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CN-122012767-A - Primer group, kit and method for LAMP-LFD detection of cryptosporidium parvum

CN122012767ACN 122012767 ACN122012767 ACN 122012767ACN-122012767-A

Abstract

The invention discloses a primer group, a kit and a method for LAMP-LFD detection of cryptosporidium parvum, which belong to the technical field of molecular biological detection, wherein the primer group comprises an LAMP inner primer pair and/or an LAMP outer primer pair for detecting a target MEDLE-5 gene, the LAMP inner primer pair comprises primers MEDLE-5-FIP and MEDLE-5-BIP, the nucleotide sequences of the primers are respectively shown as sequence table SEQ ID NO:11-12, and the LAMP outer primer pair comprises primers MEDLE-5-F3 and MEDLE-5-B3, and the nucleotide sequences of the primers are respectively shown as sequence table SEQ ID NO: 9-10. The primer group provided by the invention is designed by taking the cryptosporidium MEDLE-5 gene as a target gene, can realize the rapid detection of cryptosporidium parvum, and plays an important role in timely preventing and controlling cryptosporidium parvum.

Inventors

  • LI JIANHUA
  • ZHANG NAN
  • SUN YAWEI
  • LI XIN
  • WANG XIAOCEN
  • LIU ZHENZHEN
  • ZHANG XU
  • ZHENG YUAN
  • Dao Jiahao
  • GONG PENGTAO

Assignees

  • 吉林大学

Dates

Publication Date
20260512
Application Date
20260413

Claims (8)

  1. 1. The primer set for LAMP-LFD detection of the cryptosporidium parvum is characterized by comprising an LAMP inner primer pair and an LAMP outer primer pair for detecting a target MEDLE-5 gene, wherein the LAMP inner primer pair comprises primers MEDLE-5-FIP and MEDLE-5-BIP, the nucleotide sequences of the primers are respectively shown as SEQ ID NO. 11-12 of a sequence table, and the LAMP outer primer pair comprises primers MEDLE-5-F3 and MEDLE-5-B3, and the nucleotide sequences of the primers are respectively shown as SEQ ID NO. 9-10 of the sequence table.
  2. 2. The primer set for LAMP-LFD detection by Cryptosporidium parvum as set forth in claim 1, wherein the primers MEDLE-FIP and MEDLE-5-BIP are labeled with a biotin group and 6-carboxyfluorescein, respectively.
  3. 3. Use of the primer set for LAMP-LFD detection of cryptosporidium parvum as set forth in claim 1 or 2 for preparing a product for detecting cryptosporidium parvum.
  4. 4. The use according to claim 3, wherein the product is a kit or a test strip.
  5. 5. A kit for LAMP-LFD detection of Cryptosporidium parvum, comprising the primer set for LAMP-LFD detection of Cryptosporidium parvum as set forth in claim 1 or 2.
  6. 6. The kit for LAMP-LFD control detection of Cryptosporidium parvum as set forth in claim 5, further comprising a DNA polymerase, a polymerase buffer, an amplification product dilution, a detection test strip, a positive control reagent and a negative control reagent.
  7. 7. The kit for LAMP-LFD detection of Cryptosporidium parvum as set forth in claim 6, wherein the DNA polymerase is Bst II DNA polymerase, the polymerase buffer is LAMP premix reaction solution, the amplification product diluent is ddH 2 O, and the detection test strip is a lateral side stream single-nucleotide detection test strip.
  8. 8. The kit for LAMP-LFD control of Cryptosporidium parvum as set forth in claim 6, wherein the positive control reagent is genomic DNA of Cryptosporidium parvum and the negative control reagent is ddH 2 O.

Description

Primer group, kit and method for LAMP-LFD detection of cryptosporidium parvum Technical Field The invention relates to the technical field of molecular biological detection, in particular to a primer group, a kit and a method for LAMP-LFD detection of cryptosporidium parvum. Background Cryptosporidium parvum (Cryptosporidium parvum) is an opportunistic pathogenic protozoan of the phylum apicomplexa, and is mainly parasitic on brush-like edges of host intestinal epithelial cells, causing Cryptosporidium parvum disease which is mainly characterized by watery diarrhea. The disease is an important zoonosis, and the world health organization ranks the zoonosis as one of six diarrhea diseases in the world. The pathogen of cryptosporidiosis is mainly transmitted through a feces-mouth path, and oocysts have strong resistance to environment and conventional chlorine disinfectants, are easy to cause water source outbreaks, and form a continuous threat to public health safety. The patients with normal immune function usually have a self-limiting course after infection, but for infants, immunocompromised or defective patients (such as AIDS patients), the infection can lead to severe and persistent diarrhea, even life threatening. MEDLE-5 is a secreted protein encoded by the cryptosporidium parvum specific gene cgd6_5480, and belongs to members of the cryptosporidium parvum specific MEDLE protein family. Researches show that MEDLE family proteins are closely related to host cell invasion process and host specificity of the insect bodies, and are important potential virulence and immunogenicity factors. The kinetics of gene expression of MEDLE exhibited a significant difference compared to other members of the family, with transcript levels peaking at 2 hours after sporophytes invade the host, followed by a rapid decline, suggesting that its primary function may be concentrated in the early stages of host cell contact and adhesion of invasion. Protein localization studies showed that MEDLE-5 was widely distributed over the entire surface of sporozoites and merozoites, which is in clear contrast to MEDLE-3 members, which are primarily concentrated in the front of the worm, suggesting that their mechanism of action may not rely on typical apical invasion organs. In addition, MEDLE-5 has minimal cross-reactivity with antibodies to other family members, indicating its unique antigenicity. There are significant differences in the copy number of MEDLE genes among cryptosporidium species, for example, cryptosporidium parvum with broad-spectrum host adaptability possesses 6 MEDLE genes including MEDLE-5, whereas human cryptosporidium with a narrower host range contains only 1, which can be used as a reliable molecular marker for developing a specific nucleic acid detection method capable of identifying cryptosporidium species. In view of the secretion characteristics, early expression patterns and inter-species distribution differences, MEDLE-5 can be used as an important object for researching the interaction mechanism of cryptosporidium and a host, and has potential to become a molecular target for differentiating insect species or developing a novel diagnostic detection method. Loop-mediated isothermal amplification (LAMP) is a novel isothermal amplification of nucleic acids. This technique designs 4 to 6 specific primers (including outer primer F3/B3, inner primer FIP/BIP and optional loop primer LF/LB) for 6 specific regions of the target gene. Under the action of Bst DNA polymerase with strand displacement activity, the reaction can be carried out at a constant temperature of 60 to 65℃without a complicated thermal cycling process. The amplification efficiency is extremely high, and the exponential amplification of 10 9 to 10 10 times can be realized in 15 to 60 minutes. Therefore, the LAMP technology has the remarkable advantages of high sensitivity, strong specificity, rapid amplification and only needs a simple constant-temperature device such as a water bath kettle or a metal bath, and the dependence on precise instruments is greatly reduced. The lateral flow assay test strip (Lateral Flow Dipstick, LFD) is a well-established chromatographic immunoassay platform that moves the sample across the membrane by capillary action and specifically binds to pre-immobilized capture probes, ultimately presenting the results in the form of macroscopic bands. The LFD has the advantages of visual and clear result interpretation, simple and quick operation, no need of additional instruments and convenience for realizing simultaneous detection of multiple targets, and is an ideal terminal display technology for on-site quick detection. At present, LAMP-LFD detection aiming at cryptosporidium parvum MEDLE gene detection has not been reported yet, so that a method for detecting cryptosporidium parvum MEDLE-5 gene LAMP-LFD needs to be established to realize rapid judgment on whether a detection sample contains cryptosporidium parvum, and