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CN-121992150-A - Taqman probe primer combination for detecting double RT-qPCR of group A porcine rotavirus G3 and G9 strains and application thereof

CN121992150ACN 121992150 ACN121992150 ACN 121992150ACN-121992150-A

Abstract

The invention relates to a Taqman probe primer combination for detecting double RT-qPCR of group A porcine rotavirus G3 and G9 strains and application thereof. The probe primer combination comprises an upstream primer PoRVA G-F and a downstream primer PoRVA G-R for amplifying the VP7 gene conservation region of the G3 type porcine rotavirus, an upstream primer PoRVA G-F and a downstream primer PoRVA G-R for amplifying the VP7 gene conservation region of the G9 type porcine rotavirus, a probe PoRVA G-P for specifically targeting the VP7 gene conservation region of the G3 type porcine rotavirus, and a probe PoRVA G-P for specifically targeting the VP7 gene conservation region of the G3 type porcine rotavirus. The double RT-qPCR detection system can effectively solve the problems that two subtypes cannot be detected synchronously and efficiently in the detection of the existing porcine rotavirus epidemic strain G3 and G9 subtype, the sensitivity is insufficient and the quantitative accuracy is poor, and has the advantages of quick synchronous detection, high sensitivity and accurate quantification.

Inventors

  • HUANG SHUJIAN
  • Luo Haojian
  • YANG AOBING
  • HU MEIRONG
  • WANG YIQIAO
  • LIN JUNJIE
  • LIU YINGYING
  • LI WENJUN

Assignees

  • 佛山大学
  • 广东永顺生物制药股份有限公司

Dates

Publication Date
20260508
Application Date
20260129

Claims (10)

  1. 1. A primer combination for simultaneously amplifying a G3 type porcine rotavirus and a G9 type porcine rotavirus, which is characterized by comprising an upstream primer PoRVA G-F and a downstream primer PoRVA G-R for amplifying a VP7 gene conservation region of the G3 type porcine rotavirus, and an upstream primer PoRVA G-F and a downstream primer PoRVA G-R for amplifying a VP7 gene conservation region of the G9 type porcine rotavirus.
  2. 2. The primer combination of claim 1, wherein the nucleotide sequence of the upstream primer PoRVA G-F is shown in SEQ ID No.5, the nucleotide sequence of the downstream primer PoRVA G-R is shown in SEQ ID No.6, the nucleotide sequence of the upstream primer PoRVA G-F is shown in SEQ ID No. 15, and the nucleotide sequence of the downstream primer PoRVA G-R is shown in SEQ ID No. 16.
  3. 3. A duplex RT-qPCR detection system, comprising probe PoRVA G-P specifically targeting the conserved region of the G3-type porcine rotavirus VP7 gene, probe PoRVA G-P specifically targeting the conserved region of the G3-type porcine rotavirus VP7 gene, and the primer combination of claim 1 or 2.
  4. 4. The duplex RT-qPCR detection system according to claim 3, wherein the nucleotide sequence of the probe PoRVA G-P is shown as SEQ ID No. 7, and the nucleotide sequence of the probe PoRVA G-P is shown as SEQ ID No. 17.
  5. 5. The duplex RT-qPCR assay system according to claim 3 or 4, wherein the 5 'end of the probe PoRVA G-P and the probe PoRVA G-P are modified with different reporter fluorophores, and the 3' end of the probe PoRVA G-P and the probe PoRVA G-P are modified with quencher groups.
  6. 6. The duplex RT-qPCR detection system according to claim 5, wherein the reaction concentrations of the upstream primer PoRVA G-F, the downstream primer PoRVA G-R, the upstream primer PoRVA G-F and the downstream primer PoRVA G-R are all 0.2-0.5. Mu. Mol/L, and the reaction concentrations of the probe PoRVA G-P and the probe PoRVA G-P are all 0.2-0.3. Mu. Mol/L.
  7. 7. The duplex RT-qPCR assay system of claim 6, further comprising a G3 positive control plasmid and a G9 positive control plasmid.
  8. 8. The duplex RT-qPCR assay system of claim 6, further comprising dNTPs, magnesium ions, reaction buffer, reverse transcriptase, taq DNA polymerase and RNase inhibitor.
  9. 9. The duplex RT-qPCR detection system according to claim 8, wherein the reaction procedure comprises a reverse transcription phase at 55℃for 5-15 minutes, a pre-denaturation phase at 95℃for 30 seconds, an amplification phase at 95℃for 5-10 seconds, and 60+ -2℃for 20-30 seconds for 45 cycles.
  10. 10. Use of a primer combination according to any one of claims 1 to 2 or a duplex RT-qPCR detection system according to any one of claims 3 to 9 for porcine rotavirus detection or porcine rotavirus typing identification for non-disease diagnosis or treatment purposes.

Description

Taqman probe primer combination for detecting double RT-qPCR of group A porcine rotavirus G3 and G9 strains and application thereof Technical Field The invention relates to the technical field of nucleic acid detection, in particular to a Taqman probe primer combination for detecting double RT-qPCR of group A porcine rotavirus G3 and G9 strains and application thereof. Background Porcine rotavirus (Porcine rotavirus, poliv) is one of the main enteropathogens causing severe dehydration, slow growth and development and even death of piglets, and its widespread popularity causes significant economic loss to the world pig industry, severely hampering the sustainable development of the pig industry. According to recent epidemiological investigation reports of porcine diarrhea pathogens, rotaviruses show a remarkable high-onset situation in the global scope, wherein group A porcine rotaviruses are taken as main gene groups and are widely popular in the world, the viruses can be further divided into a plurality of subtypes according to the antigenicity difference of VP7 (G genotype) and VP4 (P genotype), wherein the G3 type and the G9 type are two dominant subtypes which are most widely popular and most pathogenic in the global scope, the two dominant subtypes are often mixed to infect or alternately epidemic, and the cross protection among serotypes is weaker, so that the clinical diagnosis and prevention and control are extremely challenging. At present, the detection method for the group A porcine rotavirus mainly comprises traditional methods such as virus separation culture, electron microscope observation, ELISA, RT-PCR and the like, but the methods have obvious limitations such as low sensitivity, poor specificity, complicated operation, long time consumption and the like, and are difficult to meet the requirements of clinical rapid and accurate diagnosis. Especially under the current situation of mixed infection or alternate epidemic, the traditional detection method often cannot effectively distinguish G3 type strains from G9 type strains, so that the diagnosis result is inaccurate, and the establishment and implementation of prevention and control measures are further affected. Therefore, the detection method capable of simultaneously, rapidly and accurately detecting the group A porcine rotavirus G3 and G9 strains is developed, and has important significance for the healthy development of pig industry. Disclosure of Invention The invention aims to disclose a Taqman probe primer combination for detecting double RT-qPCR of group A porcine rotavirus G3 and G9 strains and application thereof, so as to solve one or more technical problems in the prior art and provide at least one beneficial selection or creation condition. In order to achieve the above purpose, the present invention is realized by the following technical scheme: The first aspect of the invention is to provide a primer combination for amplifying G3 type porcine rotavirus and G9 type porcine rotavirus simultaneously. The primer combination comprises an upstream primer PoRVA G-F and a downstream primer PoRVA G-R for amplifying the VP7 gene conservation region of the G3-type porcine rotavirus, and an upstream primer PoRVA G-F and a downstream primer PoRVA G-R for amplifying the VP7 gene conservation region of the G9-type porcine rotavirus. In a further embodiment of the first aspect of the present invention, the nucleotide sequence of the upstream primer PoRVA G-F is shown in SEQ ID No. 1, the nucleotide sequence of the downstream primer PoRVA G-R is shown in SEQ ID No. 2, the nucleotide sequence of the upstream primer PoRVA G-F is shown in SEQ ID No. 4, and the nucleotide sequence of the downstream primer PoRVA G-R is shown in SEQ ID No. 5. The two primer pairs have good specificity and have no cross reaction with each other, and can realize the same-tube compound amplification of the G3 type porcine rotavirus and the G9 type porcine rotavirus. The second aspect of the invention is to provide a dual RT-qPCR detection system. The double RT-qPCR detection system comprises a probe PoRVA G-P for specifically targeting the VP7 gene conservation region of the G3 type porcine rotavirus, a probe PoRVA G-P for specifically targeting the VP7 gene conservation region of the G3 type porcine rotavirus, and the primer combination disclosed by the first aspect of the invention. In a further embodiment of the second aspect of the present invention, the nucleotide sequence of the probe PoRVA G-P is shown as SEQ ID No. 3 and the nucleotide sequence of the probe PoRVA G-P is shown as SEQ ID No. 6. In a further embodiment of the second aspect of the present invention, the 5 'ends of the probes PoRVA G-P and PoRVA G-P are modified with different reporter fluorophores, respectively, and the 3' ends of the probes PoRVA G-P and PoRVA G-P are modified with a quencher. Different probes can detect a plurality of target genes simultaneously in the same reaction tube by ma