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CN-122012810-A - TaqMan primer probe combination for detecting newcastle disease virulent and attenuated viruses, application thereof and kit

CN122012810ACN 122012810 ACN122012810 ACN 122012810ACN-122012810-A

Abstract

The invention belongs to the technical field of biology, and discloses a TaqMan primer probe combination for detecting Newcastle disease virulent and attenuated viruses, which comprises a virulent primer RT, a virulent probe RT-probe, a universal primer SRT and a universal probe SRT-probe, wherein the virulent primer RT comprises an upstream primer RT-F and a downstream primer RT-R, the universal primer SRT comprises an upstream primer SRT-F and a downstream primer SRT-R, the TaqMan primer probe combination is used for establishing an NDV virulent and attenuated virus TaqMan probe fluorescent quantitative RT-PCR detection method, the method has no cross reaction on common avian viruses AIV, FADV, IBV, IBDV amplification, has good specificity, has the minimum detection concentration of 10 1 copies/uL on the Newcastle disease virulent and attenuated viruses, improves 100 times compared with a conventional RT-PCR method, has good sensitivity, is compared with the conventional RT-PCR method for detection, has 100% of detection rate, and is superior to the conventional RT-PCR method, and the detection result is consistent with the sequencing result.

Inventors

  • REN TAO
  • CHEN LIBIN
  • QIU XUSHENG
  • ZHU HE
  • YAN XIAOTONG
  • LIN QIUYAN

Assignees

  • 华南农业大学
  • 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心)

Dates

Publication Date
20260512
Application Date
20260116

Claims (6)

  1. 1. The TaqMan primer probe combination for detecting the Newcastle disease virulent and attenuated viruses is characterized by comprising a virulent primer RT, a virulent probe RT-probe, a universal primer SRT and a universal probe SRT-probe, wherein the virulent primer RT comprises an upstream primer RT-F and a downstream primer RT-R, and the universal primer SRT comprises an upstream primer SRT-F and a downstream primer SRT-R; The nucleotide sequence of the RT-F is shown as SEQ ID NO.1, the nucleotide sequence of the RT-R is shown as SEQ ID NO.2, and the nucleotide sequence of the virulent probe RT-probe is shown as SEQ ID NO. 3; the nucleotide sequence of the SRT-F is shown as SEQ ID NO.4, the nucleotide sequence of the SRT-R is shown as SEQ ID NO.5, and the nucleotide sequence of the universal probe SRT-probe is shown as SEQ ID NO. 6.
  2. 2. The TaqMan primer probe combination for detecting newcastle disease virulent and attenuated viruses according to claim 1, wherein the 5 'end of the virulent probe RT-probe is marked with a fluorescent group FAM, the 3' end of the virulent probe RT-probe is marked with a quenching group BHQ1, and the 5 'end of the universal probe SRT-probe is marked with a fluorescent group Cy5, and the 3' end of the universal probe SRT-probe is marked with the quenching group BHQ1.
  3. 3. Use of the TaqMan primer probe combination according to any one of claims 1-2 in the preparation of an RT-PCR detection kit.
  4. 4. A kit, characterized in that the kit comprises a reaction solution containing the TaqMan primer probe combination of any one of claims 1-2; the reaction solution contains TaqMan primer probe combinations for detecting strong and weak viruses of newcastle disease.
  5. 5. The kit according to claim 4, wherein the concentration of the primer and the probe contained in the reaction solution is 0.2 to 0.25. Mu.M/L.
  6. 6. The kit according to claim 4, wherein the minimum detection concentration of the kit for Newcastle disease virulent and attenuated viruses is 10 1 copies/uL.

Description

TaqMan primer probe combination for detecting newcastle disease virulent and attenuated viruses, application thereof and kit Technical Field The invention relates to the technical field of medicines, in particular to a TaqMan primer probe combination for detecting strong and weak viruses of newcastle disease, application thereof and a kit. Background Newcastle disease (NEWCASTLE DISEASE, ND) is a rapid, septic and highly contagious avian epidemic disease caused by newcastle disease virus (NEWCASTLE DISEASE virus, NDV), has high pathogenicity and rapid transmission characteristics, and constitutes a serious threat to the avian farming industry. Since ND was confirmed, the diagnostic system was continuously upgraded with virus evolution, and I, II, III, VI, VII, VIII, IX, XII class II genotypes were isolated, wherein type VII had gradually become the dominant genotype, and most of the genotypes causing ND pathogenesis were type VII. In recent years, the epidemic characteristics of the epidemic disease become more complex, and strong and weak strain co-infection is frequently generated in non-scale farms, so that clinical symptoms and pathological damage are aggravated, immunosuppression and immune failure are caused, interference diagnosis and epidemic situation judgment are carried out, and the identification of virulence of the strain becomes a key link for epidemic situation prevention and control. The primary detection mainly adopts serological detection, and is mainly detected through ELISA, HA and HI tests due to lower cost and convenient operation, but specific genotype of the NDV cannot be further judged, so that the specific genotype is prevented in a targeted manner. Although the present method has better effect on the prevention and control of NDV, a certain amount of ND epidemic situation still exists due to the continuous expansion of virus host range, virus evolution, vaccine immunity failure, mixed infection and other reasons. The traditional detection means such as chick embryo lethal time measurement, chick in-brain pathogenicity test, intravenous inoculation virulence index evaluation and the like are complex in operation and long in time consumption, and the traditional national standard GB/T16550-2020 RT-PCR detection method only covers VI and VII virulent strains, cannot effectively detect other epidemic strains, and has the sensitivity of only 10 3 copies/mu L. Based on the detection, the invention aims to provide a TaqMan primer probe combination capable of simultaneously detecting strong and weak viruses of newcastle disease, which has wider coverage of the detectable strains, lower detectable lowest copy number and higher sensitivity, can be used for clinical sample detection, provides technical reserve for preventing and controlling NDV, is favorable for timely diagnosing and finding ND epidemic situation, and takes targeted measures for the NDV strong and weak virus result of differential diagnosis. Disclosure of Invention The invention aims to provide a TaqMan primer probe combination for detecting newcastle disease virulent and attenuated viruses, which comprises a virulent primer RT, a virulent probe RT-probe, a universal primer SRT and a universal probe SRT-probe, wherein the virulent primer RT comprises an upstream primer RT-F and a downstream primer RT-R, and the universal primer SRT comprises an upstream primer SRT-F and a downstream primer SRT-R. Meanwhile, the invention also provides application of the TaqMan primer probe combination and a kit containing the TaqMan primer probe combination. In order to achieve the above purpose, the present invention provides the following technical solutions: The TaqMan primer probe combination for detecting the Newcastle disease virulent and attenuated viruses comprises a virulent primer RT, a virulent probe RT-probe, a universal primer SRT and a universal probe SRT-probe, wherein the virulent primer RT comprises an upstream primer RT-F and a downstream primer RT-R, and the universal primer SRT comprises an upstream primer SRT-F and a downstream primer SRT-R; The nucleotide sequence of the RT-F is shown as SEQ ID NO.1, the nucleotide sequence of the RT-R is shown as SEQ ID NO.2, and the nucleotide sequence of the virulent probe RT-probe is shown as SEQ ID NO. 3; the nucleotide sequence of the SRT-F is shown as SEQ ID NO.4, the nucleotide sequence of the SRT-R is shown as SEQ ID NO.5, and the nucleotide sequence of the universal probe SRT-probe is shown as SEQ ID NO. 6. Preferably, the 5 'end of the virulent probe RT-probe is marked with a fluorescent group FAM, the 3' end is marked with a quenching group BHQ1, the 5 'end of the universal probe SRT-probe is marked with a fluorescent group Cy5, and the 3' end is marked with the quenching group BHQ1. In addition, the invention discloses application of the TaqMan primer probe combination in preparation of an RT-PCR detection kit. Finally, the invention also discloses a kit, which comprises a