MNP marker combination, primer group and kit for identifying Xinjiang hemorrhagic fever virus and application of MNP marker combination, primer group and kit
By screening multiple nucleotide polymorphic sites for Xinjiang hemorrhagic fever virus genome and designing corresponding primer groups for multiple PCR amplification, combined with a second-generation sequencing platform for detection, the problems of low detection efficiency and insufficient sensitivity in the existing technology are solved, efficient, accurate and sensitive detection of Xinjiang hemorrhagic fever virus is achieved, and viral mutation monitoring and cross-species traceability are supported.
Patent Information
- Application Number
- CN202411909834.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-03-28
AI Technical Summary
The prior art has problems such as low efficiency, complex operation, inaccurate detection of variants and insufficient sensitivity when detecting Xinjiang hemorrhagic fever virus.
A MNP tag combination, primer set and kit are provided. Multiple nucleotide polymorphic sites are screened through the analysis of the genome of Xinjiang hemorrhagic fever virus, corresponding primer sets are designed for multiple PCR amplification, and a second-generation sequencing platform is used to sequence the amplified products, achieving efficient, accurate and sensitive detection of Xinjiang hemorrhagic fever virus.
It has achieved efficient, accurate and sensitive detection of Xinjiang hemorrhagic fever virus, breaking through the limitations of low efficiency and insufficient sensitivity caused by detecting one marker at a time in the existing technology, and can detect mutations at the same time, providing technical support for virus mutation monitoring and cross-species traceability.
Smart Images
Figure CN119710088A_ABST
Abstract
Description
Technical Field
[0001] The present disclosure relates to the field of biotechnology, and in particular to an MNP marker combination, a primer set, a kit and applications thereof for identifying Xinjiang hemorrhagic fever virus. Background Art
[0002] Xinjiang hemorrhagic fever virus (XHFV) is the pathogen of Xinjiang hemorrhagic fever and is an RNA virus. It was named because it was first isolated from the blood of hemorrhagic fever patients in the Tarim Basin of Xinjiang, my country and the hard ticks caught locally. The host range of Xinjiang hemorrhagic fever virus includes: livestock such as cattle, sheep, horses and camels, as well as wild animals such as gerbils and Tarim rabbits. The Asian Hyalomma tick is its transmission medium and storage host. People are infected by the bites of virus-carrying ticks or through skin wounds. The incubation period is about 7 days, and the onset is acute, with symptoms such as fever, headache, drowsiness, fatigue, vomiting, etc. Severe patients have epistaxis, hematemesis, hematuria, proteinuria and even shock. The identification, genetic variation detection and cross-host tracing of Xinjiang hemorrhagic fever virus play an important role in the prevention of the virus.
[0003] Traditional detection methods mainly include nucleic acid detection technologies based on PCR (Polymerase Chain Reaction). These technologies have their own advantages, but they also have one or more limitations in terms of duration, operational complexity, detection throughput, accuracy and sensitivity of detecting mutations, and cost. PCR detection technology only detects one marker of the virus in one reaction, making its detection efficiency low. During detection, mutations in the primer region may lead to detection failure, resulting in false negatives, and mutations cannot be detected at the same time.
[0004] Therefore, how to provide efficient, accurate and sensitive detection for Xinjiang hemorrhagic fever virus is an urgent problem that needs to be solved.
[0005] Public Content
[0006] In order to solve the problems of the prior art, the present disclosure provides an MNP marker combination, a primer set, a kit and its application for identifying Xinjiang hemorrhagic fever virus. The technical solution is as follows:
[0007] On the one hand, an embodiment of the present disclosure provides an MNP marker combination for identifying Xinjiang hemorrhagic fever virus, the MNP marker combination comprising at least one of marker site MNP-1, marker site MNP-2, and marker site MNP-3, the MNP marker combination being a genomic region screened on the Xinjiang hemorrhagic fever virus genome that is distinguished from other species and has multiple nucleotide polymorphisms within the species, and the nucleotide sequences corresponding to the MNP marker combination in sequence are shown in SEQ ID NO: 1 to SEQ ID NO: 3 in the sequence listing.
[0008] On the other hand, the disclosed embodiment provides a primer set for identifying the MNP marker combination of the Xinjiang hemorrhagic fever virus, the primer set comprising: the first primer pair to the third primer pair, each of the primer pairs comprising a forward primer and a reverse primer, the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the third primer pair, and the reverse primer of the third primer pair are respectively as shown in SEQ ID NO: 4 to SEQ ID NO: 9 in the sequence listing.
[0009] On the other hand, the embodiments of the present disclosure provide a kit for identifying the MNP marker combination of Xinjiang hemorrhagic fever virus, wherein the kit includes the primer set.
[0010] Furthermore, the kit also includes a multiplex PCR premix.
[0011] On the other hand, the embodiments of the present disclosure provide an application of the MNP marker combination, the primer set or the kit, wherein the application includes using the MNP marker combination, the primer set or the kit to identify the Xinjiang hemorrhagic fever virus.
[0012] Furthermore, the application includes: using the MNP marker combination, the primer set or the kit for mutation monitoring and database construction of Xinjiang hemorrhagic fever virus.
[0013] The technical solution provided by the embodiments of the present disclosure has the following beneficial effects:
[0014] The present invention provides an MNP marker combination, a primer set, a kit and applications of Xinjiang hemorrhagic fever virus. The present invention screens a total of 3 MNP marker combinations by analyzing the genome sequence of Xinjiang hemorrhagic fever virus, and designs 3 pairs of primer sets according to the sequence information of the 3 MNP marker combinations; multiple PCR amplification is performed using the designed primer sets, and the amplification products are sequenced by integrating the second-generation sequencing platform, so as to meet the requirements of one-time detection of the 3 MNP marker combinations of Xinjiang hemorrhagic fever virus, break through the limitation of low efficiency caused by one marker of Xinjiang hemorrhagic fever virus detected by the existing PCR-based technology at one time, the limitation of false positive and false negative easily appearing when judging the result based on the detection of one marker, the limitation of low sensitivity caused by the failure of detection of one marker and the limitation of being unable to monitor sequence variation, etc., realize the efficient, accurate and sensitive identification of Xinjiang hemorrhagic fever virus, and provide technical support for the variation monitoring and cross-species tracing of Xinjiang hemorrhagic fever virus. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0016] Figure 1 A flow chart of the screening of Xinjiang hemorrhagic fever virus MNP marker combinations and the design of primer sets provided in the embodiments of the present disclosure;
[0017] Figure 2 A schematic diagram of the polymorphism principle of the MNP labeling site provided in the embodiments of the present disclosure;
[0018] Figure 3 A flow chart for detecting the MNP-labeled combination provided in the embodiments of the present disclosure. DETAILED DESCRIPTION
[0019] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be further described in detail below with reference to the accompanying drawings.
[0020] Embodiment 1
[0021] The disclosed embodiments provide an MNP marker combination for identifying Xinjiang hemorrhagic fever virus, the MNP marker combination comprising at least one of a marker site MNP-1, a marker site MNP-2, and a marker site MNP-3, the MNP marker combination being a genomic region screened on the Xinjiang hemorrhagic fever virus genome that is distinguished from other species and has multiple nucleotide polymorphisms within the species, and the nucleotide sequences corresponding to the MNP marker combination in sequence are shown in SEQ ID NO: 1 to SEQ ID NO: 3 in the sequence list.
[0022] The specific steps for screening MNP marker combinations are:
[0023] The genome sequence of a representative strain of Xinjiang hemorrhagic fever virus was selected as a reference genome, and the other genome sequences of the virus were compared with the reference genome to obtain the single nucleic acid polymorphism sites of the virus;
[0024] On the reference genome, a window of 100 bp to 150 bp was used and a window shift was performed with a step length of 1 bp to screen and obtain multiple candidate MNP marker regions, wherein the candidate MNP marker region was required to contain ≥ 2 single nucleotide variant sites and no single nucleic acid polymorphism sites existed in the 30 bp sequences at both ends;
[0025] After obtaining the candidate MNP markers for the virus, sequence alignment is performed in a public database to screen no less than 3 marker sites specific to the virus as the MNP marker combination.
[0026] Embodiment 2
[0027] The present disclosure embodiment provides a primer set for the MNP marker combination of Xinjiang hemorrhagic fever virus for identification, the primer set includes: the first primer pair to the third primer pair, each primer pair includes a forward primer and a reverse primer, the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the third primer pair, and the reverse primer of the third primer pair are shown in sequence as SEQ ID NO: 4 to SEQ ID NO: 9 in the sequence table. The specific sequence is shown in Table 1.
[0028] Table 1 shows the MNP marker combinations and their corresponding primer pairs
[0029]
[0030] The primer pairs in the primer set provided in this embodiment do not conflict with each other and can be amplified efficiently by multiplex PCR.
[0031] The design of the primer set provided in this embodiment is as follows:
[0032] like Figure 1As shown, multiplex PCR amplification primers for candidate MNP markers are designed by primer design software. The primer design follows the principle that primers do not interfere with each other. All primers can be combined into a primer pool for multiplex PCR amplification, that is, all designed primers can be amplified normally in one amplification reaction.
[0033] Evaluation of detection efficiency of primer sets:
[0034] The positive samples of Xinjiang hemorrhagic fever virus were detected by the primer set provided in this example, and a total of 3 replicates were performed; the sequencing data were analyzed, and 3 pairs of highly compatible and product-specific primer sets were screened out for the specific identification of the 3 MNP markers of Xinjiang hemorrhagic fever virus.
[0035] like Figure 2 As shown, based on the genome sequence of Xinjiang hemorrhagic fever virus publicly available on the Internet, through sequence alignment, the three MNP marker combinations screened for Xinjiang hemorrhagic fever virus are shown in Table 1 above.
[0036] Using positive samples of Xinjiang hemorrhagic fever virus with known copy numbers identified by digital PCR (provided by the Xinjiang Uygur Autonomous Region Center for Disease Control and Prevention), positive samples with copy numbers of Xinjiang hemorrhagic fever virus of 1 copy / reaction, 10 copies / reaction, and 100 copies / reaction were prepared, and an equal volume of sterile water was set as a negative control sample. Three replicate libraries were tested for each sample for 4 consecutive days, that is, 12 sets of sequencing data were obtained for each sample; according to the data analysis results of each sample shown in Table 2, the reproducibility and accuracy of the detection method were evaluated, and the thresholds for quality control system contamination and target Xinjiang hemorrhagic fever virus detection were established. At the same time, the detection process of the MNP marker combination was as follows Figure 3 shown.
[0037] Table 2 is an analysis of the sensitivity and stability of Xinjiang hemorrhagic fever virus identified by the kit
[0038]
[0039] As can be seen from Table 2, in the positive samples with 10 copies / reaction and 100 copies / reaction, the three MNP marker sites of Xinjiang hemorrhagic fever virus were detected, and the sequence was specifically aligned with the reference sequence of the virus, indicating that the primer set provided in this example has good technical stability, high specificity and sensitivity as low as 10 copies / reaction.
[0040] Evaluation of reproducibility and accuracy of detection of Xinjiang hemorrhagic fever virus:
[0041] Based on whether the genotypes of the common detection sites in the two replicates are reproducible, the reproducibility and accuracy of the primer set for identifying Xinjiang hemorrhagic fever virus were evaluated. Specifically, the genotypes of each MNP marker site generated by 12 sets of data from 100 copies / reaction positive samples were compared pairwise, and the results are shown in Table 3.
[0042] Table 3 shows the reproducibility and accuracy evaluation of the primer set
[0043] Repeat 1 Repeat 2 Number of common sites Number of repeatable sites Reproducibility Accuracy S-1 S-2 3 3 100％ 100％ S-1 S-3 3 3 100％ 100％ S-1 S-4 3 3 100％ 100％ S-1 S-5 3 3 100％ 100％ S-1 S-6 3 3 100％ 100％ S-1 S-7 3 3 100％ 100％ S-1 S-8 3 3 100％ 100％ S-1 S-9 3 3 100％ 100％ S-1 S-10 3 3 100％ 100％ S-1 S-11 3 3 100％ 100％ S-1 S-12 3 3 100％ 100％
[0044] As can be seen from Table 3, the number of MNP markers with differences in the main genotype is 0; according to the principle that the reproducible genotype between two repeated experiments is considered accurate, the accuracy rate a=1-(1-r) / 2=0.5+0.5r, r represents the reproducibility, that is, the ratio of the number of reproducible sites of the main genotype to the number of common sites. In the reproducibility test of this embodiment, the difference logarithm of the main genotype of MNP markers between different libraries and different library batches of each sample is 0, that is, the reproducibility r=100%, and the accuracy rate a=100%.
[0045] In the positive samples with 10 copies / reaction and 100 copies / reaction, the primer set of this embodiment can stably detect 3 MNP marker sites of Xinjiang hemorrhagic fever virus, and at most 1 MNP marker site is detected in the negative control; therefore, the judgment standard for Xinjiang hemorrhagic fever virus positivity provided in this embodiment is: when no less than 2 MNP marker combinations of Xinjiang hemorrhagic fever virus are detected in the sample to be tested and the detected sequence abundance (i.e., the ratio of the number of sequences detected in the sample to be tested that are aligned to the MNP marker combination of Xinjiang hemorrhagic fever virus and the total number of sequenced sequences of the sample to be tested) is at least 10 times that of the blank control, it is determined that the nucleic acid of Xinjiang hemorrhagic fever virus is detected in the sample to be tested.
[0046] Embodiment 3
[0047] The disclosed embodiments provide a kit for identifying the MNP marker combination of Xinjiang hemorrhagic fever virus, and the kit includes the primer set provided in Embodiment 2.
[0048] Furthermore, the kit may also include a multiplex PCR premix.
[0049] Embodiment 4
[0050] The disclosed embodiments provide an application of an MNP marker combination, a primer set or a kit, wherein the application includes using the MNP marker combination, the primer set or the kit to identify Xinjiang hemorrhagic fever virus.
[0051] MNP labeling combinations, primer sets or kits were used to identify 6 nucleic acid samples provided by the Xinjiang Uygur Autonomous Region Center for Disease Control and Prevention. Among the 6 nucleic acid samples, 4 were human samples and 2 were tick samples, named S1 to S6 (S1 was a negative sample for Xinjiang hemorrhagic fever virus, and S2 to S6 were positive samples for Xinjiang hemorrhagic fever virus). The identification results are shown in Table 4.
[0052] Table 4 is an analysis of the detection of Xinjiang hemorrhagic fever virus in 6 nucleic acid samples
[0053]
[0054] As can be seen from Table 4, the primer set and the kit can accurately detect the three MNP marker sites of the Xinjiang hemorrhagic fever virus contained in each sample in one reaction, and the simultaneous detection of the three MNP marker sites ensures the true positive of the Xinjiang hemorrhagic fever virus in the sample. For the same detection, the method based on fluorescence PCR detects one marker in one reaction, and the result is determined based on the detection of one marker, which is prone to false positives and false negatives. If the identification based on three markers is to be achieved, the method based on fluorescence PCR needs to be detected three times. This shows that the primer set and the kit provided in this embodiment identify the Xinjiang hemorrhagic fever virus with high accuracy and efficiency.
[0055] Embodiment 5
[0056] The MNP marker combination, primer set or kit is used for mutation monitoring of Xinjiang hemorrhagic fever virus.
[0057] As shown in Table 4, the primer set and kit provided in this embodiment can detect all three MNP marker sites of Xinjiang hemorrhagic fever virus, and the average sequencing coverage of each MNP marker site is 4000 times, as shown in Table 5. The strains in the five samples in Table 4 were compared in pairs at the three marker genotypes. The results are shown in Table 5. The genotypes of the strains in the three human samples are exactly the same, but there is a difference in the genotype of one MNP marker site with the strains in the two tick-source samples. The genotypes of the three MNP marker sites of the strains in the two tick-source samples are exactly the same.
[0058] Table 5 shows the detection and analysis of 4 copies of a Xinjiang hemorrhagic fever virus sample
[0059] Strain 1 Strain 2 Number of commonly detected markers Number of major genotype difference markers S2 S3 3 0 S2 S4 3 0 S2 S5 3 1 S2 S6 3 1 S5 S6 3 0
[0060] As shown in Table 5, the primer set and kit provided in this embodiment can directly identify the genetic variation between strains in real samples without culture by detecting the sequence of MNP markers, and can be used to discover the variation between strains in epidemic prevention monitoring; it can be used in scientific research to ensure the genetic consistency of the same named strains in different laboratories, thereby ensuring the comparability of research results. Therefore, this is of great significance for epidemic prevention monitoring, precision treatment and scientific research of pathogens.
[0061] Embodiment 6
[0062] This embodiment provides an application, which includes using the MNP marker site, primer set or kit to construct a database of Xinjiang hemorrhagic fever virus.
[0063] As shown in Table 3, after the sample to be tested is detected using the primer set, the main genotype of the MNP marker site of the Xinjiang hemorrhagic fever virus in each sample to be tested is obtained, the main genotypes of the strains obtained in all samples are compared, and the main genotypes with differences are entered into the database file to form the MNP fingerprint database of the Xinjiang hemorrhagic fever virus. After the main genotype of the MNP marker site of each sample or strain to be tested is compared with the constructed MNP fingerprint database, the MNP fingerprint map of the strain with differences in the main genotype can be entered into the constructed MNP fingerprint database. Therefore, in theory, the constructed MNP fingerprint database can be continuously updated and enriched. Because the constructed database is based on the gene sequence of the strain detected, it is compatible with all high-throughput sequencing data, and has the characteristics of being fully co-constructed and shared, and updatable at any time.
[0064] The MNP marker combination provided in this embodiment has high specificity in identifying Xinjiang hemorrhagic fever virus; the primer set provided in this embodiment can satisfy the amplification of three MNP marker sites in one reaction, and integrate the second-generation sequencing platform to sequence the amplified products, thereby realizing sequence analysis of the three MNP marker sites of Xinjiang hemorrhagic fever virus in one reaction. Compared with the fluorescence PCR-based method that only uses a pair of primers to detect one site, and what is detected is the fluorescence signal, the primer set provided in this embodiment is efficient, accurate and sensitive in identifying Xinjiang hemorrhagic fever virus, providing technical support for identification.
[0065] The kit provided in this embodiment can detect all MNP marker sites in one reaction. In the reproducibility test, the three MNP marker sites can be stably detected, and the difference logarithm of the MNP marker main genotype between different libraries and different library batches of each sample detected by the kit is 0, the reproducibility r=100%, and the accuracy a=100%, indicating the high accuracy and stability of the kit in detecting Xinjiang hemorrhagic fever virus.
[0066] During identification, samples are tested through high-throughput sequencing. By adding a unique label to each sample, hundreds or thousands of samples can be tested at one time, further improving the detection efficiency. Viruses can be identified by the detected MNP-labeled base sequence without the need for parallel testing of standard samples. By obtaining the detected MNP-labeled base sequence, not only can the virus be identified in one reaction, but the genetic variation of pathogens between samples can also be detected simultaneously.
[0067] The above description is only an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the present disclosure.
Claims
1. A MNP marker combination for identifying Xinjiang hemorrhagic fever virus, characterized in that: The MNP marker combination includes at least one of the marker site MNP-1, the marker site MNP-2 and the marker site MNP-3. The MNP marker combination is a genomic region that is screened on the Xinjiang hemorrhagic fever virus genome and is distinguished from other species and has multiple nucleotide polymorphisms within the species. The nucleotide sequences corresponding to the MNP marker combination in sequence are shown in SEQ ID NO: 1 to SEQ ID NO: 3 in the sequence list.
2. A primer set for identifying the MNP marker combination of Xinjiang hemorrhagic fever virus as claimed in claim 1, characterized in that: The primer set includes: the first primer pair to the third primer pair, each of the primer pairs includes a forward primer and a reverse primer, and the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the third primer pair and the reverse primer of the third primer pair are respectively shown as SEQ ID NO: 4 to SEQ ID NO: 9 in the sequence listing.
3. A kit for identifying the MNP marker combination of Xinjiang hemorrhagic fever virus, characterized in that: The kit comprises the primer set as claimed in claim 2.
4. The kit according to claim 3, characterized in that The kit also includes a multiplex PCR premix.
5. A use of the MNP labeling combination according to claim 1, the primer set according to claim 2 or the kit according to claim 3, characterized in that: The application includes using the MNP marker combination, the primer set or the kit to identify the Xinjiang hemorrhagic fever virus.
6. The use according to claim 5, characterized in that: The application includes: using the MNP marker combination, the primer set or the kit for variation monitoring and database construction of Xinjiang hemorrhagic fever virus.