MNP marker combination, primer group and kit for identifying severe fever with thrombocytopenia syndrome virus and application of MNP marker combination, primer group and kit

Abstract

The invention discloses an MNP marker combination, a primer group and a kit for identifying severe fever with thrombocytopenia syndrome virus and application of the MNP marker combination, the primer group and the kit, the MNP marker combination comprises at least one of a marker site MNP-1, a marker site MNP-2 and a marker site MNP-3, and the primer group comprises a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair and a sixth primer pair, each primer pair comprises a forward primer and a reverse primer, and 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 sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 6 in a sequence table. According to the MNP marker combination, the primer group and the kit for identifying the severe fever with thrombocytopenia syndrome virus and the application of the MNP marker combination, the primer group and the kit, efficient, accurate and sensitive identification and variation monitoring on the severe fever with thrombocytopenia syndrome virus are realized on the basis of a multi-target detection scheme.

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 fever with thrombocytopenia syndrome virus. Background Art

[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 glycoprotein precursors Gn / Gc), and S (encoding nucleoprotein NP and nonstructural protein NSs). The virus is primarily transmitted through tick bites (e.g., Haemaphysalis longicornis) 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 has become endemic in many countries in East Asia and is classified 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 SFTSV detection technologies that combine genetic variation detection and cross-host tracing has significant clinical and public health value.

[0003] Traditional detection methods are mainly based on PCR (Polymerase Chain Reaction) nucleic acid detection technology. PCR detection technology only detects one marker of the virus in one reaction, which has low detection efficiency. During detection, it may also fail due to variations in the primer region, resulting in false negatives. Moreover, it cannot detect the sequence of the marker at the same time, and therefore cannot perform variation monitoring and cross-host tracing. Therefore, there is an urgent need for a method that can efficiently and accurately detect SFTSV and take into account both genetic variation detection and cross-host tracing. Summary of the Invention

[0004] To address the problems of the prior art, the present disclosure provides an MNP labeling combination, primer set, kit, and application thereof for identifying fever with thrombocytopenia syndrome virus. The technical solution is as follows:

[0005] In one aspect, an embodiment of the present disclosure provides an MNP marker combination for identifying fever with thrombocytopenia syndrome virus, wherein the MNP marker combination includes at least one of a marker site MNP-1, a marker site MNP-2, and a marker site MNP-3. The MNP marker combination is a genomic region that is screened on the fever with thrombocytopenia syndrome virus genome and is distinguished from other species and has multiple nucleotide polymorphisms within the species. The positions of the marker sites MNP-1 to MNP-3 on the reference sequence are shown in the following table:

[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, an embodiment of the present disclosure provides a primer set for identifying the MNP marker combination of the fever with thrombocytopenia syndrome virus, the primer set comprising: a first primer pair to a 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: 1 to SEQ ID NO: 6 in the sequence listing.

[0008] In yet another aspect, the present disclosure provides a kit for identifying MNP marker combinations for fever with thrombocytopenia syndrome virus, the kit comprising the above primer set.

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

[0010] 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 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 monitoring mutations of the 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 constructing a database of fever with thrombocytopenia syndrome virus.

[0014] The technical solutions provided by the embodiments of the present disclosure have the following beneficial effects:

[0015] The present disclosure provides an MNP marker combination, primer set, kit, and application thereof for the fever with thrombocytopenia syndrome virus. The present disclosure screened a total of three MNP marker combinations by analyzing the genome sequence of the fever with thrombocytopenia syndrome virus, and designed three pairs of primer sets based on the sequence information of the three MNP marker combinations; the MNP marker combination and primer set have high specificity in identifying the fever with thrombocytopenia syndrome virus. Multiplex PCR amplification is performed using the designed primer set, and the amplified products are sequenced using a second-generation sequencing platform. This can meet the requirements of the one-time detection of the three MNP marker combinations of the fever with thrombocytopenia syndrome virus. Compared with the fluorescence PCR-based method, which only uses a pair of primers to detect one site and detects a fluorescent signal, the primer set provided in this embodiment is efficient, accurate, and sensitive in identifying the fever with thrombocytopenia syndrome virus, providing technical support for identification. It also provides technical support for the variation monitoring, cross-species tracing, and database construction of the fever with thrombocytopenia syndrome virus. The kit provided in this embodiment can meet the requirements of detecting all MNP marker sites in a single reaction. In the reproducibility test, the three MNP-labeled sites can be stably detected, and the logarithm of the difference in the main genotype of the MNP marker between different libraries and different library construction batches for 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 the fever with thrombocytopenia syndrome virus. During identification, the samples are tested by high-throughput sequencing. By adding a unique label to each sample, hundreds of thousands of samples can be tested at one time, further improving the detection efficiency. The virus is identified by the detected MNP-labeled base sequence, without the need for parallel testing of standard samples. By obtaining the base sequence of the detected MNP marker, not only can the virus be identified in a single reaction, but the genetic variation and traceability of the pathogen between samples can also be detected simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the description of the embodiments. 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 any creative work.

[0017] Figure 1 Schematic diagram of the polymorphism principle of MNP labeling sites provided in the embodiments of the present disclosure;

[0018] Figure 2 Flow chart of the detection of MNP-labeled combinations 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] Example 1

[0021] The present disclosure provides an MNP marker combination for identifying fever with thrombocytopenia syndrome virus. 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 screened on the fever with thrombocytopenia syndrome virus genome that is different from other species and has multiple nucleotide polymorphisms within the 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 labeling 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] The present disclosure provides a primer set for identifying a combination of MNP markers for 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 the first primer pair, the reverse primer of the first primer pair, the forward primers 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. The specific sequences are shown in Table 2.

[0026] Table 2 shows the primer pairs corresponding to the MNP labeling combinations

[0027]

[0028] 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.

[0029] Evaluation of detection efficiency of primer sets:

[0030] The primer set provided in this example was used to detect positive samples of fever with thrombocytopenia syndrome virus, with a total of three replicates; the sequencing data was analyzed to screen out three pairs of highly compatible and product-specific primer sets for the specific identification of the three MNP markers of fever with thrombocytopenia syndrome virus.

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

[0032] Using a positive sample of fever with thrombocytopenia syndrome virus with a known copy number identified by digital PCR (provided by the Hubei Provincial Center for Disease Control and Prevention), positive samples with a copy number of 1, 10, and 100 copies / reaction of the fever with thrombocytopenia syndrome virus were prepared, and an equal volume of sterile water was set as a negative control sample. Three replicate libraries were tested for each sample, and the test was continued for 4 consecutive days, that is, 12 sets of sequencing data were obtained for each sample; based on the data analysis results of each sample shown in Table 3, the reproducibility and accuracy of the detection method were evaluated, and the thresholds for quality control system contamination and target fever with thrombocytopenia syndrome virus detection were established. At the same time, the detection process of the MNP labeling combination is as follows Figure 2 shown.

[0033] Table 3 is an analysis of the sensitivity and stability of the primer set for identifying fever with thrombocytopenia syndrome virus

[0034]

[0035]

[0036] As can be seen from Table 3, in the positive samples with 10 copies / reaction and 100 copies / reaction, the three MNP marker sites of the fever with thrombocytopenia syndrome 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.

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

[0038] The reproducibility and accuracy of the primer set for identifying FEBT virus were evaluated based on the reproducibility of the genotypes at the commonly detected sites across two replicates. Specifically, the genotypes at each MNP-labeled site generated from 12 sets of data from positive samples with 100 copies / reaction were compared pairwise. The results are shown in Table 4.

[0039] Table 4 shows the reproducibility and accuracy evaluation of the primer set

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

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

[0042] In positive samples with 10 copies / reaction and 100 copies / reaction, the primer set of this example can stably detect three MNP marker sites of fever with thrombocytopenia syndrome virus, and in the negative control, at most one MNP marker site is detected; therefore, the criterion for determining whether fever with thrombocytopenia syndrome virus is positive provided in this example is: when at least 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 detected in the test sample that are aligned to the MNP marker combination of fever with thrombocytopenia syndrome virus to the total number of sequenced sequences in the test sample) is at least 10 times that of the blank control, it is determined that fever with thrombocytopenia syndrome virus nucleic acid has been detected in the test sample.

[0043] Example 3

[0044] The disclosed embodiments provide a kit for identifying MNP marker combinations of fever with thrombocytopenia syndrome virus, and the kit includes the primer set provided in Example 2.

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

[0046] Example 4

[0047] 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 for identifying 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 labeling combinations, primer sets, or kits. Among the eight nucleic acid samples, six were human samples and two were tick samples, designated S1 to S8 (S1 was a negative sample for FETV, and S2 to S8 were positive samples for FETV). The identification results are shown in Table 5.

[0049] Table 5 is an analysis of the detection of HFTS virus in 6 nucleic acid samples

[0050]

[0051] As shown in Table 5, the primer set and kit can accurately detect the three MNP-labeled sites of the HFST virus in each sample through a single reaction. The simultaneous detection of the three MNP-labeled sites ensures the true positive of the HFST virus in the sample. Figure 1As can be seen, the fluorescent PCR-based method detects one marker per reaction, and judging the results of the detection of a single marker is prone to false positive and false negative results. If identification based on three markers is to be achieved, the fluorescent PCR-based method requires three detections. This shows that the primer set and kit provided in this example have high accuracy and efficiency in identifying FETV.

[0052] Example 5

[0053] The MNP marker combination, primer set or kit is used for monitoring mutation of fever with thrombocytopenia syndrome virus.

[0054] As shown in Table 5 provided in Example 3, the primer set and kit provided in this Example can detect all three MNP marker sites of the fever with thrombocytopenia syndrome virus, and the average sequencing coverage of each MNP marker site is 4000 times. The genotypes of the strains in the seven positive samples (S2 to S8) in Table 5 were compared in pairs at the three markers. The results are shown in Table 6. The genotypes of the strains in the five human samples (S2 to S6) and one tick-derived sample (S7) are exactly the same, but they differ from the strains in the other tick-derived sample (S8) at the genotype of one MNP marker site.

[0055] Table 6 shows the detection and analysis of 7 positive samples of FSTS virus

[0056]

[0057]

[0058] As shown in Table 6, the primer set and kit provided in this example can directly identify genetic variation between strains in real samples without culture by detecting the sequence of the MNP marker. This can be used to identify variation between strains in epidemic prevention monitoring and to ensure genetic consistency of identically named strains in different laboratories in scientific research, thereby ensuring comparability of research results. Therefore, this is of great significance for epidemic prevention monitoring, precision treatment, and scientific research of pathogens.

[0059] Example 6

[0060] This embodiment provides an application, which includes using the MNP marker site, primer set or kit for cross-host tracing of the fever with thrombocytopenia syndrome virus.

[0061] As shown in Table 6 of Example 5, the viruses in the seven positive samples all detected three MNP marker sites, but the genotypes of the MNP sites were different. The genotypes of the tick-derived sample S7 and the five human samples (S2-S6) were exactly the same, while the genotypes of the tick-derived sample S8 and the five human samples all differed at one marker site, indicating that the virus in the human sample was more likely transmitted by the tick in the S7 sample rather than the S8 sample.

[0062] Example 7

[0063] This embodiment provides an application, which includes using the MNP labeling site, primer set or kit for constructing a database of fever with thrombocytopenia syndrome virus.

[0064] As shown in Table 5 provided in Example 2, after the primer set is used to detect the sample to be tested, the main genotype of the MNP marker site of the fever with thrombocytopenia syndrome 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 fever with thrombocytopenia syndrome 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 patterns of the strains 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 tested, 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.

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

Claims

1. An MNP marker combination for identifying fever with thrombocytopenia syndrome virus, characterized in that: The MNP marker combination includes at least one of the marker sites MNP-1, MNP-2, and MNP-3. The MNP marker combination is a genomic region that is screened on the genome of the fever with thrombocytopenia syndrome virus and is distinguished from other species and has multiple nucleotide polymorphisms within the species. The positions of the marker sites MNP-1 to MNP-3 on the reference sequence are shown in the following table:

2. A primer set for identifying the MNP marker combination of the fever with thrombocytopenia syndrome virus according to claim 1, characterized in that: The primer set includes: a first primer pair to a 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 shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 6 in the sequence listing.

3. A kit for identifying MNP-labeled combinations of fever with thrombocytopenia syndrome virus, characterized in that: The kit comprises the primer set according to claim 2.

4. The kit according to claim 3, wherein 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 for identifying the fever with thrombocytopenia syndrome 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 monitoring the variation of fever with thrombocytopenia syndrome virus.

7. The use according to claim 5, characterized in that The application includes: using the MNP marker combination, the primer set or the kit for tracing the source of fever with thrombocytopenia syndrome virus.

8. The use according to claim 5, characterized in that The application includes: using the MNP marker combination, the primer set or the kit for constructing a database of fever with thrombocytopenia syndrome virus.

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