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Internal-reference-containing double isothermal nucleic acid amplification method for quickly detecting respiratory syncytial virus

An isothermal nucleic acid amplification and syncytial virus technology, applied in the field of molecular biology, can solve the problems of poor DNA polymerase activity, incorrect mixture, improper temperature, etc., and achieve low cost, short detection time, and good specificity.

Active Publication Date: 2019-05-17
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it could also mean that the reaction was inhibited due to incorrect temperature, incorrect RAA reaction mix, poor DNA polymerase activity, or the presence of inhibitory substances in the sample matrix, resulting in a false negative result

Method used

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  • Internal-reference-containing double isothermal nucleic acid amplification method for quickly detecting respiratory syncytial virus
  • Internal-reference-containing double isothermal nucleic acid amplification method for quickly detecting respiratory syncytial virus
  • Internal-reference-containing double isothermal nucleic acid amplification method for quickly detecting respiratory syncytial virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Design and determination of specific primer pairs, target probes, and internal reference probes suitable for isothermal nucleic acid amplification detection of universal respiratory syncytial virus

[0037] Download the entire genome sequence of all RSV viruses, perform sequence comparison, find a conserved region with high homology, and determine a target sequence suitable for detecting type A and type B universal RSV, and its nucleotide sequence is shown as SEQ ID NO. 5. Multiple specific primers and target probes were designed in this conserved region.

[0038] RAA primer design principles: First, the primer length, RAA primers are longer than typical PCR primers, generally required to be 30-35bp; second, the primer sequence, avoid repeated G at the 5' end (3-5bp), preferably C or T; preferably G and C at the 3' end (last 3 bases); GC content should not be greater than 70% or less than 30%; avoid formation of secondary structures, primer dimers, etc. betwe...

Embodiment 2

[0068] Embodiment 2 detects the double isothermal nucleic acid amplification method (1) containing internal reference of respiratory syncytial virus

[0069] 1. Sample source and RSV RNA extraction

[0070] Virus samples were samples containing RSV live virus collected from different patients' lavage fluid by Hebei Children's Hospital. Tianlong extraction kit was used for RNA extraction, and the RNA extraction equipment was Tianlong automatic nucleic acid extractor.

[0071] 2, primer and probe adopt the primer and the probe (SEQ ID NO.1-4) that are applicable to the detection RSV virus of isothermal nucleic acid amplification method determined in embodiment 1, wherein target probe marks FAM fluorescent group, internal reference Needle labeled HEX fluorophore.

[0072] 3. Prepare the amplification system: prepare the isothermal nucleic acid amplification system in a 200 μL centrifuge tube according to the following ratio (volume is 50 μL):

[0073]

[0074] The above-prep...

Embodiment 3

[0077] Embodiment 3 detects the double isothermal nucleic acid amplification method (2) containing internal reference of respiratory syncytial virus

[0078] Refer to Example 2 for the method, the difference is that in the 50 μL isothermal nucleic acid amplification system, the concentration of the target probe is 160 nM, the concentration of the internal reference probe is 160 nM, the concentration of the forward and reverse primers is 450 nM respectively, and other parameters and steps Same as Example 2. The results showed that the amplified fluorescent signal began to appear within 1 min, and the peak value was high. Such as Figure 3A-Figure 3B shown. By repeating the above embodiments, the same amplified fluorescent signal can be obtained with good repeatability.

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Abstract

The invention provides an internal-reference-containing double isothermal nucleic acid amplification method for quickly detecting a respiratory syncytial virus, and belongs to the technical field of isothermal nucleic acid amplification. The method comprises the steps that firstly, through respiratory syncytial virus (RSV) gene sequencing and comparison, a specific primer pair and probe for detecting the RSV are designed, an internal reference probe is designed, the nucleotide sequences of the specific primer pair and the probes are shown as SEQ ID NO.1-4 respectively, on this basis, an internal-reference-containing double isothermal nucleic acid amplification system is established, and a kit for detecting the respiratory syncytial virus is further established. The method is implemented under isothermal conditions, the simultaneous amplification of the respiratory syncytial virus and internal reference DNA can be achieved within 5-30 min, the sensitivity and the specificity are high, the false-negative and ineffective results are excluded through the addition of the internal reference, and the method is more suitable for being applied to detection of a large number of samples and applicable to the quick detection of the respiratory syncytial virus.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to target sequences, specific primers and target probes for detecting respiratory syncytial virus. The present invention also relates to double isothermal nucleic acid amplification using specific primers, probes and internal reference probes. Methods and kits for detecting respiratory syncytial virus. Background technique [0002] Respiratory syncytial virus (RSV) is an enveloped, single-stranded, negative-sense RNA virus belonging to the genus Pneumovirus in the family Paramyxoviridae. There is a single antigen type of RSV, which is divided into two subgroups, type A and type B, according to the analysis of antigen and genetic variation, and usually only one subtype is dominant in the epidemic. Respiratory syncytial virus has been identified as the most common and important cause of acute respiratory infection, leading to increased morbidity and mortality in infants and a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
Inventor 马学军申辛欣王智宏应清界李鑫娜齐菊菊
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT