阅读说明：本技术 Ebv病毒检测试剂盒 (EBV virus detection kit ) 是由 *** 王志新 于 2019-12-16 设计创作，主要内容包括：本发明涉及分子生物技术和基因检测领域,具体而言,涉及一种EBV病毒检测试剂盒,所述试剂盒包括a)引物对；b)上游探针、下游探针、发夹探针；以及c)两种纳米金探针。上述引物和探针能够实现在闭管条件下对EBV进行高灵敏、高分辨、低成本检测,能够有效的避免扩增产物的交叉污染。(The invention relates to the field of molecular biotechnology and gene detection, in particular to an EBV virus detection kit, which comprises a) a primer pair; b) an upstream probe, a downstream probe, a hairpin probe; and c) two kinds of nano-gold probes. The primers and the probes can realize high-sensitivity, high-resolution and low-cost detection of EBV under a closed tube condition, and can effectively avoid cross contamination of amplification products.)

An EBV virus detection kit comprising:

a) SEQ ID NO: 1 and SEQ ID NO: 2, and (b) a primer pair shown in the figure;

b) SEQ ID NO: 3, and the upstream probe shown in SEQ ID NO: 4, and the downstream probe shown in SEQ ID NO: 5; and

c) two nanogold probes, each comprising SEQ ID NO: 6 and SEQ ID NO: 7.

2. The EBV virus detection kit of claim 1, SEQ ID NO: 6, the eighth position at the 5' end of the nucleic acid fragment is locked nucleic acid, and the nucleotide sequence shown in SEQ ID NO: 7 is locked nucleic acid at the sixth position of the 5' end.

3. The EBV virus detection kit according to claim 1 or 2, SEQ ID NO: 6 and SEQ ID NO: 7 is independently connected with the gold nanoparticle through a connecting segment, and the connecting segments do not hybridize with each other and a) and b).

4. The EBV virus detection kit according to claim 3, wherein the length of the connecting fragment is 1nt to 15 nt.

5. The EBV virus detection kit according to claim 4, wherein the linker fragment is Olig N, wherein N is deoxyribonucleic acid selected from A, T, C, G.

6. The EBV virus detection kit of claim 1 or 2, further comprising one or more of DNA polymerase, endonucleases, dntps, buffers or buffer salts, soluble magnesium salts, Tween-20 and water.

7. The EBV virus detection kit of claim 6, wherein the DNA polymerase is selected from any one of Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4DNA polymerase, Klenow fragment.

8. The EBV virus detection kit according to claim 6, wherein the endonuclease is selected from any one of TaqPol, TthPol, TaqExo, AffFEN, PfuFEN, MjaFEN and MthFEN.

9. The composition is prepared by mixing the EBV virus detection kit of any one of claims 1-8.

10. The composition of claim 9, wherein the concentration of the primers in a) is independently selected from 0.5 μ Μ to 1.5 μ Μ, the concentration of the probes in b) is independently selected from 0.5 μ Μ to 1.5 μ Μ, the concentration of the nanogold probes in c) is independently selected from 0.03 μ Μ to 0.2 μ Μ, and the composition further comprises the target nucleic acid.

Technical Field

The invention relates to the field of molecular biotechnology and gene detection, in particular to an EBV (Epstein-Barr Virus) detection kit.

Background

Epstein-Barr virus (EBV) is a double-stranded DNA virus of lymphotropic cells, belongs to subfamily Gaps Virus gamma, EBV is found in 1964, diseases caused by confirmed EBV infection are more and more, the infection rate of EBV in normal population is more than 90%, and the EBV is carried throughout life. The conventional detection technology aiming at the EBV is mature day by day, the commonly used detection methods mainly comprise culture, immunoassay, EBV DNA load detection and the like, and EBV infected persons usually have the increase of peripheral blood lymphocytes and the increase of accompanied heterotypic lymphocytes and transaminase, but the detection technology has very little help on diagnosis of the EBV infection. Detection of antibodies by serum reflects the trend of disease to some extent, but does not present a specific number of active viruses. With the continuous development of Polymerase Chain Reaction (PCR) technology, the fluorescence PCR technology is gradually and widely applied to the detection of EBV, and through the detection of the EBV DNA loading capacity, the risk of nasopharyngeal carcinoma can be evaluated, the infection of unknown reasons can be distinguished, and meanwhile, the fluorescence PCR technology also has positive significance on the dynamic monitoring and prognosis evaluation of related tumors.

Disclosure of Invention

According to the invention, the detection of the EBV nucleic acid is realized by rapidly detecting the hybridization condition of the nanogold probe and the template through the visible light wavelength, and a simpler and faster mode is provided for EBV detection. The implementation process of the kit has low requirement on facility conditions, quick and simple result interpretation and low cost.

Specifically, the invention relates to an EBV virus detection kit, which comprises:

a) SEQ ID NO: 1 and SEQ ID NO: 2, and (b) a primer pair shown in the figure;

b) SEQ ID NO: 3, and the upstream probe shown in SEQ ID NO: 4, and the downstream probe shown in SEQ ID NO: 5; and

c) two nanogold probes, each comprising SEQ ID NO: 6 and SEQ ID NO: 7.

The primers and the probes can realize high-sensitivity, high-resolution and low-cost detection of EBV under a closed tube condition, and can effectively avoid cross contamination of amplification products.

According to still another aspect of the present invention, the present invention also relates to a composition prepared by mixing the EBV virus detection kit as described above.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the result of examining the hybridization efficiency of modified LNA gold nanoprobes according to one embodiment of the present invention;

FIG. 2 shows the results of a sensitivity test of an EBV nucleic acid detection reagent according to one embodiment of the present invention;

FIG. 3 is a graph showing the result of an experiment on the influence of interfering substances in the EBV nucleic acid detecting reagent according to one embodiment of the present invention;

FIG. 4 shows the result of verifying the specificity of the EBV nucleic acid detection reagent according to one embodiment of the present invention;

FIG. 5 shows the results of an actual clinical sample of the EBV nucleic acid detecting reagent according to one embodiment of the present invention;

FIG. 6 shows the real-time fluorescence PCR detection of EBV clinical samples according to one embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

The invention relates to an EBV (Epstein-Barr virus) virus detection kit, which comprises:

a) SEQ ID NO: 1 and SEQ ID NO: 2, and (b) a primer pair shown in the figure;

b) SEQ ID NO: 3, and the upstream probe shown in SEQ ID NO: 4, and the downstream probe shown in SEQ ID NO: 5; and

c) two nanogold probes, each comprising SEQ ID NO: 6 and SEQ ID NO: 7.

The EBV nucleic acid detection kit provided by the invention detects the BamHI gene region sequence of the genome, and each sample can be read by only one tube of detection reagent without additional operations such as tube opening and the like. The detection steps mainly comprise the following three stages: the first stage is template amplification, and high-sensitivity amplification of a target template is realized through participation of a primer pair and an amplification enzyme; the second stage is signal generation and accumulation, and high-efficiency conversion of the target template to signal molecules is realized through specific binding of the probe and the amplification template, participation of endonuclease and hybridization of the nanogold probe and the signal molecules; and the third stage is signal interpretation, and the EBV is rapidly detected through the interpretation of the visible light intensity difference. In order to increase the hybridization efficiency of the nanogold probe, LNA (low-case letters in the sequence are LNA modified bases) is added on the sequence of the nanogold probe to improve the hybridization efficiency of the nanogold probe. The probe is added to ensure that the EBV is detected with high sensitivity, high resolution and low cost under the condition of closed tube by the three stages.

In some embodiments, the average particle size of the gold nanoparticles in the gold nanoparticle probe is 1nm to 200 nm.

In some embodiments, the average particle size of the gold nanoparticles in the gold nanoparticle probe is 5nm to 80 nm.

In some embodiments, the average particle size of the gold nanoparticles in the gold nanoparticle probe is 10nm to 30 nm.

In some embodiments, the nucleic acid sequence of SEQ ID NO: 6, the eighth position of the 5' end of the nucleic acid fragment is locked nucleic acid, and the sequence shown in SEQ ID NO: 7 is locked nucleic acid at the sixth position of the 5' end.

Locked Nucleic Acids (LNA) can increase the melting temperature of the probe and enhance the stability of these test substances. Compared with the nano-gold probe without LNA modification, the reaction system containing the nano-gold probe modified by LNA has smaller reading of negative results, and the nano-gold probe modified by LNA participates in hybridization reaction more effectively, so that the signal value of the negative reaction result is lower, and the result differentiation is more remarkable finally.

In some embodiments, the nucleic acid sequence of SEQ ID NO: 6 and SEQ ID NO: 7 is independently connected with the gold nanoparticle through a connecting segment, and the connecting segments do not hybridize with each other and a) and b).

In the present invention, the criterion for the evaluation of "hybridization" means that nucleic acids do not hybridize under stringent conditions. Such "stringent conditions" are well known to those skilled in the art and include, for example, hybridization at 60 ℃ for 12 to 16 hours in a hybridization solution containing 400mM NaCl, 40mM PIPES (pH6.4) and 1mM EDTA, followed by washing with a washing solution containing 0.1% SDS and 0.1% SSC at 65 ℃ for 15 to 60 minutes. Alternatively, two nucleic acid fragments are cloned in a molecule such as Sambrook et al: the experimental manuals (1989) (Cold spring Lane laboratory Press, New York, USA) "expression of cloned genes in E.coli" section described under standard hybridization conditions with each other. Such conditions as hybridization at 45 ℃ in 6.0 XSSC, followed by a washing step at 50 ℃ in 2 XSSC. To select stringency, the salt concentration in the washing step can be chosen, for example, between 2.0 XSSC at 50 ℃ for low stringency and 2.0 XSSC at 50 ℃ for high stringency. In addition, the temperature in the washing step may vary between about 22 ℃ for low stringency at room temperature and 65 ℃ for high stringency. In a specific embodiment, the stringent conditions are those in the PCR reaction of the present application.

In some embodiments, the linking fragment is 1nt to 15nt in length; for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 nt.

In some embodiments, the linker fragment is Olig N, wherein N is deoxyribonucleic acid and is selected from A, T, C, G.

In some embodiments, the kit further comprises one or more of a DNA polymerase, an endonuclease, dntps, a buffer or buffer salt, a soluble magnesium salt, Tween-20, and water.

The term "buffer" as used herein refers to an aqueous solution or composition that resists changes in pH when an acid or base is added to the solution or composition. This resistance to pH changes is due to the buffer properties of such solutions. Thus, a solution or composition that exhibits buffering activity is referred to as a buffer or buffer solution. Buffers generally do not have the unlimited ability to maintain the pH of a solution or composition. Rather, they are generally capable of being maintained at a pH within a specified range, such as pH 7-pH 9. Generally, Buffers are capable of maintaining a pH at their pKa and within the next logarithm (see, e.g., Mohan, Buffers, A guide for the preparation and use of Buffers in biological systems, CALBIOCHEM, 1999). Buffers and buffer solutions are generally prepared from buffered salts or preferably non-ionic buffer components such as TRIS and HEPES. The buffer which can be used in the method of the invention is preferably selected from the group consisting of phosphate buffer, phosphate buffered saline buffer (PBS), 2-amino-2 hydroxymethyl-1, 3-propanediol (TRIS) buffer, TRIS buffered saline solution (TBS) and TRIS/edta (te). The buffer can be obtained after dissolution of the buffer salt in a solvent, usually water.

In some embodiments, the soluble magnesium salt is MgCl₂。

In some embodiments, the water is generally free of nucleic acids and nucleases, such as double distilled or deionized water. The water is distilled water, deionized water or reverse osmosis water.

In some embodiments, the DNA polymerase is selected from any of Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4DNA polymerase, Klenow fragment.

Taq is preferably used in the present invention.

In some embodiments, the endonuclease is selected from any one of tapol, TthPol, TaqExo, AfuFEN, PfuFEN, mqifen, or MthFEN.

Afufen is preferably used in the present invention.

The invention also relates to a composition which is prepared by mixing the EBV virus detection kit.

In some embodiments, the composition is a solution, wherein the concentration of the primers in a) is independently selected from 0.5 μ M to 1.5 μ M, the concentration of the probes in b) is independently selected from 0.5 μ M to 1.5 μ M, and the concentration of the nanogold probes in c) is independently selected from 0.03 μ M to 0.2 μ M, and the composition further comprises the target nucleic acid.

In some embodiments, the target nucleic acid is extracted by a saturated phenol-chloroform method, a silica gel adsorption column method, a resin extraction method, or a magnetic bead extraction method; in some embodiments, the kit further comprises a target nucleic acid extraction system for performing the DNA extraction method described above.

In some embodiments, the composition has a pH of 8 to 9, preferably 8.5.

The invention also relates to a method for detecting EBV virus, which comprises the following steps:

a) obtaining a composition as described above;

b) PCR reaction was performed, and the presence or absence of EBV virus was determined by observing the color change of the reaction system.

Embodiments of the present invention will be described in detail with reference to examples.