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CN-121975979-A - Freeze-dried eugenic four-item limit amplification four-way detection kit and application thereof

CN121975979ACN 121975979 ACN121975979 ACN 121975979ACN-121975979-A

Abstract

The invention relates to the technical field of biology, in particular to a freeze-dried eugenic four-item limit amplification tetrad detection kit and application thereof. The invention develops a limiting amplification reaction system suitable for the eugenic tetrad-toxoplasma gondii, rubella virus, human cytomegalovirus and herpes simplex virus by optimizing a primer, a reaction reagent and a reaction system and by means of a hydrogel limiting amplification technology, and the limiting amplification system is used for detecting toxoplasma gondii, rubella virus, human cytomegalovirus and herpes simplex virus, has simple and rapid steps, low equipment requirement, can effectively prevent and treat aerosol pollution, has good storage stability, and has high sensitivity and high specificity.

Inventors

  • DING JIANWEN
  • XIAO FANG
  • XU LIKUI

Assignees

  • 湖南爱威医学检验所有限公司

Dates

Publication Date
20260505
Application Date
20260211

Claims (10)

  1. 1. The primer combination for the LAMP amplification of four eugenic items is characterized by comprising a primer combination for toxoplasma gondii detection, a primer combination for rubella virus detection, a primer combination for human cytomegalovirus detection and a primer combination for type I and type II herpes simplex virus detection; The primer combination for toxoplasma gondii detection comprises an F3 primer with a nucleotide sequence shown as SEQ ID NO. 6, a B3 primer with a nucleotide sequence shown as SEQ ID NO. 7, a FIP primer with a nucleotide sequence shown as SEQ ID NO. 8, a BIP primer with a nucleotide sequence shown as SEQ ID NO. 9, an LF primer with a nucleotide sequence shown as SEQ ID NO. 10 and an LB primer with a nucleotide sequence shown as SEQ ID NO. 11; The primer combination for rubella virus detection comprises an F3 primer with a nucleotide sequence shown as SEQ ID NO. 18, a B3 primer with a nucleotide sequence shown as SEQ ID NO. 19, a FIP primer with a nucleotide sequence shown as SEQ ID NO. 20, a BIP primer with a nucleotide sequence shown as SEQ ID NO. 21, an LF primer with a nucleotide sequence shown as SEQ ID NO.22 and an LB primer with a nucleotide sequence shown as SEQ ID NO. 23; the primer combination for detecting the human cytomegalovirus comprises an F3 primer with a nucleotide sequence shown as SEQ ID NO. 39, a B3 primer with a nucleotide sequence shown as SEQ ID NO. 40, a FIP primer with a nucleotide sequence shown as SEQ ID NO. 41, a BIP primer with a nucleotide sequence shown as SEQ ID NO. 42, an LF primer with a nucleotide sequence shown as SEQ ID NO. 43 and an LB primer with a nucleotide sequence shown as SEQ ID NO. 44; The primer combination for detecting the type I and type II herpes simplex viruses comprises an F3 primer with a nucleotide sequence shown as SEQ ID NO. 71 and/or SEQ ID NO. 77, a B3 primer with a nucleotide sequence shown as SEQ ID NO. 72 and/or SEQ ID NO. 78, a FIP primer with a nucleotide sequence shown as SEQ ID NO. 73 and/or SEQ ID NO. 79, a BIP primer with a nucleotide sequence shown as SEQ ID NO. 74 and/or SEQ ID NO. 80, an LF primer with a nucleotide sequence shown as SEQ ID NO. 75 and/or SEQ ID NO. 81, and an LB primer with a nucleotide sequence shown as SEQ ID NO. 76 and/or SEQ ID NO. 82.
  2. 2. The limiting amplification reagent is characterized by comprising a reagent A and a reagent B, The reagent A comprises a buffer solution, divalent cations, fluorescent dye, dimercapto polyethylene glycol, a first reducing agent and a freeze-drying protective agent A; The reagent B comprises the primer combination of claim 1 or 2, dNTP, bst DNA polymerase, a second reducing agent, eight-arm polyethylene glycol acrylate and a lyoprotectant B; The freeze-drying protective agent A comprises pullulan, PVP, PEG20000, hydroxypropyl-beta-cyclodextrin and glutathione; The freeze-drying protective agent B comprises mannitol, BSA, trehalose, glycine, hydroxypropyl-beta-cyclodextrin and PVP.
  3. 3. The limiting amplification reagent of claim 2, wherein, In the reagent B, the final concentration of the F3 primer is 0.04-0.08 mu M, the concentration ratio of the FIP primer to the LF primer to the F3 primer is 8:4:1, and the concentration of the BIP primer is the same as that of the FIP primer; the divalent cations include magnesium ions; The first reducing agent comprises TCEP; the second reducing agent includes DTT.
  4. 4. The limiting amplification reagent of claim 3, wherein, The reagent A comprises 1 XBst Buffer, 4-6 mM magnesium ion, 0.5-1 mM TCEP, 1 Xnucleic acid dye, 8-11 mM dimercapto polyethylene glycol, 4-6 g/L pullulan, 1.5-2.5 g/L PVP, 4-6 g/L PEG20000, 6-10 g/L hydroxypropyl-beta-cyclodextrin and 0.25-0.75 mM glutathione; The reagent B comprises 0.04-0.06 mu M F of a primer, 0.04-0.06 mu M B of a primer, 0.16-0.0.2 mu M of a LF primer, 0.16-0.2 mu M of a LB primer, 0.32-0.4 mu M of a FIP primer, 0.32-0.4 mu M of a BIP primer, 0.27-0.37U/mu LBst of enzyme, 2.0-2.75 mM of an eight-arm polyethylene glycol acrylate, 0.2-0.6 mM of DTT, 0.8-1.25 mM of dNTP, 1-3 g/L of mannitol, 0.8-1.2 g/L of BSA, 2-4 g/L of trehalose, 14-16 mM of glycine, 2.4-2.6 g/L of hydroxypropyl-beta-cyclodextrin and 1-3 g/L of PVP.
  5. 5. The limiting amplification reagent of claim 4, wherein the reagent B further comprises a reverse transcriptase and an RNA protector.
  6. 6. Application of at least one of the following I) to II) in preparation of four detection products for eugenic use: I) The primer combination of claim 1; II) the limiting amplification reagent according to any one of claims 2 to 5.
  7. 7. The kit for detecting the eugenic four items is characterized by comprising a sample releasing agent and the limiting amplification reagent according to any one of claims 3-5.
  8. 8. The kit of claim 7, wherein the sample releasing agent comprises a resin, EDTA and Triton X-100.
  9. 9. The method for detecting the four eugenic items for the non-diagnosis purpose is characterized by detecting a sample by using at least one of the following A) to C): a) The primer combination of claim 1 or 2; b) The limiting amplification reagent of any one of claims 3 to 5; C) A kit according to claim 7 or 8.
  10. 10. The method of detecting according to claim 9, comprising the steps of: step 1, mixing a sample with a sample releasing agent to obtain a pretreatment liquid; Step 2, mixing the pretreatment liquid with the limiting amplification reagent according to any one of claims 3-5, and then carrying out limiting amplification; and 3, carrying out negative and positive interpretation of the sample according to the limit amplification result.

Description

Freeze-dried eugenic four-item limit amplification four-way detection kit and application thereof Technical Field The invention relates to the technical field of biology, in particular to a freeze-dried eugenic four-item limit amplification tetrad detection kit and application thereof. Background Toxoplasma gondii (TOX), rubella Virus (RV), human Cytomegalovirus (HCMV) and Herpes Simplex Virus (HSV) are the core pathogens for four-term eugenic (TORCH) screening. Infection of pregnant women with these pathogens can have serious consequences. Currently, clinical laboratory tests of toxoplasma, rubella, cytomegalovirus and herpes simplex virus mostly adopt serological tests, such as ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), IFA (indirect immunofluorescence method) and the like, to detect specific antibodies in serum. virus-IgM positivity in serum demonstrated recent or acute infection. A positive virus-IgG in serum suggests a past infection. Serological tests only detect antibodies, have the limitations of window period, missed detection of immunosuppressed population, incapability of distinguishing recent/past infection and the like, and need antigen binding or nucleic acid detection. The fluorescent quantitative PCR method with high sensitivity and good specificity is also used for clinical detection of toxoplasma, rubella, cytomegalovirus and herpes simplex virus. However, the method has the defects of dependence on precise instruments (such as a thermal cycler and a fluorescence detection module), complex operation, long time consumption (usually more than 1-2 hours) and high cost, so that the method is limited in application in basic medical institutions or resource-deficient areas. Therefore, it is particularly necessary to develop a TOX, RV, HCMV, HSV screening method that is highly sensitive, rapid, and convenient. Disclosure of Invention In view of the above, the invention aims to provide a freeze-dried eugenic four-item limit amplification four-way detection kit and application thereof. The invention provides a primer combination for four-item LAMP amplification, which comprises a primer combination for toxoplasma gondii detection, a primer combination for rubella virus detection, a primer combination for human cytomegalovirus detection and a primer combination for type I and type II herpes simplex virus detection; The primer combination for toxoplasma gondii detection comprises an F3 primer with a nucleotide sequence shown as SEQ ID NO. 6, a B3 primer with a nucleotide sequence shown as SEQ ID NO. 7, a FIP primer with a nucleotide sequence shown as SEQ ID NO. 8, a BIP primer with a nucleotide sequence shown as SEQ ID NO. 9, an LF primer with a nucleotide sequence shown as SEQ ID NO. 10 and an LB primer with a nucleotide sequence shown as SEQ ID NO. 11; The primer combination for rubella virus detection comprises an F3 primer with a nucleotide sequence shown as SEQ ID NO. 18, a B3 primer with a nucleotide sequence shown as SEQ ID NO. 19, a FIP primer with a nucleotide sequence shown as SEQ ID NO. 20, a BIP primer with a nucleotide sequence shown as SEQ ID NO. 21, an LF primer with a nucleotide sequence shown as SEQ ID NO.22 and an LB primer with a nucleotide sequence shown as SEQ ID NO. 23; the primer combination for detecting the human cytomegalovirus comprises an F3 primer with a nucleotide sequence shown as SEQ ID NO. 39, a B3 primer with a nucleotide sequence shown as SEQ ID NO. 40, a FIP primer with a nucleotide sequence shown as SEQ ID NO. 41, a BIP primer with a nucleotide sequence shown as SEQ ID NO. 42, an LF primer with a nucleotide sequence shown as SEQ ID NO. 43 and an LB primer with a nucleotide sequence shown as SEQ ID NO. 44; The primer combination for detecting the type I and type II herpes simplex viruses comprises an F3 primer with a nucleotide sequence shown as SEQ ID NO. 71 and/or SEQ ID NO. 77, a B3 primer with a nucleotide sequence shown as SEQ ID NO. 72 and/or SEQ ID NO. 78, a FIP primer with a nucleotide sequence shown as SEQ ID NO. 73 and/or SEQ ID NO. 79, a BIP primer with a nucleotide sequence shown as SEQ ID NO. 74 and/or SEQ ID NO. 80, an LF primer with a nucleotide sequence shown as SEQ ID NO. 75 and/or SEQ ID NO. 81, and an LB primer with a nucleotide sequence shown as SEQ ID NO. 76 and/or SEQ ID NO. 82. Further, the primer combination of the invention further comprises a primer combination for detecting an internal reference, wherein the internal reference is beta-actin. The invention provides a limiting amplification reagent, which comprises a reagent A and a reagent B, The reagent A comprises a buffer solution, divalent cations, fluorescent dye, dimercapto polyethylene glycol, a first reducing agent and a freeze-drying protective agent A; The reagent B comprises a primer combination, dNTP, bst DNA polymerase, a second reducing agent, eight-arm polyethylene glycol acrylate and a freeze-drying protective agent B; the freeze-drying protective ag