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CN-121992151-A - Composition for detecting I, II type monkey pox virus typing, and typing detection kit and application thereof

CN121992151ACN 121992151 ACN121992151 ACN 121992151ACN-121992151-A

Abstract

The invention discloses a composition for detecting I, II type monkey pox virus typing, a typing detection kit and application thereof, comprising an LNA-Taqman probe for detecting monkey pox virus I, an upstream primer for monkey pox virus typing detection, and a downstream primer for monkey pox virus typing detection, wherein the nucleic acid sequence of the LNA-Taqman probe is shown as SEQ ID NO. 1, the nucleic acid sequence of the upstream primer is shown as SEQ ID NO. 2, and the nucleic acid sequence of the downstream primer is shown as SEQ ID NO. 3. According to the invention, LNA monomers are precisely designed at key SNP loci of the monkey pox virus typing of the probe by utilizing the characteristic that hybridization stability of a Locked Nucleic Acid (LNA) probe is rapidly reduced when single base is misplaced. The design can remarkably amplify the parting signal difference and effectively distinguish highly homologous virus branches. The problems of cross reaction and false positive caused by high mismatch tolerance of the traditional DNA probe are avoided, and the accuracy of typing identification is ensured.

Inventors

  • ZHAO XIN
  • ZHANG SHENGNAN
  • GUO JUN
  • HU KONGXIN
  • WANG JING
  • CONG HAOLONG

Assignees

  • 中国质量检验检测科学研究院
  • 天津科技大学

Dates

Publication Date
20260508
Application Date
20260203

Claims (10)

  1. 1. A composition for detecting I, II type monkey pox virus typing is characterized by comprising an LNA-Taqman probe for detecting the monkey pox virus I, wherein the nucleic acid sequence of the LNA-Taqman probe is shown as SEQ ID NO. 1; The nucleic acid sequence of the monkey pox virus typing detection upstream primer is shown as SEQ ID NO. 2; And, monkey pox virus typing detection downstream primer, its nucleic acid sequence is shown in SEQ ID NO. 3.
  2. 2. Use of the composition of claim 1 for the preparation of a kit for detecting the genotyping of the I, II type monkey poxvirus.
  3. 3. A kit for detecting I, II-type monkey pox virus typing, which is characterized in that the kit comprises the composition of claim 1.
  4. 4. The kit of claim 3, further comprising a fluorescent quantitative PCR premix, a positive control, a negative control, and water.
  5. 5. The kit according to claim 4, wherein the positive control comprises a monkey pox virus I plasmid having a copy number of 6.90X10 7 copies/. Mu.L and the negative control comprises a monkey pox virus type II negative test plasmid having a copy number of 6.57X10 7 copies/. Mu.L, respectively.
  6. 6. The kit of claim 5, wherein the fluorescent quantitative PCR premix has a concentration of 12.5. Mu.L, an upstream primer of 10. Mu.M, a volume of 1. Mu.L, a concentration of 10. Mu.M LNA-Taqman probe for detecting monkey pox virus I, a volume of 1. Mu.L, a positive control volume of 5. Mu.L, 4.5. Mu.L water, and a total volume of 25. Mu.L.
  7. 7. The kit according to claim 5, wherein the detection limit of the kit is up to 6.90 copies/. Mu.L.
  8. 8. A using method of a kit for detecting I, II-type monkey pox virus typing is characterized by comprising the steps of assembling raw materials into the kit, and carrying out fluorescent quantitative PCR reaction with the reaction program of 95 ℃ for 20S, 95 ℃ for 15S, 62 ℃ for 45S for 40 cycles.
  9. 9. A method for typing and detecting a type I, II of a monkey pox virus based on LNA aiming at non-therapeutic diseases is characterized by comprising the steps of typing and detecting the type I, II of the monkey pox virus by a fluorescent quantitative PCR technology; Wherein the reaction system comprises 2X Probe qPCR Mix MultiPlus 12.5.5 mu L, 10 mu M of upstream primer 1 mu L, 10 mu M of downstream primer 1 mu L, 10 mu M of LNA-Taqman probe 1 mu L, DNA template 5 mu L, ROX REFERENCE DYE II (100X) 0.5 mu L, sterile and asepsis water 4 mu L and the total reaction system is 25 mu L; The reaction procedure was 95℃20S, 95℃15S, 62℃45S for a total of 40 cycles.
  10. 10. The method of typing detection according to claim 9, wherein the DNA template includes a monkey poxvirus I plasmid and a monkey poxvirus II plasmid having copy numbers of 6.90X10 7 copies/μL、6.57×10 7 copies/. Mu.L, respectively.

Description

Composition for detecting I, II type monkey pox virus typing, and typing detection kit and application thereof Technical Field The invention belongs to the technical field of molecular biology detection, and particularly relates to a composition for detecting I, II type monkey pox virus typing, a typing detection kit and application thereof. Background Monkey pox is a zoonotic disease caused by the monkey pox virus, which belongs to the genus orthopoxvirus (Orthopoxvirus) of the family poxviridae (Poxviridae). The World Health Organization (WHO) has listed it as a potential public health emergency. Based on the significant differences in genomics and pathogenicity, monkey poxviruses are largely divided into two distinct evolutionary branches. Branch I (middle non-basin branch) and branch II (west non branch), respectively. The I branch is highly pathogenic. Historical data shows that their mortality rate (CFR) can be as high as 10%, especially in children and immunocompromised populations that are not vaccinated with smallpox vaccine. Patients infected with the virus often develop symptoms such as high fever, severe headache, generalized lymphadenectasis, and extensive and intensive rash. Serious cases are often accompanied by serious complications such as secondary bacterial infection, pneumonia and encephalitis. While branch II is relatively weak in virulence. The mortality rate is usually less than 1%. Clinical manifestations are usually lighter and may be accompanied by symptoms of precursor fever or mild symptoms. The number of rashes is small and often limited to genital, perianal or oral areas. At present, most of the detection methods for the monkey pox viruses are universal detection methods, such as guidelines and standards of the technical guidelines for controlling monkey pox (2022) and the like are universal detection methods for the monkey pox viruses, and the specific types of the monkey pox viruses cannot be further distinguished. The current detection means for the monkey pox virus typing comprises real-time fluorescence quantitative PCR (qPCR), and the method has the advantages of rapidness and sensitivity as a gold standard for routine laboratory detection, however, the detection effect is highly dependent on the specificity of primers and probes, false negatives can occur when the viruses have genetic variation, and the relative quantitative mode based on a standard curve has the defects of accurate typing and micro-difference distinction. The loop-mediated isothermal amplification (LAMP) technology has simple equipment requirements and is suitable for on-site rapid screening, but the reaction is easy to generate nonspecific amplification, possibly causes false positive results, is currently used for qualitative or semi-quantitative detection, and has limited application in complex scenes requiring accurate typing. The liquid drop type digital PCR (ddPCR) has absolute quantitative capability and high tolerance to inhibitors, is particularly suitable for accurately quantifying low-load viruses, but has high instrument and consumable cost, relatively complex operation flow and low flux, and is difficult to be used as a conventional means for large-scale typing screening. The high-throughput whole-gene sequencing technology (NGS) can provide the most comprehensive genome information, is an ultimate tool for typing, tracing and mutation research, but has extremely high cost, complex data analysis and long time consumption, has high requirements on sample quality and virus load, is usually used for important cases or scientific research analysis, and cannot meet the requirement of rapid and high-throughput conventional typing diagnosis. In view of different virulence and clinical manifestations of different branches of the monkey pox virus, the method capable of detecting the monkey pox virus in a parting way is developed, can be used for accurate clinical diagnosis and treatment, disease prevention and risk assessment, and is helpful for relevant departments to formulate differential isolation, vaccination and resource allocation strategies. Therefore, developing a parting detection method capable of rapidly and accurately distinguishing the monkey pox virus branches I and II is significant for realizing clinic individual accurate treatment and improving public health prevention and control efficiency. Disclosure of Invention This section is intended to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. The present invention has been made in view of the above and/or problems occurring in the prior art. It is therefore an object of the present invention to overcome the deficiencies of the prior art and to provide a composition for detecting the genotyping of the I, II type monkey poxvirus. In order to solve the technical problems, the invention provides a composition for detecting I, II type monkey pox virus typing, which comprises an LNA