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CN-122012757-A - Primer probe combination for simultaneously detecting streptococcus suis virulence genes sley, epf and mrp based on fluorescence type MIRA method and application thereof

CN122012757ACN 122012757 ACN122012757 ACN 122012757ACN-122012757-A

Abstract

The invention belongs to the technical field of microorganism detection, and discloses a primer probe combination, a detection kit and application thereof for simultaneously detecting key virulence genes sle, epf and mrp of streptococcus suis. Compared with the traditional PCR and real-time fluorescent quantitative PCR (q-PCR) detection method, the MIRA-qPCR detection method established by the invention can detect 3 virulence genes simultaneously in one reaction system, is more rapid and simple, and has good sensitivity and specificity. Therefore, the method is simpler and easier to implement, consumes less time, has various result presentation modes, is more suitable for timely detecting, preventing and controlling epidemic disease pathogens in a basic layer, and provides a good path for on-site rapid detection of streptococcus suis sly, epf, mrp genes and discrimination of high-virulence streptococcus suis.

Inventors

  • HE BING
  • CHEN QIUYONG
  • ZHOU LUNJIANG
  • WANG LONGBAI
  • WU XUEMIN
  • CHEN RUJING
  • WU RENJIE
  • QIU JINGLI
  • CHEN YUHONG

Assignees

  • 福建省农业科学院畜牧兽医研究所

Dates

Publication Date
20260512
Application Date
20260122

Claims (10)

  1. 1. A primer probe combination for simultaneous detection of streptococcus suis virulence genes sle, epf and mrp, said combination comprising: i) A first pair of primers and a first probe for detecting sly genes, wherein: the sequence of the sly gene upstream primer is shown as SEQ ID NO. 1; The sequence of the sly gene downstream primer is shown as SEQ ID NO. 2; the sequence of sly gene probe is shown in SEQ ID NO. 3; ii) a second pair of primers and a second probe for detecting epf genes, wherein: The sequence of the epf gene upstream primer is shown as SEQ ID NO. 4; the sequence of the epf gene downstream primer is shown in SEQ ID NO. 5; the sequence of epf gene probe is shown in SEQ ID NO. 6; iii) A third pair of primers and a third probe for detecting the mrp gene, wherein: the sequence of the primer upstream of the mrp gene is shown as SEQ ID NO. 7; the sequence of the mrp gene downstream primer is shown in SEQ ID NO. 8; the sequence of the mrp gene probe is shown in SEQ ID NO. 9.
  2. 2. A MIRA-qPCR assay kit for simultaneous detection of streptococcus suis virulence genes sle, epf and mrp comprising the primer probe combination of claim 1.
  3. 3. The MIRA-qPCR detection kit as claimed in claim 2, wherein the concentration of the primer is 5-20. Mu.M.
  4. 4. The MIRA-qPCR detection kit according to claim 2, wherein the probe for detecting sly gene is labeled with a CY5 reporter fluorophore, the probe for detecting epf gene is labeled with a FAM reporter fluorophore, and the probe for detecting mrp gene is labeled with a VIC reporter fluorophore.
  5. 5. The MIRA-qPCR assay kit of claim 2, further comprising buffers, enzyme mixtures and dNTPs required for performing the multi-enzyme isothermal rapid amplification reaction.
  6. 6. A MIRA-qPCR method for simultaneously detecting streptococcus suis virulence genes sley, epf and mrp is characterized by comprising the following steps: s1, extracting genome DNA of a sample to be detected; S2, carrying out real-time fluorescence multi-enzyme isothermal rapid amplification reaction under isothermal conditions by using the DNA obtained in the step S1 as a template and using the primer probe combination of claim 1; S3, judging whether streptococcus suis slot, epf and mrp virulence genes are contained in the sample according to fluorescent signals in the amplification process.
  7. 7. The method according to claim 6, wherein the constant temperature condition is 37 ℃ to 42 ℃.
  8. 8. The method of claim 6, wherein the time for the real-time fluorescent MIRA-qPCR reaction is 10-40 minutes.
  9. 9. Use of a primer probe combination according to claim 1 or a kit according to claim 2 for the detection of streptococcus suis sle, epf and mrp virulence genes, streptococcus suis virulence typing, epidemiological investigation of streptococcus suis related diseases or evaluation of streptococcus suis vaccine efficacy, wherein the use is for non-diagnostic and therapeutic purposes.
  10. 10. Use of the primer probe combination according to claim 1 or the kit according to claim 2 for the preparation of a product for detecting streptococcus suis slot, epf and mrp virulence genes.

Description

Primer probe combination for simultaneously detecting streptococcus suis virulence genes sley, epf and mrp based on fluorescence type MIRA method and application thereof Technical Field The invention belongs to the technical field of microorganism detection, and relates to a primer probe combination for simultaneously detecting streptococcus suis virulence genes sley, epf and mrp based on a fluorescence type MIRA method and application thereof. Background Streptococcus suis (Streptococcus suis) is an important zoonotic agent, and mainly infects pig groups, and can cause serious diseases such as meningitis, arthritis, septicemia and the like of pigs, thereby causing great economic loss. In addition, the bacteria can infect human beings through direct contact or eating pollution products, and cause suppurative meningitis, toxic shock syndrome and even death. Different streptococcus suis can be classified into 29 species according to capsular polysaccharide antigens, with the dominant serotypes that prevail being predominantly types 2,3, 7, 9. However, not all streptococcus suis are pathogenic, clinically common subclinical infected recessive carrier animals, which increases the difficulty of clinical identification and epidemiological judgment of streptococcosis. The streptococcus suis carries more virulence factors, mainly comprises Fibronectin Binding Protein (FBPs), lysozyme release protein (MRP) virulence related sequence (PRF 2), extracellular factors (EPF), hemolysin (SLY) and the like, wherein SLY is a key factor for causing tissue injury, meningitis, inflammatory reaction and apoptosis, and MRP and EPF are commonly present together to promote bacterial diffusion and are markers of strong strains. In clinical practice, the rapid and accurate determination of whether streptococcus suis carried by animals is a high virulent strain is an important basis for confirming pathogenicity and further designing a targeted medication scheme. Therefore, there is a significant need to establish a method that can rapidly and sensitively detect the key virulence genes of streptococcus suis in clinical assays. The multi-enzyme isothermal nucleic acid rapid amplification technology (Multienzyme Isothermal Rapid Amplification, MIRA) requires only 2 primers, and a thermal cycler is not needed, so that the kit is a convenient and rapid pathogen detection tool. The invention establishes a fluorescence quantitative MIRA (MIRA-qPCR) rapid detection method aiming at key virulence factors of streptococcus suis, and provides technical support for rapid and simple detection of the streptococcus suis sly, epf, mrp genes. At present, detection of the streptococcus suis sly, epf, mrp gene based on MIRA-qPCR is not reported. Disclosure of Invention The primary object of the invention is to provide a primer probe combination for simultaneously detecting the sly, epf, mrp genes of streptococcus suis. A second object of the present invention is to provide a kit for primer probe combinations that can detect the Streptococcus suis sly, epf, mrp gene simultaneously. The third object of the invention is to provide a real-time fluorescence MIRA detection method based on the primer probe combination or the kit. The fourth object of the invention is to provide the application of the primer probe combination or the kit, and the primer probe combination provided by the invention can specifically and rapidly detect the streptococcus suis sly, epf, mrp genes in the same reaction system and has the advantage of high sensitivity. In order to achieve the above object, the present invention provides the following technical solutions: the invention provides a primer probe combination for simultaneously detecting streptococcus suis virulence genes sley, epf and mrp, which comprises the following components: i) A first pair of primers and a first probe for detecting sly genes, wherein: the sequence of the sly gene upstream primer is shown as SEQ ID NO. 1; The sequence of the sly gene downstream primer is shown as SEQ ID NO. 2; the sequence of sly gene probe is shown in SEQ ID NO. 3; ii) a second pair of primers and a second probe for detecting epf genes, wherein: The sequence of the epf gene upstream primer is shown as SEQ ID NO. 4; the sequence of the epf gene downstream primer is shown in SEQ ID NO. 5; the sequence of epf gene probe is shown in SEQ ID NO. 6; iii) A third pair of primers and a third probe for detecting the mrp gene, wherein: the sequence of the primer upstream of the mrp gene is shown as SEQ ID NO. 7; the sequence of the mrp gene downstream primer is shown in SEQ ID NO. 8; the sequence of the mrp gene probe is shown in SEQ ID NO. 9. The invention also provides a MIRA-qPCR detection kit for detecting virulence genes sle, epf and mrp of streptococcus suis, which comprises the primer probe combination. Further, the concentration of the primer is 5-20 mu M. Further, the probe for detecting sly gene is labeled with CY5 reporter fluorophore,