Rubella virus detection and diagnosis kit and application thereof

A diagnostic kit and detection kit technology, applied in the fields of biology and medicine, can solve the problems of complicated operation, false positive reaction, false negative reaction, etc., and achieve the effect of high sensitivity, strong specificity and improved specificity

Inactive Publication Date: 2013-01-02
刘昊智
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Virus isolation is a very accurate method, but the culture period is long, the operation is complicated, and there are many biological influence factors, so it is not suitable for general laboratories.
Immunological method is a commonly used method. It does not require pretreatment of serum samples. It is easy to operate in clinical application and is suitable for laboratory diagnosis and large-scale field investigation of RV infection. However, this method is easily limited by the time of antibody production. False negative reactions can be caused by improper blood collection time; and some other viruses such as Epstein-Barr virus can also cause false positive reactions

Method used

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  • Rubella virus detection and diagnosis kit and application thereof
  • Rubella virus detection and diagnosis kit and application thereof
  • Rubella virus detection and diagnosis kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The preparation of embodiment 1 rubella virus fluorescent quantitative PCR detection kit

[0038] (1) RNA extraction solution,

[0039] dNTPs (25mM),

[0040] Reverse Transcriptase Superscript III (200U / μl),

[0041] RNase inhibitor (40U / μl),

[0042] TaqDNA polymerase (5U / μl),

[0043] MgCl 2 (100mM).

[0044] (2) Composition of fluorescent PCR 5×buffer:

[0045] 50 mM Tris-HCl (PH8.3), 250 mM KCl, 1 mg / ml gelatin.

[0046] (3) Mixed enzyme group salty:

[0047] 1.5U / ul Reverse Transcriptase Superscript III, 1.5U / ul Taq DNA Polymerase, 5U / ul RNase Inhibitor.

[0048] (4) Fluorescent quantitative PCR reaction solution:

[0049] PCR 5×buffer 6μl, P1, P2 each 0.3μl (25μM), fluorescent probe FP 0.2μl (25μM), MgCl 2 0.9μl (100mM), dNTPs 0.24μl (25mM), DEPC-treated water 15.86μl.

Embodiment 2

[0050] Example 2: Detection of rubella virus with fluorescent quantitative PCR kit

[0051] (1) Take 500 μl of centrifuged gargle or throat swab extract, add 1ml of RNA extract, then add 200 μl of chloroform, shake for 15 seconds, and incubate at room temperature for 10 minutes. Centrifuge at 12000g for 10min at 4°C, and the RNA is located in the upper layer. Replace the upper layer with a new 1.5ml EP tube, add 500μl isopropanol, incubate at -20°C for 10min, and then centrifuge at 12000g for 10min at room temperature. Discard the supernatant, add 1ml of 75% ethanol to wash the RNA pellet, shake and mix well, and centrifuge at 12000g for 5min at 4°C. The supernatant was discarded and dried at 37°C for 5 minutes to obtain viral RNA.

[0052] (2) Dilute the standard positive template 10 times to l×10 6 Copy number / μl, l×10 5 Copy number / μl, l×10 4 Copy number / μl.

[0053] (3) Take 24 μl of fluorescent quantitative PCR reaction solution, add 2 μl of mixed enzyme respectivel...

Embodiment 3

[0056] Example 3 Detection of kit linear range, linearity, specificity, sensitivity, accuracy, precision, repeatability

[0057] (1) specificity

[0058] Specificity evaluation: The assembled rubella virus detection kit was used to detect rubella virus and other viruses, and the results showed that the specificity of the kit was high.

[0059] Table 1 Detecting Rubella Virus Antigen Content Method Specificity Test 450nm Absorbance Results

[0060]

[0061]

[0062] The results show that the detection kit has high specificity and has no crossover with HSV, adenovirus, RSV, measles virus, HAV and other viruses.

[0063] (2) Sensitivity

[0064] Kit for detection of rubella virus

[0065] Table 2 Sensitivity of rubella virus antigen detection methods

[0066]

[0067] The results show that: when the antigen content is 1000-5ng / ml, the OD value ≥ negative control * 2.1, and the detection sensitivity of this method is 5ng / ml

[0068] (3) Detection range and linearity:...

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Abstract

The invention discloses a rubella virus (RV) detection and diagnosis kit and application thereof. The kit adopts the fluorescent polymerase chain reaction (PCR) technique and comprises ribonucleic acid (RNA) extracting solution, reverse transcriptase (RT)-PCR reaction liquid, mixed enzyme, an RV positive standard template, positive control and negative control, wherein primers (P1 and P2) and probes (RVFP1 and RVFP2) in the RT-PCR reaction liquid are gene segments of a rubella virus E2. The sequence of the primer P1 is 5'-ATCTGCGGGCTGTCCACCA-3', the sequence of the primer P2 is 5'-AGCAAGTCGCACGTCCCAT-3', the sequence of an RV fluorescent mark probe RVFP is 5'-AAGACGGCTGGACTTGTCGT-3'. The kit can detect rubella virus RNA on patients infecting the rubella virus quickly, accurately and effectively, and accordingly, a new way for diagnosing and preventing the rubella virus infection is provided. Therefore, the RV detection and diagnosis kit has quite wide application prospects.

Description

technical field [0001] The invention relates to a fluorescent detection kit and its application, in particular to a fluorescent qualitative and quantitative PCR detection kit for detecting rubella virus, which belongs to the fields of medicine and biotechnology. Background technique [0002] Rubella virus (Rubella virus, RV) is an RNA virus belonging to the togavirus family (togavirus) and is restricted to humans. Under the electron microscope, most of them are irregular spherical cores with a diameter of 50-70nm. The antigenic structure of rubella virus is quite stable, and only one serotype is known. Rubella virus is prone to vertical infection. After a pregnant woman is infected with rubella virus for the first time in early pregnancy, the virus can enter the fetus through the placental barrier, often causing miscarriage or stillbirth. It can also cause congenital rubella syndrome and fetal malformation. The viability of the virus in vitro is weak, and it is sensitive to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 刘昊智
Owner 刘昊智
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