An MNP marker combination, primer set, reagent kit, and its application for identifying fever with thrombocytopenia syndrome virus.

Abstract

This invention discloses an MNP marker combination, primer set, kit, and application for identifying fever-associated thrombocytopenia syndrome virus (FATS). The MNP marker combination includes at least one of marker sites MNP-1, MNP-2, and MNP-3. The primer set includes primer pairs 1 to 3, each primer pair including a forward primer and a reverse primer. The forward primer of the first primer pair, the reverse primer of the first primer pair, the forward primer of the third primer pair, and the reverse primer of the third primer pair are shown sequentially as SEQ ID NO: 1 to SEQ ID NO: 6 in the sequence listing. This MNP marker combination, primer set, kit, and application for identifying FATS, based on a multi-target detection scheme, achieves efficient, accurate, and sensitive identification and mutation monitoring of FATS.

Description

Technical Field

[0001] This disclosure relates to the field of biotechnology, and in particular to an MNP marker combination, primer set, kit, and application for identifying fever with thrombocytopenia syndrome virus. Background Technology

[0002] Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) is a single-stranded negative-sense RNA virus. Its genome consists of three segments: L (encoding RNA polymerase), M (encoding the glycoprotein precursor Gn / Gc), and S (encoding the nucleoprotein NP and the non-structural protein NSs). This virus is primarily transmitted through tick bites (such as those of the Haemaphysalis longhorn tick), but can also be transmitted from person to person via blood or aerosols. The incubation period is 5–14 days. Clinical features include high fever, thrombocytopenia, leukopenia, and multiple organ failure, with a high mortality rate. SFTSV is prevalent in many East Asian countries and is listed as a high-risk pathogen by the World Health Organization. Early and accurate detection can guide isolation and treatment, reducing the risk of nosocomial transmission. Therefore, developing highly sensitive detection technologies for SFTSV, while also considering genetic variation detection and cross-species tracing, has significant clinical and public health value.

[0003] Traditional detection methods primarily rely on PCR (Polymerase Chain Reaction) nucleic acid detection technology. PCR detection targets only one viral marker per reaction, resulting in low efficiency. Furthermore, detection failures due to primer region variations can lead to false negatives, and it cannot simultaneously detect marker sequences, thus hindering mutation monitoring and cross-host tracing. Therefore, a method is urgently needed that can efficiently and accurately detect SFTSV while also enabling genetic mutation detection and cross-host tracing. Summary of the Invention

[0004] To address the problems of existing technologies, this disclosure provides an MNP marker combination, primer set, reagent kit, and application for identifying fever-associated thrombocytopenia syndrome virus. The technical solution is as follows:

[0005] On one hand, this disclosure provides an MNP marker combination for identifying fever with thrombocytopenia syndrome virus (FPSV). The MNP marker combination includes at least one of marker sites MNP-1, MNP-2, and MNP-3. The MNP marker combination is a genomic region screened on the FPSV genome that is distinct from other species and possesses multiple nucleotide polymorphisms within said species. The positions of marker sites MNP-1 to MNP-3 on the reference sequence are shown in the table below:

[0006] MNP Marker Number Reference sequence MNP starting point MNP endpoint MNP-1 MT114317.1 749 849 MNP-2 OM453236.1 1730 1824 MNP-3 MT114317.1 1133 1274 .

[0007] On the other hand, embodiments of this disclosure provide a primer set for identifying MNP marker combinations of the fever with thrombocytopenia syndrome virus, the primer set comprising: a first primer pair to a third primer pair, each primer pair 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 being as shown in SEQ ID NO: 1 to SEQ ID NO: 6 in the sequence listing.

[0008] In another aspect, embodiments of this disclosure provide a kit for identifying MNP marker combinations for fever with thrombocytopenia syndrome virus, the kit comprising the aforementioned primer set.

[0009] Furthermore, the kit also includes a multiplex PCR premix.

[0010] In another aspect, embodiments of this disclosure provide an application of the MNP marker combination, the primer set, or the kit described herein, the application including using the MNP marker combination, the primer set, or the kit for the identification of the fever with thrombocytopenia syndrome virus.

[0011] Furthermore, the application includes using the MNP marker combination, the primer set, or the kit for mutation monitoring of fever with thrombocytopenia syndrome virus.

[0012] Furthermore, the application includes using the MNP marker combination, the primer set, or the kit for tracing the source of the fever with thrombocytopenia syndrome virus.

[0013] Furthermore, the application includes using the MNP marker combination, the primer set, or the kit for the construction of a database of fever with thrombocytopenia syndrome virus.

[0014] The beneficial effects of the technical solutions provided in this disclosure are:

[0015] This disclosure provides an MNP marker combination, primer set, kit, and application for fever with thrombocytopenia syndrome virus (FPSV). Through analysis of the FPSV genome sequence, three MNP marker combinations were screened, and three primer sets were designed based on the sequence information of these three MNP marker combinations. Both the MNP marker combinations and primer sets exhibit high specificity in identifying FPSV. Multiplex PCR amplification using the designed primer sets, followed by sequencing of the amplified products using a next-generation sequencing platform, allows for the simultaneous detection of all three MNP marker combinations for FPSV. Compared to fluorescence PCR methods that use only one primer pair to detect one site and detect only the fluorescence signal, the primer set provided in this embodiment offers high efficiency, accuracy, and sensitivity in identifying FPSV, providing technical support for identification. It also provides technical support for FPSV mutation monitoring, cross-species tracing, and database construction. The kit provided in this embodiment can detect all MNP marker sites in a single reaction. In the reproducibility test, all three MNP marker sites were stably detected, and the logarithmic difference in major genotypes of MNP markers between different libraries and different batches of libraries tested by the kit was 0, with a reproducibility rate (r) of 100% and an accuracy (a) of 100%, demonstrating the high accuracy and stability of the kit in detecting fever with thrombocytopenia syndrome virus. For identification, high-throughput sequencing was used to detect samples. By adding a unique tag to each sample, hundreds or thousands of samples could be tested simultaneously, further improving detection efficiency. Virus identification was performed using the base sequences of the detected MNP markers, eliminating the need for parallel testing of standard samples. Obtaining the base sequences of the detected MNP markers allows for not only virus identification but also simultaneous detection of genetic variations and source tracing between samples in a single reaction. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram illustrating the polymorphism principle of MNP marker sites provided in the embodiments of this disclosure;

[0018] Figure 2 A flowchart illustrating the detection process of MNP marker combinations provided in this embodiment of the disclosure. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this disclosure clearer, the embodiments of this disclosure will be described in further detail below with reference to the accompanying drawings.

[0020] Example 1

[0021] This disclosure provides an MNP marker combination for identifying fever with thrombocytopenia syndrome virus. The MNP marker combination includes at least one of marker site MNP-1, marker site MNP-2, and marker site MNP-3. The MNP marker combination is a genomic region screened on the genome of fever with thrombocytopenia syndrome virus that is distinct from other species and has multiple nucleotide polymorphisms within a species. The positions of marker sites MNP-1 to MNP-3 on the reference sequence are shown in Table 1.

[0022] Table 1 shows the MNP marker combinations.

[0023] Site ID Reference sequence MNP starting point MNP endpoint length MNP-1 MT114317.1 749 849 100 MNP-2 OM453236.1 1730 1824 94 MNP-3 MT114317.1 1133 1274 141

[0024] Example 2

[0025] This disclosure provides a primer set for identifying MNP-labeled combinations of fever with thrombocytopenia syndrome virus. The primer set includes primer pairs 1 to 3, each primer pair including a forward primer and a reverse primer. The forward primer of primer pair 1, the reverse primer of primer pair 1, the forward primer of primer pair 3, and the reverse primer of primer pair 3 are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 6 in the sequence listing. The specific sequences are shown in Table 2.

[0026] Table 2 lists the primer pairs corresponding to MNP marker combinations.

[0027]

[0028] The primer pairs in the primer set provided in this embodiment do not conflict with each other, and can be efficiently amplified by multiplex PCR.

[0029] Detection efficiency evaluation of primer sets:

[0030] The primer set provided in this embodiment was used to detect positive samples of fever with thrombocytopenia syndrome virus (FPSV), with a total of 3 replicates. The sequencing data were analyzed, and 3 pairs of highly compatible and product-specific primer sets were selected for specific identification of 3 MNP markers of FPSV.

[0031] The three MNP marker combinations for screening viruses for fever with thrombocytopenia syndrome disclosed in this embodiment are as follows: Figure 1 As shown.

[0032] Positive samples of fever with thrombocytopenia syndrome virus (FPSV) with known copy numbers, identified by digital PCR and provided by the Hubei Provincial Center for Disease Control and Prevention, were used to prepare FPSV positive samples with copy numbers of 1 copy / reaction, 10 copies / reaction, and 100 copies / reaction. An equal volume of sterile water was also provided as a negative control. Three replicate libraries were analyzed for each sample over four consecutive days, resulting in 12 sequencing data sets per sample. Based on the data analysis results for each sample shown in Table 3, the reproducibility and accuracy of the detection method were evaluated, and thresholds for quality control system contamination and the detection of the target FPSV were established. The detection procedure for MNP marker combinations is as follows: Figure 2 As shown.

[0033] Table 3 shows the sensitivity and stability of primer sets in identifying the virus associated with fever with thrombocytopenia syndrome.

[0034]

[0035]

[0036] As shown in Table 3, in the positive samples with 10 copies / reaction and 100 copies / reaction, all three MNP marker sites of fever with thrombocytopenia syndrome virus were detected, and the sequence was specifically aligned to the reference sequence of the virus, indicating that the primer set provided in this embodiment has good technical stability, high specificity and sensitivity as low as 10 copies / reaction.

[0037] Assessment of the reproducibility and accuracy of detecting fever with thrombocytopenia syndrome virus:

[0038] The reproducibility and accuracy of primer set identification for fever with thrombocytopenia syndrome virus were evaluated based on whether the genotypes of common detection sites could be reproduced in two replicates. Specifically, the genotypes of each MNP marker site generated from 12 sets of data from 100 copies / reaction positive samples were compared pairwise, and the results are shown in Table 4.

[0039] Table 4 shows the reproducibility and accuracy of the primer sets.

[0040] Repeat 1 Repeat 2 Number of common sites Number of repeatable sites Recurrence rate r Accuracy a 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％

[0041] As shown in Table 4, the number of MNP markers with different major genotypes was 0. Based on the principle that reproducible genotypes between two replicates are considered accurate, the accuracy a = 1 - (1 - r) / 2 = 0.5 + 0.5r, where r represents the reproducibility rate, i.e., the ratio of the number of reproducible loci to the number of shared loci. In this embodiment, the logarithm of the difference in major genotypes of MNP markers between different libraries and between different library batches for each sample in the reproducibility test was 0, i.e., the reproducibility rate r = 100%, and the accuracy a = 100%.

[0042] In positive samples with 10 copies / reaction and 100 copies / reaction, the primer set of this embodiment can stably detect the three MNP marker sites of fever with thrombocytopenia syndrome virus, while the negative control can detect a maximum of one MNP marker site. Therefore, the criterion for determining the positivity of fever with thrombocytopenia syndrome virus provided in this embodiment is: when no less than two MNP marker combinations of fever with thrombocytopenia syndrome virus are detected in the test sample and the abundance of the detected sequences (i.e., the ratio of the number of sequences in the test sample that are matched to the MNP marker combination of fever with thrombocytopenia syndrome virus to the total number of sequences in the test sample) is at least 10 times that in the blank control, it is determined that the nucleic acid of fever with thrombocytopenia syndrome virus is detected in the test sample.

[0043] Example 3

[0044] This disclosure provides a kit for identifying MNP marker combinations for fever with thrombocytopenia syndrome virus, the kit comprising the primer set provided in Example 2.

[0045] Furthermore, the kit may also include a multiplex PCR premix.

[0046] Example 4

[0047] This disclosure provides an application of an MNP marker combination, primer set, or kit, including the use of the MNP marker combination, primer set, or kit to identify fever with thrombocytopenia syndrome virus.

[0048] Eight nucleic acid samples provided by the Hubei Provincial Center for Disease Control and Prevention were identified using MNP marker combinations, primer sets, or kits. Six of the eight samples were human-derived, and two were tick-derived, named S1–S8 respectively (where S1 was a negative sample for fever with thrombocytopenia syndrome virus, and S2–S8 were positive samples for fever with thrombocytopenia syndrome virus). The identification results are shown in Table 5.

[0049] Table 5 shows the analysis of viral detection results for fever with thrombocytopenia syndrome in 6 nucleic acid samples.

[0050]

[0051] Table 5 shows that the primer set and kit can accurately detect the three MNP marker sites of fever with thrombocytopenia syndrome virus in each sample with a single reaction. The simultaneous detection of the three MNP marker sites ensures a true positive result for fever with thrombocytopenia syndrome virus in the sample. Furthermore, combined with... Figure 1It is known that fluorescent PCR-based methods detect one label at a time, and the result of detecting only one label is easily interpreted, leading to false positives and false negatives. To achieve identification based on three labels, fluorescent PCR requires three detections. This demonstrates that the primer set and kit provided in this embodiment have high accuracy and efficiency in identifying fever with thrombocytopenia syndrome virus.

[0052] Example 5

[0053] MNP marker combinations, primer sets, or kits can be used to monitor variants of fever with thrombocytopenia syndrome virus.

[0054] As shown in Table 5 of Example 3, the primer set and kit provided in this example can detect all three MNP marker sites of fever with thrombocytopenia syndrome virus, with an average sequencing coverage of 4000-fold for each MNP marker site. Pairwise comparisons were performed on the genotypes of the strains in the seven positive samples (S2-S8) in Table 5 at the three marker sites. The results are shown in Table 6. The genotypes of the strains in the five human samples (S2-S6) and one tick sample (S7) were completely identical, but they differed from the strain in the other tick sample (S8) at one MNP marker site.

[0055] Table 6 shows the detection analysis of 7 positive samples for fever with thrombocytopenia syndrome virus.

[0056]

[0057]

[0058] As shown in Table 6, the primer set and kit provided in this embodiment can directly identify genetic variations among strains in real samples without culture by detecting the sequence of the MNP marker. This can be used to detect variations among strains in disease prevention and control monitoring; and to ensure the genetic consistency of the same named strains in different laboratories in scientific research, thereby ensuring the comparability of research results. Therefore, this is of great significance for pathogen prevention and control monitoring, precision treatment, and scientific research.

[0059] Example 6

[0060] This embodiment provides an application that includes using MNP marker sites, primer sets, or kits for cross-host tracing of fever with thrombocytopenia syndrome virus.

[0061] As shown in Table 6 of Example 5, the viruses in all 7 positive samples tested positive for 3 MNP marker sites, but the genotypes of the MNP sites differed. The tick-borne sample S7 and the 5 human samples (S2-S6) had identical genotypes, while the tick-borne sample S8 and the 5 human samples showed differences in genotype at one marker site. This indicates that the virus in the human samples was more likely transmitted from the tick in sample S7 than from sample S8.

[0062] Example 7

[0063] This embodiment provides an application that includes using MNP marker sites, primer sets, or kits for the construction of a database of fever with thrombocytopenia syndrome virus.

[0064] As shown in Table 5 of Example 2, after testing the samples using this primer set, the major genotype of the MNP marker site of Severe Fever with Thrombocytopenia Syndrome (SFTS) in each sample is obtained. The major genotypes of the strains obtained from all samples are compared, and the major genotypes with differences are entered into the database file to form an MNP fingerprint database for SFTS. Each time the major genotype of the MNP marker site of a sample or strain is compared with the constructed MNP fingerprint database, the MNP fingerprint profiles of strains with different major genotypes can be entered into the constructed MNP fingerprint database. Therefore, theoretically, the constructed MNP fingerprint database can be continuously updated and enriched. Because the constructed database is based on the gene sequence of the detected strains, it is compatible with all high-throughput sequencing data and has the characteristics of being fully collaboratively built and shared, and updated at any time.

[0065] The above description is merely an optional embodiment of this disclosure and is not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. The application of a primer set for identifying MNP marker combinations of fever with thrombocytopenia syndrome virus, characterized in that, The application includes: performing multiplex PCR amplification and high-throughput sequencing on the test sample using the primer set to analyze the fever with thrombocytopenia syndrome virus in the test sample for non-therapeutic and diagnostic purposes; the primer set includes: primer pairs 1 to 3, each primer pair including 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: 1 to SEQ ID NO: 6 in the sequence listing.

2. The application according to claim 1, characterized in that, The applications include using the primer set for mutation monitoring of the virus causing fever with thrombocytopenia syndrome.

3. The application according to claim 1, characterized in that, The applications include: using the primer set for tracing the origin of the virus causing fever with thrombocytopenia syndrome.

4. The application according to claim 1, characterized in that, The applications include using the primer set for the construction of a database of fever with thrombocytopenia syndrome virus.

5. A kit for identifying MNP marker combinations for fever with thrombocytopenia syndrome virus, characterized in that, The kit includes the primer set as described in claim 1.

6. The reagent kit according to claim 5, characterized in that, The kit also includes a multiplex PCR premix.

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