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Tembusu virus nano PCR (Polymerase Chain Reaction) detection kit and detection method thereof

A technology of Tembusu virus and kit, which is applied in the detection field of Tembusu virus, can solve the problems of non-specific product amplification and insufficient amplification efficiency in complex systems, and achieve the reduction of residence time, reduction of non-specific amplification, The effect of good application prospects

Inactive Publication Date: 2015-06-17
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in practical applications, PCR technology has many limitations, such as non-specific product amplification and insufficient amplification efficiency in the amplification of complex systems.

Method used

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  • Tembusu virus nano PCR (Polymerase Chain Reaction) detection kit and detection method thereof
  • Tembusu virus nano PCR (Polymerase Chain Reaction) detection kit and detection method thereof
  • Tembusu virus nano PCR (Polymerase Chain Reaction) detection kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 A kind of nano-PCR kit best embodiment that detects Tambusu virus

[0033] The kit of this example includes: (1) Reagent for extracting Tambusu virus RNA: TRIzol LS Reagent, chloroform, isopropanol, 75% ethanol; (2) Reverse transcription reagent: 5×reverse transcription buffer, the concentration is 2.5 mM dNTP Mixture, M-MLV reverse transcriptase at a concentration of 2.5 U / μL, RNase inhibitor at a concentration of 30 U / μL, and reverse transcription primers; (3) Nano-PCR reaction reagent: 2×NanoPCR Mix , upstream primers, downstream primers; (4) Others: positive control and nuclease-free water. Among them, 2×NanoPCR Mix consists of DNA polymerase, 2×NanoPCR buffer and dNTP Mixture. The positive control is the allantoic fluid of 9-11-day-old chicken embryos inoculated with Tambusu virus and collected from dead chicken embryos at 72-96 hours , the primers are lyophilized powder, pure by HPLC. The primer sequences included in the kit of this example are show...

Embodiment 2

[0036] Embodiment 2 Non-diagnostic purpose uses nano-PCR method to detect the method of Tembusu virus

[0037] The detection method of this example uses the kit in Example 1. The ovary, fallopian tube, spleen and liver of diseased poultry were taken as samples to be tested.

[0038] The detection method of this embodiment comprises the following steps:

[0039] 1. The specific steps of virus RNA extraction are as follows: (1) After cutting the sample to be tested, add physiological saline according to the mass volume ratio of 1:5 and grind evenly, centrifuge at 3000-5000rpm for 5-10 minutes, take the supernatant, and take the supernatant clear. (2) Take 250 μL of the above-mentioned treated samples and positive control respectively, add 750 μL TRIzol LS Reagent, shake vigorously for 2 minutes, and place at room temperature for 10 minutes. Add 250 μL of chloroform, shake vigorously for 15 s, leave at room temperature for 10 min, centrifuge at 12,000 g for 10 min at 4°C, an...

Embodiment 3

[0044] Example 3 Sensitivity test of Tembusu virus nano-PCR detection method

[0045] Measured by ultra-micro spectrophotometer, the total RNA concentration of Tembusu virus was calculated to be 1.8×10 5 Copies / μL nucleic acid template for sensitivity test. At the same time, nano-PCR and ordinary PCR methods were used to detect the 10-fold diluted Tambusu virus nucleic acid template.

[0046] The nano-PCR and common PCR operation process of Tambusu virus is carried out according to embodiment 2, wherein, common PCR reaction changes 2×NanoPCR Mix in step 3 into 2×PCR reaction solution, and its composition is as follows: DNA polymerase, 2× PCR buffer, dNTP Mixture, and other operating procedures remain unchanged.

[0047] The PCR amplification products were detected by 1% agarose gel electrophoresis, and the results were as follows: figure 2 shown. It can be seen that ordinary PCR can detect at least 1.8×10 3 copies / μL Tambusu virus nucleic acid template, and nano-PCR ca...

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Abstract

The invention discloses a tembusu virus nano PCR (Polymerase Chain Reaction) detection kit and a detection method thereof; the kit comprises a 5* reverse transcription buffer solution, a dNTP Mixture, reverse transcriptase, a RNA enzyme inhibitor and a reverse transcription primer and is characterized by further comprising 2*NanoPCR Mix, an upstream primer and a downstream primer, wherein the sequence of the upstream primer is represented by SEQ ID No.1; and the sequence of the downstream primer is represented by SEQ ID No.2. The kit can be used for detecting tembusu virus. The invention further provides the detection method of tembusu virus by adopting the kit. Tembusu virus can be detected rapidly and specifically; and therefore, the detection efficiency and the specific amplification yield of tembusu virus can be greatly increased.

Description

technical field [0001] The invention relates to the technical field of detection of Tembusu virus. Background technique [0002] Since April 2010, a new acute infectious disease characterized by a serious decline in egg production of laying ducks has broken out in parts of southern my country. The clinical symptoms of the disease are mainly characterized by high fever and a sharp drop in egg production of laying ducks. Autopsy shows that the dead ducks have enlarged livers, white dots, degeneration of follicles, hyperemia and hemorrhage of the follicular membrane. The incidence rate of the disease is almost 100%, and the mortality rate can reach 5%~10%, which has caused huge economic losses to the laying duck breeding industry. It has been confirmed that the pathogen of the infectious disease is Tembusu virus (TMUV). TMUV is a member of the Flavivirus genus in the family Flaviviridae, which can also infect other waterfowl and laying hens, causing the same clinical manifest...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/70C12Q1/686C12Q2563/155
Inventor 袁万哲刘聚祥孙继国陈立功刘静王庚南
Owner HEBEI AGRICULTURAL UNIV.
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