Primer group, kit and detection method for PRNP gene variation detection

Abstract

The invention relates to a primer group, a kit and a detection method for PRNP gene variation detection. The primer group comprises at least one of the following primer groups: SEQ ID NO: 1 and SEQ ID NO: 2; sEQ ID NO: 1 and SEQ ID NO: 4; sEQ ID NO: 1 and SEQ ID NO: 6; sEQ ID NO: 1 and SEQ ID NO: 8; sEQ ID NO: 3 and SEQ ID NO: 4; sEQ ID NO: 5 and SEQ ID NO: 6; sEQ ID NO: 7 and SEQ ID NO: 8. According to the invention, the full length of the PRNP gene is amplified and enriched by adopting a long fragment amplification technology, the exon and intron of the PRNP gene are simultaneously sequenced by applying a three-generation sequencing technology, variation types such as known and unknown SNV, Indel, SV, dynamic mutation and the like are detected, and accurate, rapid and high-throughput detection on the variation of the PRNP gene is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a primer set, a kit and a detection method for detecting PRNP gene mutation. Background Art

[0002] Human prion diseases are rare pathogenic spongiform encephalopathies caused by pathogenic genetic variants in normal cellular prion protein (PrPC), which misfolds into abnormal prion protein (PrPSc). These abnormal prion protein (PrPSc) accumulates in the central nervous system and, when these levels reach a critical mass, causes prion disease. The abnormal prion protein (PrPSc) never returns to its normal form. Prion diseases encompass several distinct subtypes, including familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Schenck disease (GSS), and fatal familial insomnia.

[0003] The causative gene for human prion disease is the prion protein gene (PRNP), located on chromosome 20. It is 15.36 kb long and inherits in an autosomal dominant manner. Currently, over 40 pathogenic variants have been identified (including high-penetrance variants such as E200K, P102L, D178N, A117V, and P105L, and low-penetrance variants such as V210I, V180I, and M232R), as well as a small number of polymorphisms. Among these, two non-pathogenic polymorphisms, codons 129 and 219, have garnered the most attention. Codon 129 encodes methionine (Met) or valine (Val), and its polymorphisms are associated with susceptibility to prion disease and are important determinants of the clinical phenotype of CJD. Codon 219 encodes glutamate (Glu) or lysine (Lys) and is associated with the development of CJD. Compared with the general population, patients with sporadic CJD have a higher rate of methionine homozygosity at codon 129, whereas Glu / Lys heterozygosity at codon 219 protects against the development of sporadic CJD. Early and definitive diagnosis is crucial for timely and appropriate treatment and prevention of complications and disease progression.

[0004] For a long time, sequencing the exonic regions of the PRNP gene using the Sanger method has been the most commonly used detection method. Following the advent of next-generation sequencing (NGS) technology, panel, WES, and WGS sequencing have been used to detect pathogenic variants and polymorphisms in the exonic regions of the PRNP gene. Sanger sequencing, panel, and WES methods can only detect variants in exonic regions and are unable to detect variants that may exist in introns. Although WGS can detect variants in both exons and introns, its high cost makes it unsuitable for PRNP gene testing in prion diseases. Furthermore, a dynamic mutation in the PRNP gene, an octapeptide (PHGGGWGQ) repeat, occurs. This 24-nucleotide insertion, duplication, or deletion of the 24-bp nucleotide sequence encoding the octapeptide is associated with prion diseases and requires capillary electrophoresis for detection of this dynamic mutation. Second-generation short-read sequencing technologies, such as Sanger sequencing, panel, WES, and WGS, all have limitations in detecting dynamic mutations in the octapeptide (PHGGGWGQ) repeat, potentially leading to missed detections.

[0005] Therefore, it is very necessary to develop a simple, effective and inexpensive detection method to achieve comprehensive detection of human hereditary prion diseases. Summary of the Invention

[0006] The present invention aims to provide a primer set, a kit and a detection method for detecting PRNP gene mutations, which can achieve accurate, rapid and high-throughput detection of PRNP gene mutations.

[0007] In a first aspect of the present invention, a primer set for detecting PRNP gene mutations is provided. The primer set is designed based on the full-length nucleotide sequence of the PRNP gene using Primer3 web version 4.1.0 software to amplify the PRNP gene DNA sequence. The primer set is used to amplify the human PRNP gene and flanking sequences. The primer set specifically includes at least one of the following primer sets:

[0008] (I) SEQ ID NO: 1 and SEQ ID NO: 2;

[0009] (II) SEQ ID NO: 1 and SEQ ID NO: 4;

[0010] (III) SEQ ID NO: 1 and SEQ ID NO: 6;

[0011] (IV) SEQ ID NO: 1 and SEQ ID NO: 8;

[0012] (V) SEQ ID NO: 3 and SEQ ID NO: 4;

[0013] (VI) SEQ ID NO: 5 and SEQ ID NO: 6;

[0014] (VII) SEQ ID NO: 7 and SEQ ID NO: 8.

[0015] Preferably, the primer set is selected from any one of the following groups:

[0016] (I) SEQ ID NO: 1 and SEQ ID NO: 2;

[0017] (II) SEQ ID NO: 1 and SEQ ID NO: 4.

[0018] Preferably, a barcode sequence (DNA barcode) is added to the 5' end of the nucleotide sequence of each primer in the primer set to distinguish samples when simultaneously amplifying and detecting the PRNP gene in multiple samples, thereby achieving high-throughput simultaneous multi-sample detection. When the samples to be tested are multiple different samples, a different barcode sequence can be used for each sample.

[0019] In the present invention, there is no limitation on the barcode sequence; the barcode sequences (including Barcode-F and Barcode-R) can be identical or inconsistent. The barcode sequence can be a barcode designed by PacBio or a custom-designed barcode. Furthermore, a double barcode with barcode primers or barcode adapters can be used to achieve a wider range of barcode combinations. A protective base may or may not be added to the 5' end of the barcode sequence, and those skilled in the art can select this as needed. Preferably, a 3-5 nt protective base is added to the 5' end of the barcode sequence, and the protective base sequence is GGTAG.

[0020] Preferably, the length of the Barcode sequence is 16 nt.

[0021] In one embodiment, the barcode sequence is selected from any one of the following groups:

[0022] (A) SEQ ID NO:9 and SEQ ID NO:10;

[0023] (B) SEQ ID NO: 11 and SEQ ID NO: 12.

[0024] The second aspect of the present invention provides the use of the above primer set, wherein the primer set is used in the preparation of a diagnostic product for hereditary prion disease.

[0025] Preferably, the diagnostic product includes: a diagnostic reagent, a diagnostic kit or a diagnostic chip.

[0026] The third aspect of the present invention provides a kit for diagnosing hereditary prion disease, comprising: the above-mentioned primer set.

[0027] Preferably, the kit further comprises: one or more of a DNA extraction system, a PCR reaction buffer, ddH2O, and a DNA polymerase.

[0028] A fourth aspect of the present invention provides a method for detecting PRNP gene mutation for non-diagnostic purposes, comprising the following steps:

[0029] S1, extracting DNA from the sample to be tested;

[0030] Specifically, the sample to be tested can be any one of peripheral blood, dried blood spots and other samples;

[0031] S2. Using the above primer set or the above kit, perform PCR amplification on the DNA extracted in step S1 to generate a PCR product with a barcode sequence;

[0032] S3, construction of PacBio sequencing library;

[0033] S4. Perform PacBio library sequencing and perform bioinformatics analysis on the PacBio sequencing data to obtain PRNP gene sequencing results.

[0034] The present invention has at least the following beneficial effects:

[0035] The present invention uses long-fragment amplification technology to amplify and enrich the full length of the PRNP gene, and applies third-generation sequencing technology to simultaneously sequence the PRNP gene exons and introns, detecting all known and unknown variant types such as single nucleotide variants (SNVs), indels, SVs, and dynamic mutations. This enables accurate, rapid, and high-throughput detection of PRNP gene variants. The detection method of the present invention is simple to operate, has reliable quality, strong reproducibility, and a wide detection range, resolving the problems of existing detection methods such as incomplete detection coverage, cumbersome methods, and potential missed detections and false detections. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 Flowchart of the method for detecting PRNP gene variation for non-diagnostic purposes provided by the present invention.

[0038] Figure 2 Schematic diagram of the positions and amplification directions of the primer sets P1 and P2 provided by the present invention.

[0039] Figure 3 This is a gel electrophoresis diagram of the amplification products of multiple primer sets provided by the present invention.

[0040] Figure 4 This is the gel electrophoresis diagram of the amplified products of the primer sets P1 and P2 provided by the present invention.

[0041] Figure 5 A schematic diagram of the PRNP gene sequencing depth provided by the present invention.

[0042] Figure 6 Schematic diagram of the PRNP gene sequencing point variation IGV provided by the present invention.

[0043] Figure 7 This is a schematic diagram of the dynamic mutation of the PRNP gene octapeptide repeat provided by the present invention.

[0044] Figure 8 This is the Sanger sequencing verification diagram of the PRNP gene provided by the present invention.

[0045] Figure 9 This is a capillary electrophoresis verification diagram of the PRNP gene dynamic mutation provided by the present invention. DETAILED DESCRIPTION

[0046] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0047] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular also includes the plural. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0048] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0049] Example 1

[0050] This embodiment amplifies the full-length sequence of the PRNP gene, a pathogenic gene of human hereditary prion disease, and includes the following steps:

[0051] (1) Primer design

[0052] Primer3web version 4.1.0 software was used to analyze the full-length nucleotide sequence of the PRNP gene (its chromosomal location is shown in Figure 2). Figure 2 As shown), primer sets P1 and P2, P3 and P4, P5 and P6, P7 and P8, P1 and P4, P1 and P6, P1 and P8 were designed to amplify the PRNP gene DNA sequence, and the human PRNP gene and flanking sequences were amplified, specifically:

[0053] The nucleotide sequence of primer P1 is 5′-GGTTGCCAAAGTTCCAGAAATTGCT-3′ (SEQ ID NO: 1);

[0054] The nucleotide sequence of primer P2 is 5′-CAGGGCAAGCACCAGGAAATACA-3′ (SEQ ID NO: 2);

[0055] The nucleotide sequence of primer P3 is 5′-TACAGTCAATGAGCCACGTCAGGGAG-3′ (SEQ ID NO: 3);

[0056] The nucleotide sequence of primer P4 is 5′-CCTTCCTCCTCAGCCTTCCTCAATGT-3′ (SEQ ID NO: 4);

[0057] The nucleotide sequence of primer P5 is 5′-CATGCCTGGTTTACGCCCATTTCACT-3′ (SEQ ID NO: 5);

[0058] The nucleotide sequence of primer P6 is 5′-CCACGGGTTGGAACTGAGTCCACGA-3′ (SEQ ID NO: 6);

[0059] The nucleotide sequence of primer P7 is 5′-ATGGGAGGAAAGCAGTCGACCAGAAA-3′ (SEQ ID NO: 7);

[0060] The nucleotide sequence of primer P8 is 5'-AACATGTAGAGAATGAGTTGGAAGAT-3' (SEQ ID NO: 8).

[0061] (2) DNA extraction

[0062] This example uses a blood genomic DNA extraction kit (EE121) produced by Beijing Quanshijin Biotechnology Co., Ltd. to extract genomic DNA from peripheral blood:

[0063] Take 250 μl of EDTA anticoagulated whole blood and add it to a 2 ml EP centrifuge tube.

[0064] Add 500 μl BB3 and 20 μl Proteinase K, vortex to mix, and incubate at room temperature for 10 min.

[0065] After brief centrifugation, all the liquid was transferred to the adsorption column in the collection tube, centrifuged at 12,000 rpm for 1 min, and the filtrate was discarded.

[0066] The adsorption column was placed in a collection tube, 500 μl of CB3 was added, and the mixture was centrifuged at 12,000 rpm for 1 min, and the filtrate was discarded.

[0067] Place the adsorption column in a collection tube, add 500 μl WB3, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0068] Place the adsorption column in a collection tube, add 500 μl WB3, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0069] The adsorption column was placed in a collection tube and centrifuged at 12,000 rpm for 2 min.

[0070] Place the adsorption column in a clean 1.5ml EP centrifuge tube and leave it open for 2 minutes.

[0071] Add 70 μl of EB (Elution Buffer) preheated at 70°C to the center of the adsorption column and let it stand at room temperature for 1 min.

[0072] Centrifuge at 12,000 rpm for 1 min to elute the DNA and discard the adsorption column.

[0073] Determine DNA concentration using nanodrop and proceed with subsequent experiments. Store DNA at 2-8°C temporarily or below -18°C for long-term storage.

[0074] (3) PCR amplification

[0075] KOD FX Neo DNA Polymerase (Code No. KFX-101) from TOYOBO CO., LTD. was used to perform PCR amplification on the test sample DNA using the above primer set to generate a series of PCR products with barcode sequences.

[0076] The PCR amplification reaction mixture was prepared according to the amount of DNA in the test sample. The amounts of reagents in the amplification reaction mixture were shown in Table 1.

[0077] Table 1

[0078] Reagents Volume / μL 2×KODFXneoBuffer 12.5×n dNTPMix (2 mM each) 5×n Primer set 2.5×n KODFXneo (1.0 U / μl) 0.5×n <![CDATA[ddH2O]]> 3.5×n

[0079] Where n = number of test samples + 1.

[0080] Add 1 μl of DNA (50 ng) to 24 μl of PCR amplification reaction mixture of the sample to be tested.

[0081] Perform PCR amplification in a PCR instrument. The amplification procedure is as follows:

[0082] In a PCR instrument, first denature at 98°C for 1 minute; then run 30 cycles of the following: denaturation at 98°C for 15 seconds, annealing and extension at 68°C for 8 minutes; and finally extension at 68°C for 10 minutes; and then store at 4°C.

[0083] After the amplification was completed, 2 μl was taken and tested on a 1% DNA gel. The amplification results of the primer sets P1 and P2, P3 and P4, P5 and P6, P7 and P8, P1 and P4, P1 and P6, and P1 and P8 were as follows: Figure 3 As shown in the figure, it can be seen that the primer groups P1 and P2, and P1 and P4 have the best amplification effects.

[0084] Example 2

[0085] In this embodiment, a barcode sequence and a protective base (GGTAG) are added to the 5' end of the above primers P1 to P8 to distinguish samples when simultaneously amplifying and detecting the PRNP gene in multiple samples, thereby achieving high-throughput simultaneous multi-sample detection. In this embodiment, there are two samples to be tested: sample 1 (peripheral blood) and sample 2 (dried blood spot). Sample 1 can use bc1167-F: 5'-CAGCGCATCTCACGTC-3' (SEQ ID NO: 9) and bc1167-R: 5'-GACGTGAGATGCGCTG-3' (SEQ ID NO: 10) from group (A), and sample 2 can use bc1168-F: 5'-GTGCAGTGATCGATGA-3' (SEQ ID NO: 11) and bc1168-R: 5'-TCATCGATCACTGCAC-3' (SEQ ID NO: 12) from group (B).

[0086] When the barcode sequence is bc1167-F or bc1167-R from group (A), the specific sequences of primers Y1 to Y8 corresponding to the primers P1 to P8 after adding the barcode sequence are as follows:

[0087] The nucleotide sequence of primer Y1 is 5′-GGTAGCAGCGCATCTCACGTCGGTTGCCAAAGTTCCAGAAATTGCT-3′ (SEQ ID NO: 13);

[0088] The nucleotide sequence of primer Y2 is 5′-GGTAGGACGTGAGATGCGCTGCAG GGCAAGCACCAGGAAATACA-3′ (SEQ ID NO: 14);

[0089] The nucleotide sequence of primer Y3 is 5′-GGTAGCAGCGCATCTCACGTCTACAGTCAATGAGCCACGTCAGGGAG-3′ (SEQ ID NO: 15);

[0090] The nucleotide sequence of primer Y4 is 5′-GGTAGGACGTGAGATGCGCTGCCTTCCTCCTCAGCCTTCCTCAATGT-3′ (SEQ ID NO: 16);

[0091] The nucleotide sequence of primer Y5 is 5′-GGTAGCAGCGCATCTCACGTCCATGCCTGGTTTACGCCCATTTCACT-3′ (SEQ ID NO: 17);

[0092] The nucleotide sequence of primer Y6 is 5′-GGTAGGACGTGAGATGCGCTGCCACGGGTTGGAACTGAGTCCACGA-3′ (SEQ ID NO: 18);

[0093] The nucleotide sequence of primer Y7 is 5′-GGTAGCAGCGCATCTCACGTCATGGGAGGAAAGCAGTCGACCAGAAA-3′ (SEQ ID NO: 19);

[0094] The nucleotide sequence of primer Y8 is 5′-GGTAGGACGTGAGATGCGCTGAACATGTAGAGAATGAGTTGGAAGAT-3′ (SEQ ID NO: 20).

[0095] When the barcode sequence is bc1168-F or bc1168-R from group (B), the specific sequences of primers Y9 to Y16 corresponding to the primers P1 to P8 after adding the barcode sequence and the protection base (GGTAG) are as follows:

[0096] The nucleotide sequence of primer Y9 is: 5′-GGTAGGTGCAGTGATCGATGAGGTTGCCAAAGTTCCAGAAATTGCT-3′ (SEQ ID NO: 21);

[0097] The nucleotide sequence of primer Y10 is: 5′-GGTAGTCATCGATCACTGCACCAGGGCAAGCACCAGGAAATACA-3′ (SEQ ID NO: 22);

[0098] The nucleotide sequence of primer Y11 is: 5′-GGTAGGTGCAGTGATCGATGATACAGTCAATGAGCCACGTCAGGGAG-3′ (SEQ ID NO: 23);

[0099] The nucleotide sequence of primer Y12 is: 5′-GGTAGTCATCGATCACTGCACCCTTCCTCCTCAGCCTTCCTCAATGT-3′ (SEQ ID NO: 24);

[0100] The nucleotide sequence of primer Y13 is: 5′-GGTAGGTGCAGTGATCGATGACATGCCTGGTTTACGCCCATTTCACT-3′ (SEQ ID NO: 25);

[0101] The nucleotide sequence of primer Y14 is: 5′-GGTAGTCATCGATCACTGCACCACGGGTTGGAACTGAGTCCACGA-3′ (SEQ ID NO: 26);

[0102] The nucleotide sequence of primer Y15 is: 5′-GGTAGGTGCAGTGATCGATGAATGGGAGGAAAGCAGTCGACCAGAAA-3′ (SEQ ID NO: 27);

[0103] The nucleotide sequence of primer Y16 is: 5′-GGTAGTCATCGATCACTGCAAACATGTAGAGAATGAGTTGGAAGAT-3′ (SEQ ID NO: 28).

[0104] In this example, the primer sets P1 and P2 with the best amplification effect were selected to amplify samples 1 and 2 according to the method of Example 1. The results are as follows: Figure 4 As shown in the figure, it can be seen that the PRNP gene can be effectively amplified using different samples as templates.

[0105] Example 3

[0106] like Figure 1 As shown, this embodiment uses the method of the present invention to construct a PacBio sequencing library, including the following steps:

[0107] (1) Primer design

[0108] The method is consistent with that of Example 1.

[0109] (2) DNA extraction

[0110] The method is consistent with Example 1.

[0111] (3) PCR amplification

[0112] The method is consistent with Example 1.

[0113] (4) Construction of PacBio sequencing library

[0114] Using Pacific Biosciences PacBio library construction was performed using the prep kit 3.0 kit (PacBio, Cat# 102-141-700).

[0115] The sequencing library was quantified using Qubit 3.0.

[0116] (5) PacBio sequencing and analysis

[0117] According to the concentration of the sequencing library in the previous step, an appropriate volume of the library was reacted with the binding reagent (PacBio, Cat#101-820-200) and primers (PacBio, Cat#100-970-100) to prepare the final library that can be used for sequencing. Bioinformatics analysis of the PacBio sequencing data was performed to analyze the coverage of the PRNP gene library (such as Figure 5 As shown), point mutation (as Figure 6 As shown), dynamic mutation of octapeptide (PHGGGWGQ) repeats (as shown Figure 7 as shown), large fragment deletion and other variation results.

[0118] Example 4 Detection and Verification of PRNP Gene Mutation Detection

[0119] Utilize the detection method of the present invention, refer to embodiment 1 and embodiment 2, detect 9 examples of verification sample. Figure 8 As shown), fluorescent PCR capillary electrophoresis (as Figure 9 The results obtained by the present invention are completely consistent with those of other methods, as shown in Table 2.

[0120] Table 2

[0121]

[0122] In summary, the present invention uses long-fragment amplification technology to amplify and enrich the full length of the PRNP gene, and applies third-generation sequencing technology to simultaneously sequence the PRNP gene exons and introns, detecting all known and unknown variant types such as single nucleotide variants (SNVs), indels, SVs, and dynamic mutations, thereby achieving accurate, rapid, and high-throughput detection of PRNP gene variants. The detection method of the present invention is simple to operate, has reliable quality, strong reproducibility, and a wide detection range, solving the problems of existing detection methods such as incomplete detection coverage, cumbersome methods, and possible missed detections and false detections.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer set for detecting PRNP gene mutation, characterized in that: At least one of the following primer sets is included: (I) SEQ ID NO: 1 and SEQ ID NO: 2; (II) SEQ ID NO: 1 and SEQ ID NO: 4; (III) SEQ ID NO: 1 and SEQ ID NO: 6; (IV) SEQ ID NO: 1 and SEQ ID NO: 8; (V) SEQ ID NO: 3 and SEQ ID NO: 4; (VI) SEQ ID NO: 5 and SEQ ID NO: 6; (VII) SEQ ID NO: 7 and SEQ ID NO:

8.

2. The primer set according to claim 1, characterized in that The primer set is selected from any one of the following groups: (I) SEQ ID NO: 1 and SEQ ID NO: 2; (II) SEQ ID NO: 1 and SEQ ID NO:

4.

3. The primer set according to claim 1, characterized in that A barcode sequence is added to the 5' end of the nucleotide sequence of each primer in the primer set.

4. The primer set according to claim 3, characterized in that The length of the Barcode sequence is 16 nt.

5. The primer set according to claim 3, characterized in that A protective base is added to the 5' end of the barcode sequence.

6. Use of the primer set according to any one of claims 1 to 5, characterized in that: The primer set is used in preparing diagnostic products for hereditary prion diseases.

7. The use according to claim 6, characterized in that The diagnostic products include: diagnostic reagents, diagnostic kits or diagnostic chips.

8. A kit for detecting hereditary prion disease, characterized in that: include: The primer set according to any one of claims 1 to 5.

9. The kit according to claim 8, characterized in that Also includes: One or more of DNA extraction system, PCR reaction buffer, ddH2O, and DNA polymerase.

10. A method for detecting PRNP gene variation for non-diagnostic purposes, characterized in that: The steps include: S1, extracting DNA from the sample to be tested; S2. Perform PCR amplification on the DNA extracted in step S1 using the primer set described in any one of claims 1 to 5 or the kit described in any one of claims 8 to 9 to generate a PCR product with a barcode sequence; S3, construction of PacBio sequencing library; S4. Perform PacBio library sequencing and perform bioinformatics analysis on the PacBio sequencing data to obtain PRNP gene sequencing results.

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