SNP (Single Nucleotide Polymorphism) molecular marker and application thereof in traceability of plasmodium vivax in Chinese inland
By applying the single SNP molecular marker SNP24806 and its specific primers in malaria source tracing, combined with PCR amplification and sequencing technologies, the problems of subjectivity and resource constraints in malaria source tracing were solved, enabling rapid and accurate case tracing and supporting efficient prevention and control in grassroots laboratories.
Patent Information
- Application Number
- CN202511302631.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-12
- Publication Date
- 2025-12-05
AI Technical Summary
Existing malaria tracing technologies are subjective and lack objectivity in determining the source of Plasmodium infection, and are difficult to conduct rapid and accurate case tracing in resource-limited areas, affecting the effectiveness of malaria prevention and control.
Using the single SNP molecular marker SNP24806 and its specific primers, molecular markers of Plasmodium vivax in inland China were screened through PCR amplification and sequencing technology. Combined with gene mass spectrometry chip screening and whole genome resequencing data, a low-cost and rapid traceability product was developed.
It enables rapid and accurate tracing of the source of Plasmodium vivax in resource-limited areas, improves the sensitivity and specificity of malaria case tracing, supports rapid identification and preliminary tracing by primary laboratories, and guides the timely implementation of prevention and control measures.
Smart Images
Figure CN121065372A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of molecular detection, and particularly relates to a SNP molecular marker of Plasmodium vivax and application thereof in tracing Plasmodium vivax in inland China. BACKGROUND
[0002] Malaria is an important parasitic disease that seriously endangers human health and affects social and economic development. According to the World Health Organization (WHO) report in 2024, there were about 263 million cases of malaria worldwide in 2023, an increase of 11 million cases from 2022; and there were 597,000 deaths. In China, after several generations and decades of unremitting efforts, malaria outbreaks have been effectively controlled, and the country was certified by the WHO as having eliminated malaria in 2021.
[0003] However, with the increasing frequency of international exchanges, the long-term existence of imported malaria cases and the presence of malaria-transmitting vectors in some regions of China pose a great threat to the consolidation of malaria elimination achievements in China. If the sensitivity and effectiveness of the monitoring response system cannot be maintained, and the source of infection cannot be discovered and eliminated in a timely manner, there is still a risk of re-transmission and resurgence of the epidemic.
[0004] Molecular tracing technology has important value in determining the source of Plasmodium infection. Traditional case epidemiological investigation is relatively subjective and lacks sufficient evidence, while molecular tracing technology uses geographic strain-specific molecular markers combined with population genetics analysis for determination, resulting in more objective results and clear geographical origin and transmission path of the case, providing scientific basis for malaria prevention and control. Among them, single nucleotide polymorphism (SNP) is a commonly used molecular marker in molecular tracing research.
[0005] SNP is a single nucleotide variation in the genome and is the third generation of DNA genetic markers. As a molecular tracing marker, it has shown significant advantages in determining the source of Plasmodium infection. First, it is distributed at a high density in the Plasmodium genome, allowing SNP technology to provide extremely high resolution and significantly improve tracing accuracy. Second, SNP has a very low mutation rate and is highly stable. Third, SNP detection methods are diverse, highly automated and high-throughput, and in addition to PCR amplification, sequencing and chip technology can also be used. Fourth, SNP detection is suitable for low-quality samples and requires less DNA sample, allowing more information to be obtained from degraded DNA samples.
[0006] With the publication of the genome sequence of P. falciparum in 2002 and the complete genome sequence of P. vivax in 2012, a large number of SNP markers that can contain barcodes are contained therein. Rachel Daniels et al. applied Taqman technology to screen 24 SNP combinations based on the nuclear genome of P. falciparum for identifying and tracking isolates from the Senegalese epidemic population. Mary Lynn Baniecki et al. applied HRM technology to screen 42 SNP combinations of P. vivax for identifying the geographical origin of P. vivax strains.
[0007] Due to the characteristics of high resolution and strong anti-interference ability, the existing molecular tracing of Plasmodium is mainly based on SNP combinations. However, the molecular tracing of a single SNP marker has the following unique advantages. First, the detection cost and efficiency advantage. Single SNP detection only needs to design a pair of primers, and the reagent consumption is small, and there is no need for complex multiplex PCR or high-throughput sequencing platform. The single SNP detection process is simplified, which can be completed within 1-2 hours, and is suitable for on-site rapid screening. Second, the low technical threshold and wide practicability. Single SNP detection can be completed by a conventional PCR instrument, which can be operated by a basic laboratory, and the professional technical ability requirement of the experimental personnel is not high. Single SNP detection has low requirement for DNA template amount, and is suitable for low-quality samples such as filter paper blood spots and blood clots. Third, high specificity in specific scenarios. In the case of known Plasmodium population structure, a single highly specific SNP marker can be screened out and directly used for rapid judgment of imported cases. Fourth, simple data analysis and interpretation. The single SNP typing result is simple, and does not need complex bioinformatics analysis, and can be directly interpreted by electrophoretogram, and the professional ability requirement of the basic disease monitoring personnel is not high. Fifth, the advantage in the emergency response and resource-limited scenario. Under the premise that malaria has been eliminated in China, if a malaria epidemic occurs, in the early stage, a single SNP can quickly provide preliminary tracing clues to guide subsequent prevention and control measures. In areas with insufficient equipment, funds or manpower, single SNP detection can be used as a low-cost alternative solution to achieve basic tracing function. SUMMARY
[0008] One of the technical problems to be solved by the present application is to provide a SNP molecular marker for tracing P. vivax in China inland, wherein n in the sequence table represents the mutation site of the molecular marker: SNP 24806 : the sequence is shown as SEQ ID NO: 1, and the allele mutation site is: C / T.
[0009]
[0010] n=c or t
[0011] The second technical problem solved by the present application is to provide a method for tracing the source of the SNP molecular marker of Plasmodium vivax, comprising the following steps:
[0012] 1) Selecting the SNP site from the previous genetic mass spectrometry chip tracing result 24806 ;
[0013] 2) Taking 128 Plasmodium vivax patient whole blood samples collected in inland areas of China (Anhui, Henan, Zhejiang Province) and 150 Plasmodium vivax patient whole blood samples collected in the China-Myanmar border area, extracting DNA therefrom;
[0014] 3) Designing specific primers according to the selected SNP 24806 site, performing PCR amplification on each sample, and sending the PCR product to a sequencing company to obtain sequencing results and perform comparison.
[0015] The third technical problem solved by the present application is to provide specific primers for the SNP molecular marker of Plasmodium vivax in PCR amplification, which are respectively:
[0016] Upstream PF: 5'-CTCTCCATATAACACGAACTAC-3' (SEQ ID NO: 2).
[0017] Downstream PR: 5'-GTCCAGATTGCCACCATT-3' (SEQ ID NO: 3).
[0018] The fourth technical problem solved by the present application is the application of the SNP molecular marker and specific primers of Plasmodium vivax in tracing the source of Plasmodium vivax in inland China or preparing products for tracing the source of Plasmodium vivax in inland China. Since 2017, China has achieved the first report of no local primary infection case, and has maintained this achievement for 4 consecutive years, and officially obtained the certification of the World Health Organization for eliminating malaria on June 30, 2021. However, the continuous consolidation of the achievement of eliminating malaria is threatened. With the intensification of globalization, the number of people from or returning from malaria-endemic areas is also gradually increasing, and the Anopheles mosquito, the transmission vector of malaria, still exists in the original endemic provinces in China. If imported cases are not discovered and treated in a timely manner during the epidemic season, they are more likely to become a source of infection and cause local secondary transmission. Based on previous whole genome resequencing and genetic mass spectrometry chip screening, the present application identifies a SNP molecular marker, which can effectively identify Plasmodium vivax in inland China through PCR amplification. The SNP molecular marker can be developed into a product for tracing the source of Plasmodium vivax, so as to accurately assess the transmission risk of the epidemic site and then take effective disposal measures in a timely manner.
[0019] In recent years, our team has collected samples of imported Plasmodium vivax cases from inland China (Anhui, Henan, and Zhejiang provinces) and the China-Myanmar border. We have also performed whole-genome resequencing on some Plasmodium vivax samples and used gene mass spectrometry chips to screen and identify molecular marker combinations that can effectively identify Plasmodium vivax in inland China. Based on this work, we screened and obtained one SNP molecular marker, which, through PCR amplification, can effectively identify Plasmodium vivax in inland China. We have also established a gene tracing technology that can objectively trace the origin of Plasmodium vivax and developed products for tracing the origin of Plasmodium vivax to accurately assess the transmission risk of epidemic sites and thus take timely and effective control measures. Attached Figure Description
[0020] Figure 1 It is the SNP in Example 1 24806 A schematic diagram of PCR amplification results for molecular markers. Figure 1 In the table, M represents the molecular weight standard for DNA; 1-2 represent samples of Plasmodium vivax. Detailed Implementation
[0021] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the examples are generally performed under conventional conditions or as recommended by the manufacturer.
[0022] Example 1
[0023] 1. Materials
[0024] 1.1 Genomic DNA of Plasmodium vivax
[0025] Whole blood samples were collected from 278 patients infected with Plasmodium vivax from Henan, Anhui, Zhejiang and Yunnan provinces.
[0026] 1.2 Main Reagents
[0027]
[0028] 2 methods
[0029] 2.1 Selection of SNP sites
[0030] SNPs were obtained by screening previous research results. 24806 The locus is located on chromosome 1, as detailed in Table 1.
[0031] Table 1. Information related to SNP sites
[0032] SNP number Position Allele Chromosome SNP 24806 ]] 24806 [C / T] 1
[0033] 2.2 Amplification of Plasmodium vivax SNP molecules
[0034] Based on the selected SNP 24806The molecular upstream and downstream sequence information is used to design one pair of specific primers by using a primer design software of Sheng Wu Bioengineering (Shanghai) Co., Ltd. The primers used in the application are as follows, which are synthesized by Sheng Wu Bioengineering (Shanghai) Co., Ltd.
[0035] Upstream PF: 5'-CTCTCCATATAACACGAACTAC-3' (SEQ ID NO: 2).
[0036] Downstream PR: 5'-GTCCAGATTGCCACCATT-3' (SEQ ID NO: 3).
[0037] The genomic DNA of P. vivax is used as a template to perform gene amplification. The reaction conditions are as follows: ① 94 ℃ pre-denaturation for 2.0 min; ② 94 ℃ denaturation for 30 sec, ③ 59 ℃ annealing for 30 sec, ④ 72 ℃ extension for 1.0 min, steps ② to ④ for 35 cycles; ⑤ 72 ℃ extension for 5 min, and finally stored at 4 ℃. The DNA polymerase is purchased from TransGen Biotech, and the reaction system is 50.0 μl, specifically Supermix (containing PCR amplification enzyme, Mg + , dNTPs) 25 μl; Primer-F (10 μM each) 1 μl; Primer-R (10 μM each) 1 μl; Template DNA (DNA template) 2 μl; Nuclease-free water 21 μl.
[0038] 2.3 Sequencing and sequence analysis of P. vivax amplification product
[0039] The PCR amplification product is sent to Beijing Lihe Huada Gene Technology Co., Ltd. Shanghai Branch for sequencing, and the obtained gene sequence is analyzed by using DNAMAN7 software.
[0040] 3 Results
[0041] 3.1 Amplification and sequencing of P. vivax gene
[0042] The genomic DNA of P. vivax is used as a template to perform PCR amplification, and a target fragment with a length of 761 bp consistent with the expected length is amplified (see Figure 1 ), indicating that the amplification is successful. The PCR product is sent to Beijing Lihe Huada Gene Technology Co., Ltd. Shanghai Branch for sequencing. According to the statistics, among the 278 P. vivax samples, 275 samples have PCR products in PCR amplification, and the sequencing is successful.
[0043]
[0044] Sample source Sample number Detection rate Inland China 128 97.7％(125 / 128) China-Myanmar border region 150 100％(150 / 150) Total 278 98.9％(275 / 278)
[0045] 3.2 Sensitivity, specificity analysis
[0046] In the result of the detection signal, according to the molecular identification SNP 24806 Of the 125 P. vivax samples from inland China, 120 were determined to be from inland China, with a sensitivity of 96.0%; of the 150 P. vivax samples from the China-Myanmar border, 3 were determined to be from inland China, with a specificity of 98.0%.
[0047]
[0048]
[0049] Sample source Number of detection Specificity China-Myanmar border region 150 98.0％(147 / 150)
[0050] As can be seen from the above results, the present application screens and obtains a SNP molecular marker, and application of PCR amplification can effectively identify P. vivax from inland China, the sensitivity of the SNP is 96.0%, and the specificity is 98.0%. The method is convenient, rapid and easy to popularize at the grassroots level. Relying on the present application, the team successfully established a gene tracing technology that can objectively trace the source of P. vivax, and the above achievements can be used to trace the source of P. vivax after malaria is eliminated in China, and can also be applied to the development of P. vivax tracing products, and has a broad application prospect.
[0051] The above description of the embodiments is to facilitate the understanding and use of the present application by those of ordinary skill in the art. Those skilled in the art can obviously make various modifications to these embodiments, and apply the general principles described herein to other embodiments without having to go through creative labor. Therefore, the present application is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present application without departing from the scope of the present application should be within the scope of protection of the present application.
Claims
1. The use of a SNP molecular marker in tracing the origin of Plasmodium vivax in inland China, characterized in that: SNP molecular marker: SNP 24806 : sequence as shown in SEQ ID NO: 1, and the allelic mutation site is: C / T.
2. A method for the tracing of Plasmodium vivax using a PCR method, characterized in that: The method comprises the following steps: 1) Selecting loci SNPs from the previous gene mass spec chip trace results 24806 ; 2) Extract DNA from 128 blood samples of patients with vivax malaria collected from inland areas (Anhui, Henan, Zhejiang) and 150 blood samples of patients with vivax malaria collected from Sino-Myanmar border areas; 3) according to the selected SNPs 24806 sites, specific primers were designed, PCR amplification was performed for each sample, and the PCR products were sent to a sequencing company to obtain sequencing results and perform alignment, SNP 24806 The sequence is shown as SEQ ID NO: 1, and the allelic mutation site is: C / T.
3. The method for tracing inland vivax malaria in China by PCR method according to claim 2, characterized in that the specific primers used in step 3) are as follows: Upstream PF: 5'-CTCTCCATATAACACGAACTAC-3'; Downstream PR: 5'-GTCCAGATTGCCACCATT-3'.
4. The method for tracing inland vivax malaria in China by PCR method according to claim 2, characterized in that the gene amplification reaction conditions in step 3) are as follows: ① 94℃ pre-denaturation for 2.0 min; ② 94℃ denaturation for 30 sec, ③ 59℃ annealing for 30 sec, ④ 72℃ extension for 1.0 min, steps ② to ④ for 35 cycles; ⑤ 72℃ extension for 5 min, and finally stored at 4℃.
5. Use of a SNP molecular signature for the tracing of Chinese inland Plasmodium vivax or for the preparation of a product for the tracing of Chinese inland Plasmodium vivax, characterized in that: SNP molecular marker: SNP 24806 : sequence as shown in SEQ ID NO: 1, and the allelic mutation site is: C / T.
6. Use of the primer pair according to claim 3 in tracing inland vivax malaria in China or in preparing a product for tracing inland vivax malaria in China.