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Real-time fluorescent quantitative polymerase chain reaction (PCR) detecting kit for coxsackievirus A16

A Coxsackie virus and kit technology, applied in the field of real-time fluorescent PCR detection kits, can solve problems affecting virus diagnosis and epidemic monitoring, and achieve the effect of good sensitivity

Active Publication Date: 2009-12-09
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] CA16 is one of the main pathogens causing hand, foot and mouth disease in children. Since there is no mature virus nucleic acid detection kit for the screening and diagnosis of this virus, the diagnosis and epidemic monitoring of this virus have been seriously affected.

Method used

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  • Real-time fluorescent quantitative polymerase chain reaction (PCR) detecting kit for coxsackievirus A16
  • Real-time fluorescent quantitative polymerase chain reaction (PCR) detecting kit for coxsackievirus A16
  • Real-time fluorescent quantitative polymerase chain reaction (PCR) detecting kit for coxsackievirus A16

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Coxsackievirus A16 fluorescent PCR detection kit and its use

[0036] 1. Prepare a kit including the following components: 1 tube of RNA extraction solution (50ml / tube), 24 tubes of PCR amplification reaction solution (20μl / tube), 1 tube of negative quality control (100μl / tube), 1 tube of positive quality control 1 tube of sample (100 μl / tube), 4 tubes of quantitative reference product (50 μl / tube).

[0037] 2. Specimen collection, transportation and storage

[0038] (1) Specimen collection: The object of specimen collection is clinically diagnosed cases of HFMD at the time of the outbreak and children under 5 years old in communities (villages) near the community (village) without cases or nursery institutions where the cases occurred (as healthy control population).

[0039] Specimens for virus isolation include feces, throat swabs, herpes fluid, and cerebrospinal fluid if the patient has neurological symptoms. Clinical specimens should avoid repeated...

Embodiment 2

[0067] Example 2: Preparation and use of quantitative reference and quality control in CA16 virus nucleic acid quantitative detection kit

[0068] The quantitative reference product in the CA16 virus nucleic acid quantitative detection kit is a plasmid containing the specific sequence of the CA16 virus nucleic acid, which is used to prepare a standard curve, accurately quantify the sample to be tested, and can be directly used for RT-PCR detection; quality control products include positive quality control The product and the negative quality control product are used for quality control in clinical trials, and the operation method is the same as that of the sample to be tested.

[0069] After one-step or two-step amplification, save the detection data file. Adjust the analysis parameters according to the analyzed image to optimize the standard curve graph under the standard curve (Std curve) window (that is, the correlation (correlation) value image 3 , and the information abou...

Embodiment 3

[0071] Example 3: Quantitative detection of clinical samples using the CA16 virus fluorescent PCR detection kit

[0072] The samples were from the Guangdong Provincial Center for Disease Control and Prevention, including feces, throat swabs, serum, cerebrospinal fluid and other sample types; the RNA extraction, PCR reaction and result analysis of the samples were carried out with reference to Example 1.

[0073] After the PCR reaction, adjust the analysis parameters according to the amplification curve to make the standard curve under the standard curve (Std curve) window reach the best (ie, the absolute value of the correlation value > 0.97), and then analyze the clinical samples. The test results of clinical samples are attached Figure 7 Shown: The Ct values ​​of the amplification curves of the 5 clinically positive specimens are 24.35, 26.69, 27.67, 29.91, and 31.07, respectively. Combined with the obvious exponential growth period of the amplification curves, they can all...

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Abstract

The invention relates to a real-time fluorescent polymerase chain reaction (PCR) detecting kit, in particular to a kit for early and rapidly diagnosing coxsackievirus A16 infection by a real-time fluorescent quantitative PCR technology. Aiming at a specific nucleotide sequence designing primer and a probe of coxsackievirus A16, the kit adopts a one-step fluorescent reverse transcription-polymerase chain reaction (RT-PCR) method to amplify a sample, judges whether the sample is provided with the coxsackievirus A16 or not according to an amplification curve and quantifies virus contents by a linear quantitative reference product. The kit can detect and quantitatively analyze the coxsackievirus A16 in the samples including feces, blood serum, throat swab, cerebrospinal fluid, bleb fluid, and the like.

Description

technical field [0001] The invention relates to a real-time fluorescent PCR detection kit, in particular to a kit for early and rapid diagnosis of Coxsackievirus A16 infection by real-time fluorescent quantitative polymerase chain reaction technology. The detection and quantitative analysis of Coxsackievirus A16 in samples such as feces, serum, throat swab, cerebrospinal fluid, and herpes fluid are realized by the kit of the invention. Background technique [0002] Coxsackievirus type A16 (CA16) is a member of the genus Enterovirus of the family Picornaviridae. Coxsackieviruses are divided into two groups, A and B, of which group A is divided into 24 serotypes, and group B There are 6 serotypes. The clinical syndromes caused by each serotype are different due to the different organs invaded, and severe cases can cause death. Infection during pregnancy can cause non-paralytic poliomyelitis lesions, and cause intrauterine infection and teratogenicity of the fetus. Coxsackie ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 吴荃邓中平李明程钢何蕴韶
Owner DAAN GENE CO LTD
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