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Enterovirus 71-detecting fluorescent quantitative RT-PCR kit

A fluorescence quantitative and enterovirus technology, applied in the field of EV71 virus quantitative detection, can solve the problems of inappropriate early diagnosis, cumbersome and time-consuming operation, and achieve the effect of simple and fast detection operation

Inactive Publication Date: 2012-01-04
镇江市疾病预防控制中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The first two methods are tedious and time-consuming, not suitable for early diagnosis
The existing nucleic acid amplification methods include RT-PCR method and fluorescent quantitative RT-PCR method. RT-PCR method is cumbersome and time-consuming to operate, and the detection sensitivity is slightly low.

Method used

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  • Enterovirus 71-detecting fluorescent quantitative RT-PCR kit
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  • Enterovirus 71-detecting fluorescent quantitative RT-PCR kit

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1 TaqMan-LNA probe fluorescence quantitative RT-PCR detection kit for enterovirus 71 consists of:

[0024] (1) RNA quantitative standard. Use viral RNA extraction kits such as Roche High Pure Viral RNA Extraction Kit, Qiagen Viral RNA / DNA Extraction Kit, etc., and follow the instructions of the reagents to extract an EV71 viral RNA. Using EV71 viral RNA as a template, use the upstream primer 5′-TAATACGACTCACTATAGGGAGAACACAAACAGGA-3 '(SEQ ID NO.1) and the downstream primer 5'-TTTTTTTTTTTTTTGGCTTAATGGAGTTGCCAGCATA-3'(SEQ ID NO.2) were amplified by RT-PCR. After the PCR product was purified, the RNA was transcribed with T7 in vitro transcription kit and digested with DNase The remaining DNA, and then the transcribed RNA product was purified to obtain an RNA standard for EV71. The content of the in vitro transcription product measured by a nucleic acid protein analyzer was 2.1ng / uL, the size of the transcription product was 237bp, and the copy number of the RNA sta...

Embodiment 2

[0030] Example 2: TaqMan-LNA probe fluorescence quantitative RT-PCR detection kit for detection of enterovirus 71 application example:

[0031] (1) Extraction of viral nucleic acid Extract the viral RNA of the sample to be tested, the extraction of the sample RNA can be carried out by conventional methods, such as Roche High Pure Viral RNA Extraction Kit, Qiagen ViralRNA / DNA Extraction Kit, TaKaRa Viral RNA Extraction Kit or others Viral RNA extraction kits are commonly used, and the operation is carried out according to the reagent instructions. The virus cell culture and herpes fluid are directly extracted according to the above method, and the rectal swab and throat swab samples are extracted after the swabs are squeezed out.

[0032] (2) Draw the standard curve of RNA standard substance containing 1.567×10 10 The copy / uL transcript RNA standard was serially diluted 10-fold with water into several dilutions.

[0033] (3) Establishment of Fluorescent Quantitative RT-PCR Det...

Embodiment 3

[0046] Embodiment 3 TaqMan-LNA probe fluorescence quantitative RT-PCR detects the specificity, sensitivity and repeatability of the detection method of enterovirus 71 type kit

[0047] (1) Specificity of detection method of TaqMan-LNA probe fluorescent quantitative RT-PCR detection kit for enterovirus 71

[0048] Using the TaqMan-LNA probe fluorescent quantitative RT-PCR detection kit for detection of enterovirus 71, the detection method has good specificity for enterovirus EV71, and all 12 strains of EV71 virus RNA that have been sequenced and confirmed the vp1 sequence have corresponding The specific fluorescence amplification curve was positive. For CA16, EV72, HCV, COXB5, Influenza A virus, Influenza B virus, Influenza C virus and other viral RNAs, there is no corresponding specific fluorescence amplification curve, showing a negative reaction. The results showed that the designed primers and probes were only specific to the target virus EV71, and had no cross-reaction wi...

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Abstract

The invention relates to an enterovirus 71-detecting fluorescent quantitative RT-PCR kit and a using method. The kit comprises: (1) RNA quantitative standards of EV71; (2) detection primers; (3) a TaqMan-LNA probe; (4) RT-PCR buffer solution; (5) AMV reverse transcriptase; (6) Taq enzyme; and (7) RNase Free dH2O. The kit is high in specificity to the detection of enterovirus 71, has no cross reaction with other enteroviruses, has the sensitivity of the kit up to 102 copy, and can directly quantitatively detect enterovirus 71 nucleic acid from cerebrospinal fluid, herpes fluid, throat swab, anus swab and other specimens of suspected patients with hand-foot-and-mouth disease. By utiilizing the kit of the invention, detection is simple and rapid in operation, and only 2 to 3 hours are needed from virus nucleic acid extraction to detection completion.

Description

technical field [0001] The invention relates to a TaqMan-LNA probe fluorescent quantitative RT-PCR detection kit for enterovirus 71 (EV71) and application thereof, which is suitable for quantitative detection of EV71 virus. Background technique [0002] Hand, foot and mouth disease (HFMD) is an infectious disease caused by enterovirus, which has occurred sporadically in various places in recent years. The disease is common in preschool children, especially infants, and can cause fever, rashes, and ulcers on the hands, feet, and mouth. Generally, the prognosis is good. Individual patients can cause complications such as myocarditis, pulmonary edema, and aseptic meningoencephalitis. disease. The source of infection of HFMD is patients and latent infected persons, mainly through close contact with the virus in the feces, oral secretions, and skin herpes fluid of infected persons, through the fecal-oral route or the respiratory tract (mainly oral and nasal mucosa) )spread. Th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 茅凌翔鲍务新韩方岸徐岚葛琴娟徐顺高杨静
Owner 镇江市疾病预防控制中心
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