Kit for real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection of poliomyelitis virus

An RT-PCR and polio technology, which is applied in the field of real-time fluorescent RT-PCR detection kits for poliovirus, to achieve the effects of improving reliability and accuracy, and avoiding false negatives and false positives

Inactive Publication Date: 2013-05-22
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Relatively speaking, although the production of antibodies has diagnostic value, a more sensitive and rapid PCR method is generally used for early diagnosis of infection.

Method used

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  • Kit for real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection of poliomyelitis virus
  • Kit for real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection of poliomyelitis virus
  • Kit for real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection of poliomyelitis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: Development of poliovirus detection reagent

[0035] 1. Design of primers and probes: by comparing and analyzing the nucleic acid sequences of reported polioviruses, the poliovirus pol gene was used as the amplification target site, and a highly conserved region with no secondary structure was selected According to the basic principles of primer-probe design, multiple pairs of primers and probes are designed by software and manually.

[0036] 2. Selection of clinical samples: According to relevant domestic and foreign literature reports, samples such as feces, cerebrospinal fluid, and serum can be used

[0037] 3. Establishment and optimization of the reaction system

[0038] Sample preparation: use the constructed pseudovirus containing the target amplification region as a positive quality control for poliovirus detection; use enterovirus EV71, Coxsackie virus A16, Japanese encephalitis virus, influenza virus type A, Influenza virus type B and Echo viru...

Embodiment 2

[0047] Example 2: Poliovirus detection kit and its use

[0048]1. Prepare a kit including the following components: 1 tube of RNA extraction solution (50ml / tube), 1 tube of polio primer-probe mixture (50μl / tube), 1 tube of RT-PCR reaction solution (720μl / tube) 1 tube of RT-PCR enzyme system (150 μl / tube), 1 tube of negative quality control product (200 μl / tube), 1 tube of positive quality control product of polio.

[0049] 2. Specimen collection, transportation and storage

[0050] Specimens will be collected by clinicians according to the actual situation. Testable specimens include throat swabs, nasopharyngeal secretions, and serum. The collection methods are as follows: ① Fecal samples: Take an appropriate amount of specimens to prepare sample suspension according to the proportion, centrifuge to get the supernatant, keep it sealed, and send it for inspection at low temperature; ② Cerebrospinal fluid: The collection time is within 3 days after the onset of neurological sym...

Embodiment 3

[0065] Example 3: Application of poliovirus detection kit to detect samples

[0066] With positive quality control substance, negative quality control substance, specific reference substance in embodiment 1 as samples to be tested, detect these samples respectively according to the method of embodiment 2, positive quality control substance detection result is positive, negative quality control substance and Specific reference test results were all negative (see figure 1 ,2).

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Abstract

The invention discloses a kit for real-time fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection of poliomyelitis virus. The kit comprises an RNA (Ribonucleic Acid) extraction reagent, a poliomyelitis RT-PCR reaction system, a negative quality control material and a positive quality control material, wherein the poliomyelitis RT-PCR reaction system comprises poliomyelitis primer probe mixed liquor, RT-PCR reaction liquor and an RT-PCR enzyme system. The kit can be used for quickly and quantitatively detecting target polynucleotide in real time, and poliomyelitis viruses of I, II and III type can be detected. The poliomyelitis virus in samples of excrement, cerebrospinal fluid, serum and the like can be detected; and a reliable experimental basis can be provided for flexible and quick early diagnosis of the poliomyelitis virus infection. The kit has extremely high flexibility and specificity; and through the kit, the poliomyelitis virus in respiratory tract samples of throat swab, nasopharyngeal secretion and the like and samples of serum and the like can be quickly diagnosed in the early stage.

Description

technical field [0001] The invention relates to poliovirus detection technology, in particular to a kit for real-time fluorescent RT-PCR detection of poliovirus. Background technique [0002] Poliomyelitis is an ancient disease. It is an acute infectious disease caused by poliovirus. Its clinical manifestations are various, mainly including fever, sore throat and limb pain. Some patients may develop Flaccid paralysis. In patients with poliomyelitis, due to damage to the motor neurons in the anterior horn of the spinal cord, the related muscles lose their nerve regulation and atrophy. At the same time, the subcutaneous fat, tendons and bones also atrophy, making the whole body thinner. During the epidemic, there were many cases of occult infection and no paralysis. Because the incidence rate of children is higher than that of adults, infants and young children are mainly affected before the general vaccination, so it is also called polio (Infantile paralysis). [0003] Hum...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 陈瑶李明杜红延陈华云高秀洁
Owner SOUTHERN MEDICAL UNIVERSITY
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