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CN-121992121-A - Composition, kit and detection method for detecting burkholderia-like melitensis based on double-target RPA-lateral chromatography test strip

CN121992121ACN 121992121 ACN121992121 ACN 121992121ACN-121992121-A

Abstract

The invention belongs to the technical field of molecular diagnosis, and particularly relates to a composition, a kit and a detection method for detecting burkholderia melitensis based on a double-target RPA-lateral chromatography test strip. The composition comprises a first RPA primer pair and a first probe aiming at a first target gene of the burkholderia meliotidis, and a second RPA primer pair and a second probe aiming at a second target gene, wherein the 5' ends of the first probe and the second probe are respectively marked with different reporting groups which can be identified by a lateral chromatography test strip. The detection method comprises the steps of extracting genome DNA of a sample to be detected, performing RPA isothermal amplification reaction by using the composition, diluting an amplification product, dripping the diluted amplification product into a sample loading area of a lateral chromatography test strip, and reading a result. The method effectively avoids false negative missed detection caused by single-target gene mutation through double-target double-confirmation, improves detection accuracy, is simple and convenient to operate, low in cost, free of complex instruments, visible in result naked eyes and particularly suitable for rapid screening of basic sites.

Inventors

  • ZHANG YANJUN
  • KONG XIAOXIAO
  • WU XUEYING
  • FU JIANGUANG
  • ZHU LIGUO
  • LI ZHIFENG
  • SHAO GAOXIANG
  • WANG JING
  • ZHANG YUE
  • YANG LEI
  • MENG FANWEI
  • LIANG SHUYI
  • DU YINGXIA

Assignees

  • 浙江省疾病预防控制中心(浙江省预防医学科学院)
  • 江苏省疾病预防控制中心(江苏省预防医学科学院)
  • 苏州中科苏净生物技术有限公司

Dates

Publication Date
20260508
Application Date
20260114

Claims (9)

  1. 1. Be used for detecting type gangrene primary a composition of the gram-holdelella, characterized by comprising the following steps: the first RPA primer pair and the first probe are used for specifically amplifying and detecting a first target gene of Burkholderia melioides; a second RPA primer pair and a second probe for specifically amplifying and detecting a second target gene of burkholderia meliotidis; the first target gene is different from the second target gene; The 5' end of the first probe is marked with a first reporting group, the 5' end of the second probe is marked with a second reporting group different from the first reporting group, the 3' ends of the first probe and the second probe are respectively modified with a blocking group, and the first reporting group and the second reporting group can be specifically captured and developed by different detection lines on the lateral chromatographic test strip.
  2. 2. The composition for detecting burkholderia meliotidis according to claim 1, wherein the nucleotide sequence of the upstream primer of the first RPA primer pair is shown as SEQ ID NO. 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the first probe is shown as SEQ ID NO. 3.
  3. 3. The composition for detecting burkholderia melioides according to claim 2, wherein the first reporter group is fluorescein and the blocking group is a C3 spacer.
  4. 4. The composition for detecting burkholderia meliotidis according to any one of claims 1 to 3, wherein the nucleotide sequence of the upstream primer of the second RPA primer pair is shown in SEQ ID No. 4, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 5, and the nucleotide sequence of the second probe is shown in SEQ ID No. 6.
  5. 5. The composition for detecting burkholderia melioti of claim 4, wherein the second reporter group is digoxin and the blocking group is a C3 spacer.
  6. 6. A kit for detecting burkholderia melioides, comprising a composition according to any one of claims 1-5.
  7. 7. The kit of claim 6, further comprising a lateral chromatography test strip comprising a sample addition zone, a binding pad, a detection zone and an absorbent pad, wherein the detection zone is provided with a quality control line and at least two detection lines, the quality control line is coated with a ligand capable of binding to a label on the downstream primer, and the at least two detection lines are respectively coated with an antibody or ligand capable of specifically binding to the first reporter group and the second reporter group.
  8. 8. A method for detecting burkholderia meliotis, comprising the steps of: (1) Extracting genome DNA of a sample to be detected; (2) Mixing the genome DNA obtained in the step (1) with the composition of any one of claims 1-5, and performing a recombinase polymerase isothermal amplification reaction to obtain an RPA amplification product; (3) Diluting the RPA amplification product obtained in the step (2), and then dripping the diluted RPA amplification product into a sample adding area of a lateral chromatographic test strip to perform a chromatographic reaction; (4) And observing the color development result of the detection area of the lateral chromatography test strip, wherein the color development result comprises the steps of judging that the sample to be detected contains the meliotic Burkholderia if the quality control line and the two detection lines are both developed, judging that the sample to be detected does not contain the meliotic Burkholderia if only the quality control line is developed and neither of the two detection lines is developed, and judging that the detection is invalid if the quality control line is not developed.
  9. 9. The method according to claim 8, wherein in the step (2), the condition of the isothermal amplification reaction of the recombinase polymerase is that the reaction is carried out at 42 ℃ for 15-30min.

Description

Composition, kit and detection method for detecting burkholderia-like melitensis based on double-target RPA-lateral chromatography test strip Technical Field The invention belongs to the technical field of molecular diagnosis, and particularly relates to a composition, a kit and a detection method for detecting burkholderia melitensis based on a double-target RPA-lateral chromatography test strip (LFD). Background The berkholderia-like Bacteria (BP) is a highly pathogenic bacterium, can cause the berkholderia-like disease, is popular in southeast Asia and south China, has high disease death rate, and is important for treatment, prevention and control in early stage rapid diagnosis. Currently, molecular diagnostic methods are widely used due to their high sensitivity and specificity. Among them, the isothermal amplification technology of recombinase polymerase has been attracting attention because of its rapid reaction, requiring only isothermal equipment (37-42 ℃). In order to further improve the convenience and applicability of detection, RPA technology is often used in conjunction with different detection platforms. There are a number of RPA coupling schemes in the prior art. For example, patent document CN116083612a discloses a "composition and method for one-pot detection of melioidosis nucleic acid based on RPA-Cas12 a". The technology integrates RPA amplification and CRISPR/Cas12a detection in one reaction tube, and realizes double-signal visualization through a fluorescent probe and HNB dye. The method has the advantages of simple operation and pollution prevention in closed tube detection. However, its detection relies on the activity of Cas12a protein and fluorescence excitation/reading equipment, which is relatively costly and requires a specific light source for fluorescence interpretation in a resource-limited substrate site. More importantly, the technology mainly aims at detecting a single target point of a pathogen, and introduces human internal reference genes as internal standards in the sample extraction and reaction processes. The effect of the reference gene is quality control, false negative interpretation caused by sample quality problem is prevented, but the risk that false negative occurs in detection of the single target is caused by possible mutation or deletion of the pathogen self target gene is not solved. Another common scheme is as disclosed in patent document CN114807401a, namely a composition for visually detecting melioidosis based on RPA-LbCas a system and application thereof, which adopts a two-step method of RPA amplification and CRISPR detection, and also detects single targets based on fluorescent signals. In addition, the combination of the RPA technology and the lateral chromatography test strip is also a mature rapid detection path, and the RPA technology and the lateral chromatography test strip are low in cost, visual in result and free from complex instruments. However, most of the existing RPA-LFD detection methods are designed aiming at a single target point, and the hidden trouble of missed detection exists when the genome of the pathogen is mutated. Therefore, there is still a need in the art for a burkholderia-like detection scheme for detecting burkholderia-like jakov which has higher detection accuracy, can effectively avoid false negative omission caused by single-target gene mutation, further reduces detection cost, and is more suitable for rapid screening on a basic site with crude equipment, so as to solve the problems in the prior art. Disclosure of Invention The invention aims to provide a composition, a kit and a detection method for detecting burkholderia melioides based on a double-target RPA-lateral chromatography test strip, so as to solve the problems of false negative omission caused by single-target gene mutation and the like in the prior art. The technical scheme of the invention is that in a first aspect, a composition for detecting burkholderia meliotidis is provided, which comprises the following components: the first RPA primer pair and the first probe are used for specifically amplifying and detecting a first target gene of Burkholderia melioides; a second RPA primer pair and a second probe for specifically amplifying and detecting a second target gene of burkholderia meliotidis; wherein the first target gene is different from the second target gene; The 5' end of the first probe is marked with a first reporting group, the 5' end of the second probe is marked with a second reporting group different from the first reporting group, the 3' ends of the first probe and the second probe are respectively modified with a blocking group, and the first reporting group and the second reporting group can be specifically captured and developed by different detection lines on the lateral chromatographic test strip. Preferably, the nucleotide sequence of the upstream primer of the first RPA primer pair is shown as SEQ ID NO.1, the nucleotide sequence