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CN-122012807-A - LAMP primer composition for nucleic acid chromatography, IBV detection test strip, kit, application of LAMP primer composition and kit and method for detecting IBV

CN122012807ACN 122012807 ACN122012807 ACN 122012807ACN-122012807-A

Abstract

The invention relates to the technical field of virology and molecular biology, in particular to an LAMP primer composition for nucleic acid chromatography, an IBV detection test strip, a kit, application thereof and a method for detecting IBV, wherein the LAMP primer composition comprises an outer primer pair, an inner primer pair and a loop primer pair, the outer primer pair comprises an outer forward primer F3 and an outer reverse primer B3, the inner primer pair comprises an inner forward primer FIP and an inner reverse primer BIP, and the loop primer pair comprises a loop forward primer LF and a loop reverse primer LB. The method for detecting the IBV improves the sensitivity of IBV detection, has strong specificity and low cost, does not need professional skill training and expensive instruments, can complete the whole process from sample processing to result interpretation within 1 hour only by basic constant temperature equipment, and is suitable for on-site instant detection scenes of farms, basic-level veterinary stations and the like.

Inventors

  • SHI HAO
  • REN HONGXIN

Assignees

  • 合肥善本生物科技有限公司

Dates

Publication Date
20260512
Application Date
20251219

Claims (10)

  1. 1. A LAMP primer composition for nucleic acid chromatography, characterized in that it comprises an outer primer pair comprising an outer forward primer F3 and an outer reverse primer B3, an inner primer pair comprising an inner forward primer FIP and an inner reverse primer BIP, and a loop primer pair comprising a loop forward primer LF and a loop reverse primer LB, The external forward primer F3 has a nucleotide sequence shown as SEQ ID NO. 1; The external reverse primer B3 has a nucleotide sequence shown as SEQ ID NO. 2; The internal forward primer FIP has a nucleotide sequence shown as SEQ ID NO. 3; The internal reverse primer BIP has a nucleotide sequence shown as SEQ ID NO. 4; The loop forward primer LF has a nucleotide sequence shown as SEQ ID NO. 5; the loop reverse primer LB has a nucleotide sequence shown as SEQ ID NO. 6; the 5 'end of the FIP is modified with a C3 Spacer and DNA1, the 5' end of the LF is modified with a C3 Spacer and DNA2, wherein, The DNA1 has a nucleotide sequence shown as SEQ ID NO. 7; the DNA2 has a nucleotide sequence shown as SEQ ID NO. 8.
  2. 2. The LAMP primer composition according to claim 1, wherein the LAMP primer composition is designed with a conserved region N gene of infectious bronchitis virus as a target sequence.
  3. 3. Use of the LAMP primer composition of claim 1 or 2 in the preparation of an IBV detection product.
  4. 4. The IBV detection test strip is characterized by comprising a nitrocellulose membrane, a binding pad, a sample pad and absorbent paper; the nitrocellulose membrane is provided with a T line and a C line; The T line is prepared from a T line nucleic acid probe solution, and the T line nucleic acid probe solution contains DNA3-Biotin; the DNA3 can be complementarily paired with the DNA1 as set forth in claim 1, wherein the DNA3 has a nucleotide sequence shown as SEQ ID NO. 9; The C line is prepared from a C line quality control solution, and the C line quality control solution contains an anti-DNP monoclonal antibody; Spraying a mixed probe on the bonding pad, wherein the mixed probe comprises a detection probe and a reference probe; the volume ratio of the detection probe to the reference probe is 2:1; The detection probe is DNA4-AuNPs, The DNA4 is complementarily paired with the DNA2 as set forth in claim 1, wherein the DNA4 has a nucleotide sequence shown in SEQ ID NO. 10.
  5. 5. A kit comprising the LAMP primer composition of claim 1 or 2 and a LAMP amplification reagent.
  6. 6. The kit of claim 5, wherein the LAMP amplification reagent comprises betaine, urea, a lysate, and an enzyme for performing a LAMP reaction; The final concentration of the betaine is 1-1000 mmol/L, preferably 200-600 mmol/L; the final concentration of urea is 1-100 mmol/L, preferably 10-30 mmol/L.
  7. 7. The kit of claim 5 or 6, wherein the LAMP primer composition has a final concentration of 1-100 μmol/L, wherein the molar ratio of the outer primer pair F3/B3, the inner primer pair FIP/BIP and the loop primer pair LF/LB is 1:3-8:1-8, preferably 1:5-8:2-6.
  8. 8. A method of detecting IBV, the method comprising the steps of: 1) Extracting RNA of a sample to be detected; 2) Performing a LAMP amplification reaction using the LAMP primer composition of claim 1 or 2 or the kit of any one of claims 5 to 7 with the RNA of the sample to be tested as a template; 3) Adding the amplification product into a diluent, mixing, and then dripping the mixture into the IBV nucleic acid detection test strip disclosed in claim 4, soaking for 8-15 min for color development observation, wherein the color development of the C line and the color development of the T line are positive, the color development of the C line and the color development of the T line are not negative, and the detection result is invalid if the color development of the C line is not performed.
  9. 9. The method according to claim 8, wherein in step 1) the sample to be tested is one or more of a chicken throat swab, a chicken nasal discharge, a chicken excreta, a chicken tissue homogenate and a chicken blood supernatant, preferably a chicken throat swab.
  10. 10. The method according to claim 8 or 9, characterized in that in step 2) the molar ratio of outer primer pair F3/B3, inner primer pair FIP/BIP and loop primer pair LF/LB in the LAMP amplification reaction is 1:3-8:1-8, preferably 1:5-8:2-6; the LAMP amplification reaction conditions comprise a temperature of 60-80 ℃ and a time of 20-40 min.

Description

LAMP primer composition for nucleic acid chromatography, IBV detection test strip, kit, application of LAMP primer composition and kit and method for detecting IBV Technical Field The invention relates to the technical fields of virology and molecular biology, in particular to an LAMP primer composition for nucleic acid chromatography, an IBV detection test strip, a kit, application thereof and a method for detecting IBV. Background Infectious Bronchitis (IB) is a highly infectious acute upper respiratory disease caused by IBV, and has remarkable harm to the global chicken industry, namely, on one hand, the egg yield of laying hens is drastically reduced, the quality of eggs is poor (soft shell eggs and malformed eggs appear), on the other hand, the growth of broilers is blocked, the feed return rate is reduced, and serious economic loss is directly caused. Therefore, the rapid and accurate diagnosis of IBV is a key link for controlling epidemic situation diffusion and reducing breeding loss. At present, the method for detecting the IBV nucleic acid mainly depends on technologies such as RT-PCR, real-time fluorescence RT-PCR and the like, and has the obvious limitations that firstly, the method has high requirements on professional skills of operators and can ensure operation standardization and result accuracy only through systematic training, secondly, the method depends on expensive instruments such as a PCR instrument, a fluorescence quantitative detector and the like, has high equipment purchasing and maintenance cost, and is difficult to popularize in basic-level farms. To solve the above problems, isothermal amplification techniques (such as loop-mediated isothermal amplification, LAMP) are becoming a research hotspot for IBV field detection. The LAMP technology can realize nucleic acid amplification by simple constant-temperature equipment, and the reagent cost is lower than that of RT-PCR, and can further get rid of the dependence on a reading instrument after being matched with a colloidal gold lateral chromatography technology. However, the traditional LAMP-colloidal gold detection method has the core defects that the signal capture is realized by relying on the combination mode of Biotin-Streptavidin (SA) and antibody-Digoxin/FAM/FITC, the detection method is not only influenced by the sensitivity stability of the antibody and Biotin, but also has high difficulty in controlling the quality of the antibody in the production process, and is easy to cause fluctuation of detection results. Therefore, developing IBVLAMP-colloidal gold detection technology which does not depend on antibody/biotin, is higher in sensitivity and is more convenient to operate becomes an urgent need in the current IBV detection field. Disclosure of Invention The invention aims to overcome the defects that in the existing IBV detection, the RT-PCR method depends on professionals and expensive instruments, the traditional LAMP-colloidal gold method depends on antibodies/biotin, and has unstable sensitivity, false negative risk and high cost, so that the LAMP primer composition for nucleic acid chromatography, the IBV detection test paper strip, the kit and the application thereof and the method for detecting the IBV are provided. In order to achieve the above object, in a first aspect, the present invention provides a LAMP primer composition for nucleic acid chromatography, comprising an outer primer pair comprising an outer forward primer F3 and an outer reverse primer B3, an inner primer pair comprising an inner forward primer FIP and an inner reverse primer BIP, and a loop primer pair comprising a loop forward primer LF and a loop reverse primer LB, wherein, The external forward primer F3 has a nucleotide sequence shown as SEQ ID NO. 1; The external reverse primer B3 has a nucleotide sequence shown as SEQ ID NO. 2; The internal forward primer FIP has a nucleotide sequence shown as SEQ ID NO. 3; The internal reverse primer BIP has a nucleotide sequence shown as SEQ ID NO. 4; The loop forward primer LF has a nucleotide sequence shown as SEQ ID NO. 5; the loop reverse primer LB has a nucleotide sequence shown as SEQ ID NO. 6; the 5 'end of the FIP is modified with a C3 Spacer and DNA1, the 5' end of the LF is modified with a C3 Spacer and DNA2, wherein, The DNA1 has a nucleotide sequence shown as SEQ ID NO. 7; the DNA2 has a nucleotide sequence shown as SEQ ID NO. 8. Preferably, the LAMP primer composition is designed by taking the N gene of a conserved region of the infectious bronchitis virus as a target sequence. In a second aspect, the invention provides an application of the LAMP primer composition in preparation of IBV detection products. In a third aspect, the present invention provides an IBV test strip comprising a nitrocellulose membrane, a conjugate pad, a sample pad, and a bibulous paper; the nitrocellulose membrane is provided with a T line and a C line; The T line is prepared from a T line nucleic acid probe so