CN-122012817-A - Primer probe group based on multiplex RPA amplification and application thereof

Abstract

The invention discloses a primer probe group based on multiplex RPA amplification and application thereof. The primer probe group comprises a PHV primer, a PAdv primer, a PCV primer, a Tg primer, a Cp primer and a Ca primer, wherein the nucleotide sequence of the PHV primer is shown as SEQ ID NO. 1-2, the nucleotide sequence of the PAdv primer is shown as SEQ ID NO. 4-5, the nucleotide sequence of the PCV primer is shown as SEQ ID NO. 7-8, the nucleotide sequence of the Tg primer is shown as SEQ ID NO. 10-11, the nucleotide sequence of the Cp primer is shown as SEQ ID NO. 13-14, and the nucleotide sequence of the Ca primer is shown as SEQ ID NO. 16-17. The invention can detect PHV, PAdv, PCV, tg, cp, ca pigeon respiratory pathogens simultaneously, has low detection limit and high sensitivity, can be suitable for detection scenes of mixed infection in clinic, and avoids complex operation of multiple independent detection.

Inventors

• ZOU HENG
• MENG JIE
• CHEN QI

Assignees

• 畅启生物科技(浙江)有限公司

Dates

Publication Date

20260512

Application Date

20260316

Claims (10)

1. 1. A primer probe set based on multiplex RPA amplification, characterized in that the primer probe set comprises: PHV primer, PAdv primer, PCV primer, tg primer, cp primer and Ca primer; The PHV primer has a nucleotide sequence shown in SEQ ID NO. 1-2, the PAdv primer has a nucleotide sequence shown in SEQ ID NO. 4-5, the PCV primer has a nucleotide sequence shown in SEQ ID NO. 7-8, the Tg primer has a nucleotide sequence shown in SEQ ID NO. 10-11, the Cp primer has a nucleotide sequence shown in SEQ ID NO. 13-14, and the Ca primer has a nucleotide sequence shown in SEQ ID NO. 16-17.
2. 2. The primer probe set of claim 1, wherein the primer probe set further comprises: PHV probes, PAdv probes, PCV probes, tg probes, cp probes, and Ca probes; The nucleotide sequence of the PHV probe is shown as SEQ ID NO.3, wherein the 30 th position of the PHV probe is a FAM-marked thymine nucleotide, the 31 st position is tetrahydrofuran, and the 32 nd position is a BHQ 1-marked thymine nucleotide; The nucleotide sequence of the PAdv probe is shown as SEQ ID NO.6, wherein the 28 th position of the PAdv probe is a VIC-marked thymine nucleotide, the 30 th position is tetrahydrofuran, and the 33 th position is a BHQ 1-marked thymine nucleotide; the nucleotide sequence of the PCV probe is shown as SEQ ID NO.9, wherein the 29 th position of the PCV probe is a thymine nucleotide marked by ROX, the 31 st position is tetrahydrofuran, and the 33 rd position is a thymine nucleotide marked by BHQ 1; The nucleotide sequence of the Tg probe is shown as SEQ ID NO.12, wherein the 30 th position of the Tg probe is a FAM marked thymine nucleotide, the 31 st position is tetrahydrofuran, and the 32 nd position is a BHQ1 marked thymine nucleotide; The nucleotide sequence of the Cp probe is shown as SEQ ID NO.15, wherein the 29 th position of the Cp probe is a thymine nucleotide marked by VIC, the 30 th position is tetrahydrofuran, and the 31 st position is a thymine nucleotide marked by BHQ 1; The nucleotide sequence of the Ca probe is shown as SEQ ID NO.18, wherein the 31 st position of the Ca probe is a ROX marked thymine nucleotide, the 32 nd position is tetrahydrofuran, and the 34 th position is a BHQ1 marked thymine nucleotide; The 3' -end of the PHV probe, PAdv probe, PCV probe, tg probe, cp probe and Ca probe is provided with a modification group; The modification group is any one of C3-spacer, biotin-TEG and phosphoate.
3. 3. A lyophilized reagent comprising the primer probe set of claim 1 or 2.
4. 4. An RPA detection kit comprising the primer probe set of claim 1 or 2 and an RPA amplification reaction solution.
5. 5. The RPA detection kit according to claim 4, wherein the molar ratio of PHV primer to PHV probe is (1-3): 0.2-0.8; The mol ratio of the PAdv primer to the PAdv probe is (1-3) (0.2-0.8); The molar ratio of the primer of PCV to the PCV probe is (1-3): 0.2-0.8.
6. 6. The RPA detection kit of claim 4, wherein the molar ratio of Tg primer to Tg probe is (1-3): 0.2-0.8; the molar ratio of the Cp primer to the Cp probe is (1-3) (0.2-0.8); the molar ratio of the Ca primer to the Ca probe is (1-3): 0.2-0.8.
7. 7. A method for detecting infection of respiratory pathogens of pigeons for non-disease diagnosis and treatment purposes, comprising the steps of: Extracting to obtain genome DNA of a sample to be detected; Performing RPA detection using the primer probe set of claim 1 or 2 using the genomic DNA as a template; the respiratory pathogen of pigeon is one or more of pigeon herpesvirus PHV, pigeon adenovirus PAdv, pigeon circovirus PCV, pigeon trichomonas Tg, chlamydia psittaci Cp and candida albicans Ca.
8. 8. Use of the primer probe set of claim 1 or 2 in the preparation of RPA detection products for respiratory pathogen infection of pigeons.
9. 9. The use according to claim 8, wherein the pigeon respiratory pathogen is a combination of pigeon herpesvirus PHV, pigeon adenovirus PAdv, pigeon circovirus PCV, pigeon trichomonas Tg, chlamydia psittaci Cp and candida albicans Ca.
10. 10. The use according to claim 8, wherein the product is a kit.

Description

Primer probe group based on multiplex RPA amplification and application thereof Technical Field The invention relates to the technical field of biological detection, in particular to a primer probe group based on multiplex RPA amplification and application thereof. Background The carrier pigeon industry is a very distinctive and vitality subdivision field in the pigeon breeding industry, covers multiple directions of carrier pigeon competition, ornamental display, variety cultivation and the like, not only bears the culture value of traditional folk-custom, but also forms large-scale industrial ecology, and occupies unique positions in sports competition, culture communication and economic development. The carrier pigeons have strong environmental adaptability, but the breeding of the carrier pigeons is mostly in an intensive mode, and the carrier pigeons are often accompanied with activities such as transregional transportation, centralized training and placement and the like, so that the transmission risk of respiratory diseases is obviously higher than that of common meat pigeons and ornamental pigeons. As an important component of pigeon breeding industry, the breeding industry of carrier pigeons and other pigeons has wide breeding scale and economic value in the global scope. However, respiratory diseases of pigeons are one of the major bottlenecks restricting the healthy development of the breeding industry, and are mostly caused by mixed infection with a variety of pathogens, common pathogens include pigeon herpesviruses (Pigeon Herpesvirus, PHV), pigeon adenoviruses (Pigeon Adenovirus, PAdv), pigeon circovirus (Pigeon Circovirus, PCV), pigeon trichomonas (Trichomonas gallinae, tg), chlamydia psittaci (CHLAMYDIA PSITTACI, cp), candida albicans (Ca), and the like. Under the mixed infection condition, clinical symptoms are overlapped with each other, so that the difficulty of disease diagnosis is increased, the delay of treatment scheme formulation is easily caused, the pigeons are caused to attack and die in batches, and the serious economic loss is brought to the raisers. Currently, detection methods for pigeon respiratory pathogens mainly comprise a traditional culture separation method, a serological detection method, a conventional molecular biological detection method and the like. The traditional culture separation method is complex in operation, takes longer time, usually requires 3-7 days to obtain a detection result, is difficult to successfully separate pathogens with strict nutrition requirements, cannot meet the requirement of rapid diagnosis, has the problems of relatively simple operation, relatively poor specificity, obvious cross reaction and the like, is difficult to accurately distinguish infected pathogen types in a mixed infection scene, has limited detection accuracy, and is high in detection cost due to the fact that the conventional molecular biological detection method such as common PCR, real-time fluorescence quantitative PCR and the like has certain improvement on specificity and sensitivity, but mostly relies on a precise thermal cycle instrument, and single detection can only aim at one pathogen, and is difficult to adapt to the requirement of rapid screening in a basic culture scene. The recombinant enzyme polymerase amplification (recombinase polymerase amplification, RPA) technology is a molecular biology technology capable of completing nucleic acid amplification under the condition of constant temperature, and has the remarkable advantages of no need of a thermal cycling instrument, high amplification speed, simplicity and convenience in operation and the like. However, the sensitivity, stability and operation convenience of the basic level detection on the reagent are extremely high, and the existing RPA detection reagent aiming at single or two pathogens is difficult to meet the actual requirements of the basic level field on the rapid and accurate joint detection of three or more pathogens. Therefore, the identification method for the respiratory pathogens of the pigeons, which has the advantages of strong suitability, high specificity and excellent sensitivity, is developed, and has important significance for early warning, accurate prevention and control of diseases and quality improvement and efficiency improvement of the breeding industry. Disclosure of Invention Aiming at the defects in the prior art, the invention provides a primer probe group based on multiplex RPA amplification and application thereof. The invention provides a primer group special for identifying mixed infection of pigeon respiratory pathogens, which is designed aiming at specific conserved gene sequences of various common key pathogens (PHV, PAdv, PCV, tg, cp and Ca) of pigeon respiratory pathogens, and realizes synchronous specific amplification and detection of various pathogens in the same reaction system by optimizing sequence structures, concentration ratios and amplification reaction