阅读说明：本技术 一种用于检测鼠痘病毒的rpa试剂盒、引物、探针及方法 (RPA kit, primers, probe and method for detecting varicella virus ) 是由 熊炜 薛俊欣 魏晓锋 林颖峥 李健 于 2019-04-25 设计创作，主要内容包括：本发明属生物技术领域,具体涉及一种用于检测鼠痘病毒核酸的RPA试剂盒、引物、探针及方法。本发明的试剂盒包括RPA反应体系,所述的RPA反应体系包括RPA引物探针混合液,所述RPA引物探针混合液包括一引物对和探针,所述引物对的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示,所述探针如SEQ ID NO.3+FAM-dt+THF+BHQ1-dt+SEQ ID NO.4+C3所示。本发明的试剂盒可用于检测鼠痘病毒,反应时间短、检测速度快,且灵敏度和特异性高。(The invention belongs to the technical field of biology, and particularly relates to an RPA kit, primers, a probe and a method for detecting a varicella virus nucleic acid. The kit comprises an RPA reaction system, wherein the RPA reaction system comprises an RPA primer probe mixed solution, the RPA primer probe mixed solution comprises a primer pair and a probe, the nucleotide sequence of the primer pair is shown as SEQ ID No.1 and SEQ ID No.2, and the probe is shown as SEQ ID No.3+ FAM-dt + THF + BHQ1-dt + SEQ ID No.4+ C3. The kit can be used for detecting the varicella virus, and has the advantages of short reaction time, high detection speed, high sensitivity and high specificity.)

1. A kit for detecting a varicella virus nucleic acid comprises an RPA reaction system and is characterized in that the RPA reaction system comprises an RPA primer probe mixed solution, the RPA primer probe mixed solution comprises a primer pair and a probe, the nucleotide sequence of the primer pair is shown as SEQ ID NO.1 and SEQ ID NO.2, and the probe is shown as SEQ ID NO.3+ FAM-dt + THF + BHQ1-dt + SEQ ID NO.4+ C3.

2. The kit of claim 1, wherein the concentration of the primers in the RPA reaction system is 5-20 μ M, preferably 10 μ M; the concentration of the probe is 5-10 μ M, preferably 10 μ M.

3. The kit of claim 2, wherein the RPA reaction system further comprises a core reaction solution, a reaction buffer, an Exo reaction solution, a probe enzyme mixture, magnesium acetate and dntps; preferably: the concentration of the magnesium acetate is 260-300 mu M, preferably 280 mu M;

and/or the concentration of the dNTP is 20-220 mu M, preferably 200 mu M.

4. The kit according to claim 3, wherein the primer-probe mixture comprises the primer pair and the probe, wherein the amount of each primer in the primer pair is 2.1. mu.L at a concentration of 10. mu.M, and the amount of the probe is 0.6. mu.L at a concentration of 10. mu.M; the amount of the core reaction solution is 2.5. mu.L, the amount of the reaction buffer solution is 25. mu.L, the amount of the probe enzyme mixture is 5. mu.L, and the amount of the Exo reaction solution is 1. mu.L; the amount of the magnesium acetate is 2.5 mu L, and the concentration is 280 mu M; the amount of the dNTP is 7.2 mu L, and the concentration is 11 mM; preferably: the core reaction solution is 20 times of core reaction solution;

and/or the reaction buffer solution is 2 times of the reaction buffer solution;

and/or the probe enzyme mixture is 10 times of the probe enzyme mixture;

and/or the Exo reaction solution is 50 times of the Exo reaction solution.

5. The kit of any one of claims 1 to 4, further comprising a negative control, preferably ddH₂O。

6. A primer pair for detecting a varicella virus nucleic acid is characterized in that the nucleotide sequences of the primer pair are respectively shown as sequences SEQ ID NO.1 and SEQ ID NO. 2; preferably, the detection is RPA detection.

7. A probe for detecting a mousepox virus nucleic acid is characterized in that the probe is shown as SEQ ID NO.3+ FAM-dt + THF + BHQ1-dt + SEQ ID NO.4+ C3; preferably, the detection is RPA detection.

8. A combination for detecting a varicella virus nucleic acid comprises a primer pair and a probe, and is characterized in that the nucleotide sequences of the primer pair are respectively shown as sequences SEQ ID NO.1 and SEQ ID NO. 2; the probe is shown in SEQ ID NO.3+ FAM-dt + THF + BHQ1-dt + SEQ ID NO.4+ C3; preferably, the detection is RPA detection.

9. Use of a primer pair according to claim 6 and/or a probe according to claim 7, or a combination according to claim 8, for the preparation of a kit for the detection of a varicella virus.

10. A method for detecting a varicella virus for non-diagnostic purposes, comprising the steps of:

(1) extracting total DNA in a sample to be detected by using a DNA extraction reagent;

(2) performing an RPA reaction by using the total DNA extracted in the step (1) as a template and using an RPA reaction system in the kit according to any one of claims 1-5, wherein the time of the amplification reaction in the RPA reaction is preferably 25min, and the temperature is preferably 39 ℃;

(3) and analyzing the detection result.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an RPA kit, primers, a probe and a method for detecting a varicella virus nucleic acid.

Background

The data disclose that Mouse pox (Mouse pox) caused by the Mouse pox virus (EV) is a highly contagious, devastating virulent infectious disease that is prevalent in groups of laboratory mice worldwide with a mortality rate of 96.6%. The disease is characterized by swelling of limbs, head and tail, necrosis and even toe falling off clinically, and is one of the most serious viral diseases harming mice. Marchal in England equals 1929, the patient was first found in the England laboratory rat group and was first reported in the next year; the rat pox epidemic situation of the Beijing central biological product research institute is firstly reported in 1951 in the Tangfei province of China; wuhuixing et al (1986) and Xubei et al (1989) performed serological epidemiological investigation on the groups of rats across the country, confirming that mousepox is sporadic in the groups of rats in China. At present, the disease widely exists in all countries of the world, particularly the England American method is the most serious, and the disease still occurs in China. Research shows that the disease is often epidemic in outbreak, has high fatality rate, often causes complete group elimination, seriously influences the production and breeding of experimental animals, and extremely seriously influences the smooth performance of animal experiments and the accuracy and repeatability of scientific research data. Research shows that under natural conditions, the varicella virus can infect mice of various ages, but the fatality rate of the suckling mice and the aged mice above 1 year old is higher, while the mice of 6-12 weeks old are lower, and the susceptibility of the mice of different strains is different; because the mouse pox virus can be recessive infected, the mouse does not show clinical symptoms, but the virus can be persistently existed in the body and reproduced, and the virus is continuously discharged from excrement to become a terrible infection source, so that the early diagnosis of the mouse pox is particularly important for detecting the recessive infected mouse.

Disclosure of Invention

The invention aims to overcome the defects of low sensitivity and sensitivity, long reaction time and the like of the conventional detection of the varicella virus, and provides a Recombinase Polymerase Amplification (RPA) kit, a primer, a probe and a method for detecting EV nucleic acid based on a method for establishing real-time fluorescence RPA and a basis for verifying the specificity, sensitivity and stability of the method. The rapid detection method for detecting EV nucleic acid established by RPA has high specificity and sensitivity, and provides a rapid, simple, convenient and accurate detection method for diagnosing EV infection.

The invention mainly solves the technical problems in the prior art through the following technical scheme.

The invention provides a kit for detecting mouse pox virus (EV) nucleic acid, which comprises an RPA reaction system, wherein the RPA reaction system comprises an RPA primer probe mixed solution, the RPA primer probe mixed solution comprises a primer pair and a probe, the nucleotide sequence of the primer pair is shown as SEQ ID No.1 and SEQ ID No.2, and the probe is shown as SEQ ID No.3+ FAM-dt + THF + BHQ1-dt + SEQ ID No.4+ C3.

In order to improve the amplification efficiency, the concentration of the primers of the RPA reaction system is preferably 5-20 μ M, and more preferably 10 μ M; the concentration of the probe is preferably 5 to 10 μ M, and more preferably 10 μ M.

Preferably, the RPA reaction system further comprises a core reaction solution, a reaction buffer solution, an Exo reaction solution, a probe enzyme mixture, magnesium acetate and dNTP; wherein the concentration of the magnesium acetate is preferably 260-300 mu M, and more preferably 280 mu M. The concentration of the dNTP is preferably 20-220 μ M, and more preferably 200 μ M.

In a preferred embodiment of the present invention, the primer probe mixture comprises the primer pair and the probe, wherein the amount of each primer in the primer pair is 2.1. mu.L and the concentration is 10. mu.M, and the amount of the probe is 0.6. mu.L and the concentration is 10. mu.M. The amount of the core reaction solution is 2.5. mu.L, the amount of the reaction buffer solution is 25. mu.L, the amount of the probe enzyme mixture is 5. mu.L, and the amount of the Exo reaction solution is 1. mu.L; the amount of the magnesium acetate is 2.5 mu L, and the concentration is 280 mu M; the amount of dNTP was 7.2. mu.L, and the concentration was 11 mM. Wherein:

The core reaction solution is preferably 20 times the amount of the core reaction solution (i.e., 20 Xthe amount of the core reaction solution; the same applies hereinafter).

The reaction buffer is preferably 2 times the reaction buffer.

The probe enzyme mixture is preferably 10 times of the probe enzyme mixture;

the Exo reaction solution is preferably 50 times of Exo reaction solution

Preferably, in order to determine the detection result more intuitively and accurately, the kit further comprises a negative control, and the negative control is preferably ddH₂O。

The invention also provides a primer pair for detecting the nucleic acid of the varicella virus (EV), wherein the nucleotide sequences of the primer pair are respectively shown as SEQ ID NO.1 and SEQ ID NO.2 in the sequence table; preferably, the detection is RPA detection.

The invention also provides a probe for detecting the nucleic acid of the mousepox virus (EV), wherein the probe is shown as SEQ ID NO.3+ FAM-dt + THF + BHQ1-dt + SEQ ID NO.4+ C3; preferably, the detection is RPA detection.

The invention also provides a combination for detecting the nucleic acid of the varicella virus (EV), which comprises a primer pair and a probe, wherein the nucleotide sequences of the primer pair are respectively shown as SEQ ID NO.1 and SEQ ID NO.2 in the sequence table; the probe is shown in SEQ ID NO.3+ FAM-dt + THF + BHQ1-dt + SEQ ID NO.4+ C3; preferably, the detection is RPA detection.

The invention also provides an application of the primer pair and/or the probe or the combination in preparing a kit for detecting the varicella virus (EV).

The invention also provides a method for detecting the varicella virus (EV) for non-diagnostic purposes, which comprises the following steps:

(1) extracting total DNA in a sample to be detected by using a DNA extraction reagent;

(2) taking the total DNA extracted in the step (1) as a template, and carrying out an RPA reaction by using an RPA reaction system in the kit, wherein the time of an amplification reaction in the RPA reaction is preferably 25min, and the temperature is preferably 39 ℃;

(3) and analyzing the detection result.

The method is used for respectively detecting other viruses of mice, such as Mouse Hepatitis Virus (MHV), Mouse Parvovirus (MPV), mouse Sendai Virus (SV), mouse reovirus type III (Reo-3), mouse parvovirus (MVM) and lymphocyte choriomeningitis virus (LCMV) as controls, and is compared with a PCR method to detect the specificity of the established method, and the result proves that the RPA detection method of the mousepox virus (EV) established by the invention has high specificity and sensitivity, and provides a quick, simple, convenient and accurate detection means for diagnosing SIV infection in a common laboratory of a basic unit.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The invention has the beneficial effects that:

the invention provides a novel RPA method for detecting mouse pox virus (EV) nucleic acid, compared with the common PCR method, the RPA has the following advantages: firstly, RPA belongs to an isothermal amplification technology, has low requirements on instruments and equipment, and can complete reaction only by a constant-temperature water bath kettle; secondly, the RPA detection speed is high, the reaction time is within 40min, and the reaction time is obviously shorter than that of the conventional PCR (usually 1-2 h); thirdly, a probe sequence of a fluorescent label is added under the condition allowed by the test condition, amplification is carried out on a GENIE II constant temperature amplification instrument, and real-time monitoring of nucleic acid amplification can be realized.

Drawings

FIG. 1 shows the specificity of real-time fluorescent RPA detection of EV; wherein, well 1 is EV; well 2-well 8 are MHV, MPV, SV, Reo-3, MVM, LCMV, and blank controls, respectively.

FIG. 2 shows the specificity of PCR detection of EV; wherein M is DNA Marker DL 2000; the virus samples detected in lanes 1-8 are: EV, 2.MHV, 3.MPV, 4.SV, 5.Reo-3, 6.MVM, 7.LCMV, 8. blank control.

FIG. 3 is a graph showing the sensitivity of real-time fluorescent RPA detection of EV; wherein the amplification curves are 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg and 0.1fg of the varicella virus DNA template from left to right respectively.

FIG. 4 shows a sensitivity test for detecting EV by RT-PCR; wherein M is DNA Marker DL 2000; the cDNA template concentrations in lanes 1-8 were 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg and, 0.1fg, respectively.

FIG. 5 fluorescent RPA detection of a mouse pox positive tissue sample; wherein, the 7 positive amplification curves are respectively liver, spleen, lung, kidney, heart, blood and intestine from top to bottom, and the non-amplification curve is a blank control.

FIG. 6 PCR detection of a mouse pox positive tissue sample; wherein M is DNA Marker DL 2000; the remaining lanes are: 1. blood, 2. liver, 3. spleen, 4. lung, 5. kidney, 6. heart, 7. intestine, 8. blank control.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.