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Method and composition for detecting human metapneumovirus based on real-time PCR

A metapneumovirus, real-time fluorescence technology, applied in the biological field, can solve the problem of lack of metapneumovirus composition, etc., and achieve the effect of improving the detection effect, improving the extraction effect, and low sequence diversity

Inactive Publication Date: 2015-09-02
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although there are many studies on the diagnosis of metapneumovirus, the current clinical detection of metapneumovirus is still in the bottleneck stage, mainly due to the lack of a composition with strong specificity and high detection rate that can detect metapneumovirus. And simple and efficient detection operation method

Method used

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  • Method and composition for detecting human metapneumovirus based on real-time PCR
  • Method and composition for detecting human metapneumovirus based on real-time PCR
  • Method and composition for detecting human metapneumovirus based on real-time PCR

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Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1 research object

[0031] A total of 535 subjects were used in this study, which were divided into the infant group (54 cases) under 1 year old and the children group (481 cases) from 1 year old to 15 years old. The research objects were all patients with respiratory tract infection in the Third Hospital of Beijing Medical University, including patients in outpatient clinics and patients in inpatient departments. The study was patient-informed, and volunteers were informed about the use of their throat swab subsamples and clinical data.

Embodiment 2

[0032] Example 2 Throat test sub-acquisition and sample RNA extraction

[0033] The patient's specimen is derived from a subsample of the patient's throat test. Use the sterilized long cotton swab in the culture tube to wipe the secretions on the patient's palatine arches, pharynx, and tonsils on both sides, then insert the cotton swab into the test tube, and seal it tightly for storage. Samples can be stored in a -20°C freezer for short-term storage.

[0034] RNA in all samples was extracted using the Trizol method. First, add 1ml of normal saline to each throat test sub-sample, and then rub the cotton swab inside the test tube by hand, so that the throat extract on the cotton swab can be washed into the normal saline as much as possible. Then, pour the physiological saline in the test tube into a 1.5ml centrifuge tube, centrifuge at 4°C, 15,000rpm for 5min, and remove the supernatant. At the same time, add 1ml of normal saline to each throat test sub-sample, rub the cot...

Embodiment 3

[0035] Embodiment 3 Reverse transcription obtains cDNA

[0036] cDNA was reverse transcribed using Invitrogen's M-MLV reverse transcription kit. Mix 10ul of total RNA template with 1ul Oligo(dT) (500μg / ml) and 1ul Random decamers, 1μl dNTP (10mM), heat at 65°C for 5min, and immediately put it on ice for 1min. Then add 4 μl of 5×First-Strand Buffer and 2 μl of DTT (0.1M), mix and incubate at 37° C. for 2 minutes. Next, 1 μl of M-MLV reverse transcriptase (200 unit) was added, mixed evenly, incubated at 25°C for 10 minutes, then heated at 37°C for 50 minutes, and finally inactivated at 70°C for 15 minutes. The synthesized cDNA was stored at -70°C for future use.

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Abstract

The invention provides a method and a composition for detecting the human metapneumovirus based on the real-time PCR. According to the invention, the RNA of a to-be-tested sample is extracted through the Trizol method, and the RNA is converted into cDNA through the reverse transcription prodcess. After that, through the real-time quantitative PCR, whether the to-be-tested sample contains human metapneumovirus or not can be detected. The real-time fluorescent PCR technology is applied to the diagnosis on human metapneumovirus, so that the detection sensitivity is higher. Meanwhile, the detection singularity is better, and the detection rate of human metapneumovirus is improved. The method can be used for detecting whether a clinical diagnosis subject carries human metapneumovirus or not. Meanwhile, the method can also be used for detecting human metapneumovirus in a laboratory. By means of the method, the RNA extraction effect of human metapneumovirus and the detection effect of human metapneumovirus are improved. At the same time, the cost is saved.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically to the technology based on real-time quantitative PCR, which is used for the diagnosis method of metapneumovirus. Background technique [0002] Metapneumovirus belongs to the subfamily Paramyxoviridae and is a new member of the genus Metapneumovirus. Metapneumovirus is a virus related to respiratory tract infection of humans, especially infants, especially children under 1 year old. It has become an important source of infection in children after RSV. At the same time, respiratory tract infection is also one of the main causes of human morbidity and death worldwide. Metapneumovirus can mainly cause upper and lower respiratory tract infections in humans. The main clinical features are cough, wheezing, runny nose, headache, fever and other symptoms. Because the symptoms of infection do not differ from those of other influenza illnesses, it is difficult to screen patients with metapn...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 陈奕霖杨晗贾玉棉田新霞
Owner PEKING UNIV
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