKD in an embodiment of the present application;

[0036] Figure 2 It is the amplification map obtained by using the kit of the present application to detect a cat not carrying PKD in an embodiment of the present application. Detailed Embodiments

[0037] The following illustrates the embodiments of the present invention through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, without conflict, the following embodiments and the features in the embodiments can be combined with each other.

[0038] It should be noted that in this application, "precision" refers to the degree of conformity between the measured or calculated quantity (test report value) and its actual (or true) value. Clinical precision refers to the ratio of true outputs (true positive (TP) or true negative (TN)) to misclassified outputs (false positive (FP) or false negative (FN)), and can be expressed as sensitivity, specificity, positive predictive value (PPV) or negative predictive value (NPV), Matheus correlation coefficient (MCC), or likelihood ratio, odds ratio, receiver operating characteristic (ROC) curve, area under the curve (AUC), among other measurements.

[0039] For the diagnostic (or prognostic) intervention of this application, since each output (which may be TP, FP, TN, or FN in a disease classification diagnostic test) incurs different costs, the health economic utility function may be based on the clinical situation and the sum of individual output costs. Preferably, it favors sensitivity over specificity, or PPV over NPV. Therefore, another measurement of the health economic performance sum is provided, which may be different from the more direct clinical or analytical performance measurements. These different measurements and relative trade-offs generally converge only in the case of a perfect test with a zero error rate (also known as zero misclassification of predicted object outputs or FP and FN). All performance measurements will tend to be imperfect, but to different degrees.

[0040] "Measurement", "determination", "detection", or "examination" means evaluating the presence, absence, amount, or quantity (which can be an effective amount) of a given substance or a sample derived from an object in a clinical setting (including the derivation of qualitative or quantitative concentration levels of such a substance), or otherwise assessing the value or classification of a non-analyte clinical parameter or clinical determinant of an object.

[0041] The term "mutation" in the context of this application refers to a change in the polynucleotide sequence of the wild type, which means the addition, deletion, and / or substitution of one or several (such as several) bases in the gene sequence or DNA sequence, resulting in a variant, and the variant can be naturally occurring or non-naturally occurring. When the term "mutation" is used to describe the product or protein encoded by a gene, "mutation" refers to the addition, deletion, and / or substitution of one or several (such as several) amino acid residues in the said protein or encoded product.

[0042] The data in the context of this application all meet the statistical requirements (statistically significant). The so-called "statistically significant" means that the change is greater than what can be expected by chance alone (which can be a "false positive"). Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which represents the probability of obtaining at least the extreme value of the result at a given data point, assuming that the data point is a result of chance alone. When the p-value is 0.05 or less, the result is generally considered to be highly significant.

[0043] It should be noted that in the following examples, where specific techniques or conditions are not indicated, they are carried out according to the techniques or conditions described in the literature in this field, or according to the product specifications. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through regular commercial channels.

[0044] This application provides the use of a cat PKD1 gene mutation site in the preparation of a reagent for diagnosing or treating feline polycystic kidney disease. The mutation site is PKD1:NM_058383.1:exon29:c.9864C>A:p.Cys3288X, specifically referring to the mutation of the 9864th base C in the 29th exon of the wild type PKD1 gene to A, resulting in an early termination codon in the mRNA. Compared with the protein encoded by the wild type PKD1 gene, the amino acids after the 3288th position of the mutant protein are truncated. The said mutation is a nonsense mutation in exon29.

[0045] Based on the above situation, the present application also provides a reagent for detecting the mutation sites of the feline PKD1 gene that causes polycystic kidney in cats. This reagent includes primers for detecting the above mutation sites, specifically including the upstream primer (PKD1-F) shown in SEQ ID NO:1: 5’-CTCGGAGCCGCTTCAC-3’ and the downstream primer (PKD1-R) with the nucleotide sequence shown in SEQ ID NO:2: 5’-TCCCACGACCCCGTAC-3’.

[0046] Optionally, the above reagent further includes a wild-type probe with the nucleotide sequence shown in SEQ ID NO:3: 5’-CCACCTGTTGCGTCC-3’, and a mutant probe with the nucleotide sequence shown in SEQ ID NO:4: 5’-CCACCTGTTGAGTCCT-3’.

[0047] It can be understood that both the wild-type probe and the mutant probe carry a fluorescent group and a quenching group, and the fluorescent groups of the wild-type probe and the mutant probe are different.

[0048] By way of illustration and not limitation, the above fluorescent groups are each independently selected from any one of FAM, VIC, HEX, Cy5 or ROX, and the quenching group is preferably MGB.

[0049] Based on the above situation, the present application also provides a diagnostic kit for feline polycystic kidney. This kit includes the above reagent and a PCR amplification reaction solution.

[0050] Optionally, the above PCR amplification reaction solution includes dNTP, a PCR amplification buffer, magnesium ions and Taq polymerase.

[0051] Optionally, the kit further includes a sample preservation solution, a positive control product and a negative control product.

[0052] Based on the above situation, the present application also provides an application of the primers for detecting the above mutation sites in the preparation of a diagnostic kit for the auxiliary diagnosis of feline polycystic kidney.

[0053] Optionally, a method for the auxiliary diagnosis of feline polycystic kidney using the above kit includes the following steps:

[0054] Detect whether there are mutation sites in the feline PKD1 gene in the sample using the above kit to diagnose whether an individual has feline polycystic kidney or carries PKD;

[0055] If a c.9864C>A heterozygous mutation is detected in the genotype, the individual has feline polycystic kidney or carries PKD;

[0056] If the detected genotype is wild-type, the tested individual is diagnosed as a normal cat.

[0057] By way of illustration and not limitation, the sample is preferably at least one of feline oral mucosa, nasal mucosa, and conjunctival swabs.

[0058] In one of the embodiments, the applicant designed multiple sets of primers for the feline PKD1 gene mutation site PKD1:NM_058383.1:exon29:c.9864C>A:p.Cys3288X. After a series of creative efforts, upstream primers and downstream primers as shown in SEQ ID NO.1 and SEQ ID NO.2 were screened and obtained, and wild-type probes and mutant probes as shown in SEQ ID NO.3 and SEQ ID NO.4 were designed based on these primers. This primer-probe set can effectively amplify the feline PKD1 gene, and there is no non-specific amplification caused by secondary structures between the upstream and downstream primers and the probes.

[0059] In addition, a kit is formed by preparing or purchasing PCR amplification reaction solution, sample preservation solution, positive control product, and negative control product. Among them, the PCR amplification reaction solution also includes Taq DNA polymerase, dNTPs, PCR amplification buffer, Mg 2+ etc.; the positive control product and the negative control product contain PKD1 mutant plasmid and PKD1 wild-type plasmid, respectively.

[0060] Specifically, the sample preservation solution: contains a stabilizer for maintaining the DNA stability of the sample.

[0061] The positive control product: contains the PKD1 mutant plasmid for verifying the accuracy of the detection method.

[0062] The negative control product: contains the PKD1 wild-type plasmid for verifying the specificity of the detection method.

[0063] Other auxiliary materials: PCR tubes, pipette tips, centrifuge tubes, etc.

[0064] In this embodiment, the kit is stored at -20°C, and the kit specification is: 4 test portions / box.

[0065] First, use a special swab to collect samples from the feline oral mucosa, nasal mucosa, or conjunctiva, and ensure that the swab is moist, and gently scrape the cell samples. Then put the collected samples into the sample preservation solution containing a stabilizer for preservation to prevent DNA degradation. During this process, the sample collection should be carried out under sterile conditions to avoid contamination.

[0066] The test sample does not require pretreatment and is directly added to the PCR amplification reaction solution; the loaded PCR amplification reaction solution is added to a PCR instrument, and the corresponding amplification program is selected for amplification. The amplification program is:

[0067] Initial denaturation: 95°C for 1 minute to activate Taq DNA polymerase.

[0068] Cyclic amplification: DNA denaturation at 95°C for 10 seconds; primer annealing and probe binding at 62°C for 8 seconds; this step is repeated 45 times.

[0069] Final extension: An additional extension step can be carried out according to actual needs to ensure complete amplification of all DNA fragments.

[0070] Method for result determination:

[0071] Amplify using a real-time fluorescence PCR instrument and monitor the amplification process through the increase in fluorescence signal. Analyze the change in fluorescence signal by software and determine whether the sample contains mutant or wild-type genes according to the set threshold.

[0072] Verification of detection sensitivity and specificity:

[0073] By gradually diluting mutant plasmid DNA with known concentration, determine the lowest DNA concentration that can be detected by the method of this application to verify the sensitivity of the kit.

[0074] Then use known wild-type and mutant samples for testing to ensure that the method only produces signals for the target sequence and does not cross-react with other non-target sequences, thereby verifying specificity.

[0075] Based on the above kit, cats with and without polycystic kidney disease (PKD) were detected respectively. The reagent is pre-packaged in single tubes with single doses, and the sample can be directly added for detection without extraction, which is easy to operate. And the concentration of the sample is 0.05 pg / μL.

[0076] Figure 1 and Figure 2 respectively show the PCR amplification maps of different cat samples based on the kit of this application. According to Figure 1 It can be seen that the CT value of the amplification curve of feline polycystic kidney is 30.38, and the CT value of the amplification curve of the internal standard is 30.39, that is, the target sequence and the internal standard sequence are detected within a relatively small number of cycles, and a lower CT value indicates higher amplification efficiency. And by comparing the amplification of the internal standard and the target sequence, their amplification efficiencies are similar, indicating that the PCR reaction is effective and accurate, and there is no substance in the sample that inhibits the PCR reaction.

[0077] According to Figure 2It can be seen that the internal standard amplification curve is normal. The CT value of the amplification curve of feline polycystic kidney is always 0, and there is no visible change to the naked eye, indicating that it does not contain the target gene sequence, that is, the specific mutant or wild-type sequence of the feline polycystic kidney gene does not exist or is below the detection limit in the sample. At the same time, it shows that the kit of the present application has high specificity, only amplifies the target gene sequence, and does not react with other non-target genes.

[0078] From this, it can be seen that the present application provides an application of the feline PKD1 gene mutation site in the preparation of a reagent for diagnosing or treating feline polycystic kidney. The present application detects the polycystic kidney-related mutation sites by multiplex Taqman-MGB real-time fluorescence PCR technology, with simple operation, strong specificity, high sensitivity, strong reliability and low cost. On the one hand, by detecting whether a feline individual carries the above mutations, the present application can be used for screening or diagnosing feline polycystic kidney, and cats can be selectively bred and PKD removed from the population to improve the overall health of domestic and wild feline breeds; on the other hand, the present application is conducive to carrying out the gene mutation screening work of feline polycystic kidney. Cats identified as PKD carriers can be used as models for disease research and PKD treatment development; thirdly, the present application can provide possible drug targets for treating feline polycystic kidney.

[0079] In practical applications, the reagent and the kit can be directly added to the test sample for detection. The sample does not need to be subjected to DNA extraction, with simple operation and no need for complex training of relevant personnel, greatly increasing the detection effect. Moreover, the minimum detection limit of the DNA sample is 0.05 pg / μL, which means it has high sensitivity and high specificity and can ensure accurate detection of the sample. At the same time, the relevant detection can be completed only by 1 PCR instrument, and the detection result can be directly obtained by cooperating with the corresponding software program, with low detection cost and simple operation.

[0080] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0081] The above-described embodiments only express several implementation manners of the present invention, and the description is relatively specific and detailed, but it cannot be understood as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. A reagent for detecting the mutation sites of the feline PKD1 gene that causes polycystic kidney disease in cats, characterized in that, The reagent includes primers for detecting the mutation sites of feline PKD1 gene; The mutation sites of the feline PKD1 gene are PKD1:NM_058383.1:exon29:c.9864 C>A:p.Cys3288X; The primers include an upstream primer with a nucleotide sequence as shown in SEQ ID NO:1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:2; Among them, the reagent also includes a wild-type probe with a nucleotide sequence as shown in SEQ ID NO:3 and a mutant probe with a nucleotide sequence as shown in SEQ ID NO:

4.

2. The reagent according to claim 1, wherein Different fluorescent groups are provided on the wild-type probe and the mutant probe, and each of the fluorescent groups is independently selected from any one of FAM, VIC, HEX, Cy5 or ROX.

3. The reagent according to claim 1, wherein A quenching group is also provided on the wild-type probe and the mutant probe, and the quenching group is MGB.

4. A diagnostic kit for feline autosomal dominant polycystic kidney disease gene mutation, characterized in that, It includes the reagent according to any one of claims 1 to 3 and a PCR amplification reaction solution.

5. The diagnostic kit according to claim 4, characterized in that, The PCR amplification reaction solution includes dNTP, a PCR amplification buffer, magnesium ions and Taq polymerase.

6. The diagnostic kit according to claim 4, characterized in that, The diagnostic kit also includes a sample preservation solution, a positive control product and a negative control product.

7. Use of the diagnostic kit according to any one of claims 4 to 6 in preparing an auxiliary diagnostic kit for feline polycystic kidney.

8. The application according to claim 7, characterized in that, Based on the multiplex Taqman-MGB real-time fluorescence PCR technology.

9. The application according to claim 8, characterized in that, The conditions of the multiplex Taqman-MGB real-time fluorescence PCR technology are: Initial denaturation: 95°C, 1 minute, to activate Taq DNA polymerase; Cyclic amplification: DNA denaturation at 95°C for 10 seconds; primer annealing and probe binding at 62°C for 8 seconds; this step is repeated 45 times.

10. Use of a primer and a probe for detecting cat PKD1 gene mutation sites in the preparation of an auxiliary diagnostic kit for feline polycystic kidney, characterized in that, The mutation sites of the feline PKD1 gene are PKD1:NM_058383.1:exon29:c.9864 C>A:p.Cys3288X; The primers include an upstream primer with a nucleotide sequence as shown in SEQ ID NO:1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:2; The probes include a wild-type probe with a nucleotide sequence as shown in SEQ ID NO:3 and a mutant probe with a nucleotide sequence as shown in SEQ ID NO:4.

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