A primer set, kit, and application for detecting MNP marker sites of zaruzin virus.
By designing a primer set and kit for detecting the MNP marker site of Zarruvirus, and utilizing multiplex PCR amplification and next-generation sequencing platforms, the problems of accuracy and mutation monitoring in existing technologies for detecting Zarruvirus have been solved, achieving high-specificity, high-throughput, and high-efficiency virus detection and mutation monitoring.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- LISHUI CENT FOR DISEASE CONTROL & PREVENTION
- Filing Date
- 2026-02-12
- Publication Date
- 2026-05-26
AI Technical Summary
Existing technologies are insufficient for accurate and sensitive detection of zaruvirus, and cannot simultaneously detect strain variations of zaruvirus, leading to cross-reactivity and detection failure.
A primer set and kit for detecting MNP marker sites of zaruzin virus were designed. Three MNP marker sites were screened and detected through multiplex PCR amplification and next-generation sequencing platform, achieving high-specificity, high-throughput, and high-efficiency virus detection and mutation monitoring.
It achieves high specificity, high throughput, high efficiency, and high accuracy in the detection of Zarrovirus, can accurately identify the virus and monitor its mutations, constructs a DNA fingerprint database, and supports virus identification and mutation monitoring.
Smart Images
Figure CN122081561A_ABST
Abstract
Description
Technical Field
[0001] This disclosure relates to the field of biotechnology, and in particular to a primer set, kit, and application for detecting MNP marker sites of zaruzin virus. Background Technology
[0002] Sapovirus is a significant pathogen causing acute gastroenteritis, primarily transmitted via the fecal-oral route. It easily leads to outbreaks in crowded places such as schools and nursing homes, posing a serious threat to public health. Currently, commonly used clinical methods for sapovirus detection include PCR-based nucleic acid testing. However, this technique is prone to cross-reaction with homologous viruses such as norovirus due to insufficient specificity, and can fail due to primer region mutations. Furthermore, it cannot simultaneously detect sapovirus strain variations. Therefore, accurately and sensitively detecting sapovirus and monitoring its mutations are urgent problems that need to be solved.
[0003] Public content
[0004] To address the problems of existing technologies, this disclosure provides a primer set, a reagent kit, and their applications for detecting MNP marker sites of zaruzin virus. The technical solution is as follows:
[0005] On one hand, this disclosure provides a primer set for detecting the MNP marker site of zaruzin virus, the primer set including at least one of 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.
[0006] On the other hand, this disclosure provides a kit for detecting MNP marker sites of zaruzin virus, the kit comprising the above-described primer set.
[0007] Specifically, the kit also includes a multiplex PCR premix.
[0008] Furthermore, this disclosure provides an application of the above-described primer set or kit in the identification of the zaaruvirus, wherein the marker sites of MNP-1 to MNP-3 are as follows:
[0009] .
[0010] Specifically, the specific steps for identifying the application of the Zaruru virus are as follows:
[0011] First, total viral DNA is obtained from the sample to be tested. Using the kit provided by this invention, a first round of multiplex PCR amplification is performed on the total viral DNA and a blank control, with a cycle number not exceeding 25, to obtain the first-round amplification product. After purifying the first-round amplification product, sample tags and next-generation sequencing adapters based on the second-round PCR amplification are added to obtain the second-round amplification product. The second-round amplification product is purified and quantified. When detecting multiple strains, equal volumes of the second-round amplification products are mixed and high-throughput sequencing is performed. The sequencing results are aligned to the reference sequence of the virus to obtain the number of detection sequences and genotype data of the total viral DNA. Based on the number of sequencing sequences and the number of detected MNP marker sites obtained from the total viral DNA and the blank control, data quality control and data analysis are performed on the sequencing data of the total viral DNA to obtain the number of detected MNP sites, the number of sequencing sequences covering each MNP marker site, and the genotype data of the MNP marker sites.
[0012] When identifying zaruvirus, quality control is performed based on the number of zaruvirus sequencing sequences and the number of MNP sites detected in the test sample and the blank control, and then it is determined whether the test sample contains the nucleic acid of zaruvirus.
[0013] Specifically, when no less than two MNP marker sites of zarrovirus are detected in the sample to be tested and the abundance of the detected sequence is at least 10 times that of the blank control, it is determined that the nucleic acid of zarrovirus has been detected in the sample to be tested.
[0014] In another aspect, this disclosure provides an application of the above-described primer set or kit in the monitoring and database construction of the Zaruzon virus mutation, wherein the marker sites of MNP-1 to MNP-3 are as follows:
[0015] .
[0016] Specifically, when the application includes zaru virus mutation monitoring and database construction, the genotype data of the MNP marker sites of the zaru virus in the test samples are identified and entered into a database file to form the DNA fingerprint database of the zaru virus. Different test samples are compared with the DNA fingerprint database of the zaru virus. The comparison results are used to determine whether the zaru virus in the test samples differs from the strains in the DNA fingerprint database in terms of the main genotype at the MNP marker sites. The main genotype is the genotype supported by more than 50% of the sequencing fragments at one MNP marker site. Zaru viruses with main genotype differences at at least one MNP marker site are considered new variant types, and their genotypes are included in the DNA fingerprint database.
[0017] The beneficial effects of the technical solution provided in this disclosure are as follows: This disclosure provides a primer set, kit, and application for detecting MNP marker sites of Zarru virus. By analyzing the genome sequence of Zarru virus, this invention screened a total of 3 MNP marker sites and designed a multiplex primer set based on the sequence information of the 3 MNP marker sites. Multiplex PCR amplification was performed using the designed multiplex primer set, and the amplified products were sequenced using a second-generation sequencing platform. This can meet the requirement of detecting 3 MNP marker sites of Zarru virus at one time, breaking through the limitations of existing technologies that detect one marker of one type of Zarru virus at one time and rely on parallel detection of standard samples. This achieves high specificity, high throughput, high efficiency, and high accuracy detection of Zarru virus, providing technical support for Zarru virus identification, mutation monitoring, and database construction. Attached Figure Description
[0018] 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.
[0019] Figure 1 This is a flowchart of the screening and primer design for the Zanthoxyvirus MNP marker sites provided in Embodiment 1 of this disclosure;
[0020] Figure 2 This is a flowchart of the detection process for MNP marker sites provided in Embodiment 1 of this disclosure;
[0021] Figure 3 This is a schematic diagram of the MNP marker polymorphism principle provided in Embodiment 3 of this disclosure. Detailed Implementation
[0022] 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.
[0023] Example 1
[0024] This disclosure provides a primer set for detecting the MNP marker site of zaruzin virus. The primer set includes at least one of 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. Wherein, combining Figure 1 As shown in Table 1, based on the publicly available genome sequence of the zaruvirus, three MNP marker sites for screening the zaruvirus were identified through sequence alignment.
[0025] Table 1 shows the starting positions of MNP marker sites on the reference sequence and their corresponding primer pairs.
[0026]
[0027] Primer design:
[0028] like Figure 2 As shown, multiplex PCR amplification primers for candidate MNP marker sites were designed using primer design software. The primer design followed the principle that primers do not interfere with each other, and all primers can be combined into a primer set for multiplex PCR amplification, that is, all designed primers can be amplified normally in one amplification reaction.
[0029] 1. The detection of zarif virus exhibits technical stability, high specificity, and a sensitivity as low as 10 copies / reaction, as detailed below:
[0030] Samples of zaruvirus were prepared at copy numbers of 1 copy / reaction, 10 copies / reaction, and 100 copies / reaction. An equal volume of sterile water was used as a blank control. The detection procedure for MNP marker sites is as follows: Figure 3 As shown in Table 2, three replicate libraries were tested for each sample over four consecutive days, resulting in 12 sequencing data sets per sample. The data were then compiled into Table 2. Based on the data analysis results for each sample shown in Table 2, the reproducibility and accuracy of the detection method were evaluated, and thresholds for contamination and target virus detection in the quality control system were established.
[0031] Table 2 shows the sensitivity and stability analysis of the detection of zaruzin virus.
[0032]
[0033] As shown in Table 2, all three MNP marker sites of zajuvirus were detected in samples with 10 copies / reaction and 100 copies / reaction, and the sequence was specifically aligned to the reference sequence of the virus. This indicates that the primer set and the detection method have technical stability, high specificity and sensitivity down to 10 copies / reaction.
[0034] 2. Assessment of the reproducibility and accuracy of the detection of sarrovirus:
[0035] The reproducibility and accuracy of the MNP marker detection method for detecting zaruzinia virus were evaluated based on whether the genotypes of the 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 3.
[0036] Table 3 shows the reproducibility and accuracy assessment of genotype detection.
[0037]
[0038] As shown in Table 3, 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 example, the logarithm of the difference in major genotypes of MNP markers between different libraries and between different library preparation batches for each sample was 0, i.e., the reproducibility rate r = 100%, and the accuracy a = 100%.
[0039] In both 10-copy / reaction and 100-copy / reaction positive samples, the primer set consistently detected all three MNP marker sites of Zarrovirus, while the blank control detected at most one MNP marker site. Therefore, the criterion for determining Zarrovirus positivity in this embodiment is: when at least two Zarrovirus MNP marker sites are detected in the test sample and the abundance of detected sequences (i.e., the ratio of the number of sequences aligned to Zarrovirus MNP marker sites in the test sample to the total number of sequences in the test sample) is at least 10 times that in the blank control, Zarrovirus nucleic acid is detected in the test sample.
[0040] This demonstrates that the primer set can perform multi-target detection of Zarrovirus, thereby achieving high-throughput, high-efficiency, high-accuracy, and high-sensitivity detection and mutation monitoring of Zarrovirus, and the primer pairs in the primer set do not conflict with each other.
[0041] Example 2
[0042] This disclosure provides a kit for detecting MNP marker sites of zaruzin virus, the kit comprising the primer set provided in Example 1.
[0043] Specifically, the kit also includes a multiplex PCR premix.
[0044] The kit includes a primer set for the detection of MNP marker combinations for Zarrovirus, and the kit provided in this embodiment can accurately identify Zarrovirus.
[0045] This kit is based on the primer set provided in Example 1. The specific sequence information of the primer set can be found in the above example. Since this kit adopts some or all of the technical solutions in Example 1, it has at least all the beneficial effects brought about by the technical solutions in Example 1, which will not be elaborated here.
[0046] Example 3
[0047] This disclosure provides an application of the primer set of Example 1 or the kit of Example 2 in the identification of zaruzin virus. The marker sites of MNP-1 to MNP-3 are shown below:
[0048] .
[0049] Screening for MNP marker combinations of zanuvirus:
[0050] The genome sequence of a representative strain of Zarrovirus was selected as the 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.
[0051] On the reference genome, the region with multiple candidate MNP marker sites was screened by shifting the window in the range of 100bp to 300bp with a step size of 1bp. The candidate MNP marker site region is required to contain ≥2 single nucleotide variant sites and there are no single nucleotide polymorphism sites on the sequences at both ends of 30bp.
[0052] At least five sites with high regional discrimination (DP) values among the candidate polynucleotide polymorphism sites were selected; the selection criteria were: DP = d / t.
[0053] In the formula, t is the number of comparisons when comparing all genomes in the candidate polynucleotide polymorphism site region, and d is the number of genomes with at least two single nucleotide polymorphism differences in the candidate polynucleotide polymorphism site region.
[0054] Other step sizes can also be used in the window translation stage, not limited to 1 bp. However, this embodiment uses 1 bp, which is beneficial for comprehensive screening of polymorphic sites on the viral genome.
[0055] After obtaining the MNP marker sites of the virus, sequence alignment was performed in a public database to screen at least 5 virus-specific sites as candidate MNP marker sites.
[0056] Nucleic acid samples from six sewage samples collected by the Lishui Municipal Center for Disease Control and Prevention were analyzed using an MNP marker site detection kit. The samples were named S1-S6 (S1 being a negative sample for Zarruvirus, and S2-S6 being positive samples for Zarruvirus). First, total viral DNA was obtained from the samples. The kit provided in this invention was used to perform a first round of multiplex PCR amplification on the total viral DNA and a blank control, with a cycle count not exceeding 25, yielding the first-round amplification product. After purifying the first-round amplification product, sample tags and next-generation sequencing adapters based on the second-round PCR amplification were added to obtain the second-round amplification product. The second-round amplification product was purified and quantified. When detecting multiple strains, equal volumes of the second-round amplification product were mixed and subjected to high-throughput sequencing. The sequencing results were compared to the viral reference sequence to obtain the number of detected sequences and genotype data in the total viral DNA. Based on the number of viral sequencing sequences and the number of detected MNP marker sites obtained from the total viral DNA and blank control, data quality control and data analysis were performed on the sequencing data of the total viral DNA to obtain the number of detected MNP sites, the number of sequencing sequences covering each MNP marker site, and the genotype data of the MNP marker sites.
[0057] When identifying zarrovirus, quality control is performed based on the number of zarrovirus sequencing sequences and the number of MNP sites detected in the test sample and blank control. The result is then used to determine whether the test sample contains zarrovirus nucleic acid. The quality control protocol and determination method use zarrovirus DNA with a known copy number as the test sample to evaluate the sensitivity, accuracy, and specificity of the kit for detecting zarrovirus.
[0058] In this embodiment, all three MNP markers of zaruzin virus could be detected in samples S2 to S6, and the average sequencing coverage of each MNP marker site reached 4000-fold, as shown in Table 4.
[0059] Table 4 shows the results of the detection and analysis of roxithromycin in the samples.
[0060]
[0061] As shown in Table 4, the kit and detection method accurately detected all three MNP marker sites in each sample of zargovirus in a single reaction. In contrast, methods based on fluorescent PCR that detect one marker per reaction require three tests, demonstrating the high accuracy and efficiency of the kit and method in detecting zargovirus.
[0062] Example 4
[0063] This disclosure provides an application of the above-described primer set or kit in the monitoring and database construction of Zaruzon virus mutations. The marker sites for MNP-1 to MNP-3 are shown below:
[0064] .
[0065] Specifically, based on obtaining the number of MNP sites detected by the virus, the number of sequencing sequences covering each MNP marker site, and the genotype data of the MNP marker sites, the genotype data of the MNP marker sites of Zarrovirus are identified from the test samples and entered into the database file to form the DNA fingerprint database of Zarrovirus. Different test samples are compared with the DNA fingerprint database of Zarrovirus. The comparison results are used to identify whether the Zarrovirus in the test samples differs from the strains in the DNA fingerprint database in terms of the main genotype at the MNP marker sites. The main genotype is the genotype supported by more than 50% of the sequencing fragments at one MNP marker site. Zarrovirus with a main genotype difference at at least one MNP marker site is a new variant type, and its genotype is included in the DNA fingerprint database.
[0066] The genotypes of the three markers of zaruzin virus detected in samples S2 to S6, as shown in Table 4, were compared pairwise between samples, and the results are shown in Table 5.
[0067] Table 5 shows the detection and analysis of four copies of a single zaruzin virus strain.
[0068]
[0069] Table 5 shows that S2 and the four samples tested in the same batch all exhibited major genotypic differences at one MNP of the zaruzin virus, indicating that S2 has genetic variation compared to the other samples. Therefore, this kit can identify genetic variations between strains by detecting the sequence of MNP markers. It can be used in disease prevention and control surveillance to detect variations between strains, and also in scientific research to ensure the genetic consistency of the same named strains in different laboratories, thus ensuring the comparability of research results. This is of great significance for pathogen prevention and control surveillance, precision treatment, and scientific research.
[0070] 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. A primer set for detecting MNP marker sites of zaruzin virus, characterized in that, The primer set includes at least one of 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. A kit for detecting MNP marker sites of zaruzin virus, characterized in that, The kit includes the primer set as described in claim 1.
3. The reagent kit according to claim 2, characterized in that, The kit also includes a multiplex PCR premix.
4. The application of the primer set of claim 1 or the kit of claim 2 in the identification of the zajor virus, wherein the MNP marker site includes at least one of the marker sites MNP-1 to MNP-3, and the marker sites MNP-1 to MNP-3 are as follows: 。 5. The application of the primer set of claim 1 or the kit of claim 2 in the monitoring and database construction of the zaruzin virus mutation, wherein the MNP marker site includes at least one of the marker sites MNP-1 to MNP-3, and the marker sites MNP-1 to MNP-3 are as follows: 。