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CN-122012815-A - Synchronous detection primer, probe and application of norovirus II, group A rotavirus and adenovirus

CN122012815ACN 122012815 ACN122012815 ACN 122012815ACN-122012815-A

Abstract

The invention provides a synchronous detection primer for type II norovirus, group A rotavirus and adenovirus, which comprises specific primer pairs designed for conserved genes of three pathogens, namely type II norovirus, group A rotavirus and adenovirus, and the specific primer pairs are respectively an ORF1/ORF2 primer pair, a VP6 gene primer pair and a Hexon gene primer pair. The invention also provides a detection probe comprising an ORF1/ORF2 capture probe, a VP6 capture probe and a Hexon capture probe. Meanwhile, the invention also provides application of the primer and the probe in preparation of infectious diarrhea related virus detection products, and a specific detection kit and a specific detection method. The invention can realize multiple detection of three common diarrhea viruses, can finish detection in a short time, quickly and accurately identify amplified products and develop color, improves detection sensitivity and specificity, does not need large instruments, and has low cost.

Inventors

  • SHEN JILU
  • Yuan Lufei
  • SONG LELE
  • Ma Zhoujie
  • ZHU XIANGLING
  • JIANG RONG

Assignees

  • 安徽省公共卫生临床中心(安徽省传染病医院)

Dates

Publication Date
20260512
Application Date
20260309

Claims (10)

  1. 1. The synchronous detection primer for the type II norovirus, the group A rotavirus and the adenovirus is characterized by comprising a specific primer pair designed for conserved genes of three pathogens, namely the type II norovirus, the group A rotavirus and the adenovirus, and has the following sequences: type II norovirus ORF1/ORF2 primer pair: ORF1/ORF2-F: 5’-FITC-TTTACGTGCCCAGACAAGAGCCAATGTTCA-3’; ORF1/ORF2-R:5’-TAGATAGATAGATAGA/iSpC12/GTCACTCGACGCCATCTTCATTCACAAAAC-3’; Group a rotavirus VP6 gene primer pair: VP6-F: 5’-FITC-GAACGGAAATGACTTTCAAACTGGAGGAA-3’; VP6-R: 5’-CATTCTGCTTCCAAGT/iSpC12/CTCAATCGTAGTTCTGGCGGTCTCAACAT-3’; Adenovirus Hexon gene primer pair: Hexon-F:5’-FITC-GAACTACATTGGCTTTAGGGATAACTTCATCG-3’; Hexon-R:5’-TCGACTGAGAATTGAC/iSpC12/AACTGTCAACAGCCTGATTCCACATAGAA-3’。
  2. 2. the primer of claim 1, wherein the 5' end of the downstream primer R of the three primer pairs is a labeled nucleic acid sequence, and a C12 framework structure modification, namely the iSpC spacer, is designed between the nucleic acid sequence and the adjacent unmodified sequence for preventing primer dimer formation.
  3. 3. The primer set according to claim 1, wherein the ORF1/ORF2 primer set, VP6 gene primer set and Hexon gene primer set are used for detection of type II norovirus, group A rotavirus, adenovirus, respectively.
  4. 4. Use of the primer according to any one of claims 1-3 for preparing an infectious diarrhea associated virus detection product, wherein the infectious diarrhea associated virus comprises a group-a rotavirus, a group-II norovirus, an adenovirus.
  5. 5. Synchronous detection probes for norovirus type II, group A rotavirus and adenovirus are characterized by comprising an ORF1/ORF2 capture probe, a VP6 capture probe and a Hexon capture probe, and the sequences are as follows: TCTATCTATCTATCTATTTTTT-Biotin as ORF1/ORF2 capture probe; VP6 capture probe ACTTGGAAGCAGAATGTTTTTT-Biotin; hexon capture probe GTCAATTCTCAGTCGATTTTTT-Biotin.
  6. 6. The probe of claim 5, wherein the ORF1/ORF2 capture probe, VP6 capture probe and Hexon capture probe are detection line probes designed for type II norovirus, group A rotavirus and adenovirus, respectively, so that the amplified product can be combined with the capture probes during the chromatography of the test strip, thereby developing the detection line.
  7. 7. Use of a probe according to claim 5 or 6 for the preparation of a product for detection of infectious diarrhea associated viruses including norovirus type II, group a rotavirus, adenovirus.
  8. 8. A kit for simultaneous detection of norovirus type II, group a rotavirus and adenovirus, comprising: a primer according to any one of claims 1 to 3; The probe of claim 5 or 6; a lateral flow immunochromatographic strip; an RPA amplification reagent; and (3) detecting a detection system of the amplified product sample chromatographic strip.
  9. 9. The kit according to claim 8, wherein the lateral flow immunochromatographic strip is composed of a sample pad for loading a sample, a gold-labeled pad for storing an AuNP-Ab conjugate, an NC membrane for fixing a detection line and a quality control line, a water-labeled pad for collecting an aqueous phase and providing a driving force for the flow of a reaction solution on the test strip, and a bottom plate as a base sheet for integrating the sample pad, the gold-labeled pad, the NC membrane, and the water-labeled pad.
  10. 10. A method for synchronous detection of non-diagnostic norovirus type II, group a rotavirus and adenovirus, characterized by using a kit according to claim 8 or 9, the method comprising: (1) Taking RNA of a sample to be detected as a template, and forming an RPA amplification system by using the primer and the RPA amplification reagent to carry out RPA amplification; (2) Fixing the probe at the NC membrane detection line position of the lateral flow immunochromatographic strip; (3) Combining the RPA amplification product obtained in the step (1) with a loading buffer solution, and judging the infection types of the type II norovirus, the group A rotavirus and the adenovirus in the sample to be detected according to the detection result of the lateral flow immunochromatography strip detection system.

Description

Synchronous detection primer, probe and application of norovirus II, group A rotavirus and adenovirus Technical Field The invention relates to the technical field of medical detection, in particular to a synchronous detection primer, a probe and application of a type II norovirus, a group A rotavirus and adenovirus. Background Viral diarrhea is one of the common diseases threatening global public health, and has high incidence, especially in infants, the elderly and immunocompromised people, with a higher prevalence. The etiology is complex and diverse, and is usually caused by rotavirus, norovirus, adenovirus, astrovirus and other pathogens. Diarrhea symptoms caused by different viruses are similar, but there are significant differences in epidemiological characteristics, disease severity and prevention and control measures. Therefore, the timely and accurate identification of diarrhea virus pathogens is critical for accurate treatment and infection control of diseases. The traditional laboratory diagnosis method, such as virus isolation and culture, electron microscope observation, immunological detection and the like, has the problems of long time consumption (usually 3-7 days), low sensitivity, complicated operation, high requirements on experimental conditions and professional technicians and the like. In clinical application, rapid diagnosis is critical for the treatment of patients with viral diarrhea, especially infants and elderly patients, and if some pathogens (such as norovirus) are not recognized in time, serious consequences such as nosocomial infection outbreak, serious dehydration, electrolyte disorder and even death may be caused. The existing PCR detection technology has high sensitivity and high specificity, but generally requires an expensive fluorescent quantitative PCR instrument, a strict laboratory partition, a longer detection period (usually 2-4 hours) and professional operators, is difficult to widely develop in emergency departments, basic medical institutions and limited resource areas, and cannot meet the clinical requirements of rapid screening and on-site detection of diarrhea pathogens. Therefore, a new method for synchronously detecting various diarrhea viruses in a short time, which is simple and convenient to operate and has controllable cost is urgently needed at present, and the method has important practical significance for clinical differential diagnosis, hospital infection prevention and control and public health emergency response of viral diarrhea. Disclosure of Invention The invention aims to solve the technical problem of providing a synchronous detection primer and probe for II-type norovirus, group A rotavirus and adenovirus and application thereof, which are used for realizing the rapid synchronous identification and multiple detection of three main viral diarrhea pathogenic viruses, namely II-type norovirus, group A rotavirus and adenovirus by combining an RPA (recombinant enzyme polymerase amplification) -lateral flow immunochromatography technology through the specially designed primer and probe. Specifically, the primer is designed aiming at the conserved gene sequences of three viruses, and simultaneously nucleic acid modification and iSpC interval structure are introduced, so that the amplification specificity and efficiency can be improved, primer dimer formation can be effectively avoided, the accuracy and stability of an amplification product can be guaranteed, meanwhile, the rapid amplification advantage of the RPA technology and the convenient detection characteristic of a lateral flow immunochromatography strip are combined, the detection time consumption can be greatly shortened, the amplification product can be rapidly and accurately identified and developed by means of a specific capture probe on the chromatographic strip, the detection sensitivity and specificity are remarkably improved, the misdiagnosis and missed diagnosis risks are reduced, a large-scale complex instrument is not needed in the technology, the detection cost is low, the operation is simple and convenient, and the method is suitable for large-scale screening and basic medical scenes. The invention adopts the following technical scheme to solve the technical problems: Synchronous detection primers for type II norovirus, group A rotavirus and adenovirus comprise specific primer pairs designed for conserved genes of three pathogens, namely type II norovirus, group A rotavirus and adenovirus, and the sequences are as follows: type II norovirus ORF1/ORF2 primer pair: ORF1/ORF2-F: 5’-FITC-TTTACGTGCCCAGACAAGAGCCAATGTTCA-3’; ORF1/ORF2-R:5’-TAGATAGATAGATAGA/iSpC12/GTCACTCGACGCCATCTTCATTCACAAAAC-3’; Group a rotavirus VP6 gene primer pair: VP6-F: 5’-FITC-GAACGGAAATGACTTTCAAACTGGAGGAA-3’; VP6-R: 5’-CATTCTGCTTCCAAGT/iSpC12/CTCAATCGTAGTTCTGGCGGTCTCAACAT-3’; Adenovirus Hexon gene primer pair: Hexon-F:5’-FITC-GAACTACATTGGCTTTAGGGATAACTTCATCG-3’; Hexon-R:5’-TCGACTGAGAATTGAC/iSpC12/AACTGTCAACA