Lysis solution for magnetic bead method HCMV nucleic acid extraction and application of lysis solution

A technology of lysate and magnetic bead method, which is applied in the field of molecular biology, can solve the problem that the antibody cannot respond to the HCMV load, and achieve the effect of efficient extraction

Active Publication Date: 2020-02-21
SHANGHAI ZJ BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] However, the detection of antibodies cannot reflect the load of HCMV in the current sample, and there is no direct evidence for congenital infection of the fetus

Method used

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  • Lysis solution for magnetic bead method HCMV nucleic acid extraction and application of lysis solution
  • Lysis solution for magnetic bead method HCMV nucleic acid extraction and application of lysis solution
  • Lysis solution for magnetic bead method HCMV nucleic acid extraction and application of lysis solution

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preparation example Construction

[0047] The preparation method of the lysate for extracting HCMV nucleic acid by the magnetic bead method provided by the invention at least comprises the following steps:

[0048] Mix guanidine isothiocyanate, sodium lauryl sulfate, NP40, Tris-HCL, EDTA and water, adjust the pH value of the lysate, and add β-mercaptoethanol to obtain the lysate.

[0049] Further, the pH value of the lysate is adjusted to 8.5±0.1.

[0050] The invention provides a lysate for HCMV nucleic acid extraction by magnetic bead method for preparing HCMV nucleic acid extraction products in samples or for HCMV nucleic acid extraction in amniotic fluid.

[0051] The HCMV nucleic acid sample can be selected from amniotic fluid samples.

[0052] When extracting HCMV nucleic acid from a sample, the required sample volume is at least 200 μL. Further, it can be 200-250ul.

[0053] A kit for extracting HCMV nucleic acid by magnetic bead method provided by the present invention includes the aforementioned lysat...

Embodiment 1

[0078] Prepare the test kit of magnetic bead method HCMV nucleic acid extraction with following formula, described test kit comprises lysate, magnetic bead, the first washing liquid, the second washing liquid, eluent, RNA precipitation aid and proteinase K, wherein

[0079] Based on the total amount of the lysate, the content of each component of the lysate is:

[0080]

[0081]

[0082] Based on the total amount of the first washing liquid, the content of each component of the first washing liquid is: guanidine isothiocyanate: 2mol / L, sodium chloride: 0.5mol / L, triton X-100: 1 % (w / v), absolute ethanol: 50% (v / v), Tris-HCl: 2mmol / L, solvent is water.

[0083] Based on the total amount of the second washing liquid, the content of each component of the second washing liquid is: absolute ethanol: 75% (v / v), and the solvent is water.

[0084] Based on the total amount of the eluent, the content of each component of the eluent is: 40mmol / L, EDTA 10mmol / L, and the solvent is...

Embodiment 2

[0090] 1. Experimental method

[0091] 1.1 Detection object

[0092] A high-concentration HCMV-positive amniotic fluid sample was taken, and three gradients were diluted with the HCMV-negative amniotic fluid sample and normal saline respectively. Three concentration samples of HCMV-negative amniotic fluid sample dilution are defined as AF-1, AF-2, AF-3, three concentration samples of physiological saline dilution are respectively defined as NC-1, NC-2, NC-3.

[0093] 1.2 Sample nucleic acid extraction

[0094] Use the magnetic bead method HCMV nucleic acid extraction kit described in Example 1 of the present invention and the comparative extraction reagent (QIAamp circle R DNA mini Kit (QIAGEN company) extraction reagent on the market) to carry out sample extraction, and each extraction is repeated twice. The comparison extraction reagent was operated according to its instructions, and the operation of the kit for extracting HCMV nucleic acid by the magnetic bead method of t...

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Abstract

The invention provides a lysis solution for magnetic bead method HCMV nucleic acid extraction. The lysis solution contains at least the following components, based on the total amount of the lysis solution: 1-3 mol / L guanidine isothiocyanate, 0.05-0.1% (w / v) sodium lauryl sulfate, 1-5% (w / v) NP40, 10-80 mmol / L Tris-HCl, 5-15 mmol / L EDTA, and 0.1-0.5% (v / v) beta-mercaptoethanol, wherein a solvent is water. The lysis solution solves a current situation that no HCMV quantitative detection reagent for an amniotic fluid sample exists on the market, and the amniotic fluid sample has complex components and contains multiple unknown inhibitors; and the efficient extraction of the amniotic fluid sample is successfully realized by using the magnetic bead method, so that quantification of HCMV in theamniotic fluid sample becomes possible.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a lysate for extracting HCMV nucleic acid by a magnetic bead method and an application thereof. Background technique [0002] HCMV is a ubiquitous double-stranded DNA virus that is mainly transmitted through sexual contact or direct contact with the blood, urine or saliva of an infected person. The average incubation period is about 28-60 days. The decreased immunity of pregnant women during pregnancy can inhibit the body's inflammatory response and increase the mother's susceptibility to fetal antigens. On the other hand, it may increase the susceptibility of pregnant women and fetuses to certain infectious diseases. After primary infection of HCMV in pregnant women, the intrauterine fetal infection rate is as high as 30%-40%. [0003] At present, there are many kinds of HCMV detection kits on the market, which can be divided into antibody detection and nucleic acid d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1013C12Q1/6806C12Q2523/308
Inventor 曹蕾杨慧慧李越李婷婷熊磊邵俊斌
Owner SHANGHAI ZJ BIO TECH
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