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Composition and kit for detecting 23 respiratory pathogens and detection method of kit

A composition and pathogen technology, applied in the field of nucleic acid detection, can solve the problems of increased sequencing cost, cumbersome operation, long time consumption, etc.

Pending Publication Date: 2017-12-19
SUZHOU SYM BIO LIFESCI CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the detection sensitivity of the first-generation sequencing method is low, the detection cycle is long, and multiple detection cannot be achieved, and the requirements for instruments and operators are relatively high
Although the next-generation sequencing method has made a qualitative leap in detection throughput, the overall technology is very complicated, requiring high professional level of testing personnel, long detection cycle, and relatively high cost of equipment and reagents; A certain detection sensitivity requires deep sequencing of samples, which further increases the cost of sequencing
[0012] Among the two current relatively similar patents, the Chinese patent (CN102181576A) discloses a primer, probe and method for detecting pathogens of respiratory infectious diseases with a liquid chip. Performing two PCRs, the operation is cumbersome; Chinese patent (CN103205509A) discloses a high-throughput non-diagnostic detection method for 13 kinds of respiratory viruses based on a new suspension chip technology, the number of detection targets is 13, and it takes 7 hours. The number of detection targets is small, and it takes a long time

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  • Composition and kit for detecting 23 respiratory pathogens and detection method of kit
  • Composition and kit for detecting 23 respiratory pathogens and detection method of kit
  • Composition and kit for detecting 23 respiratory pathogens and detection method of kit

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Embodiment 1

[0072] This embodiment is a kit for detecting 23 kinds of respiratory pathogens, and the kit is based on liquid chip technology to detect pathogen nucleic acid. All nucleotide sequences of 23 pathogens were retrieved according to the database, homology analysis was carried out according to different pathogens, specific primers and universal primers, and specific probes were designed, and then the nucleic acid was extracted by using Pre-Nat instrument, and passed RT-PCR and After asymmetric PCR amplification (the principle of multiple amplification is as follows: figure 1 shown), the amplified product was hybridized with different magnetic beads bound to different probes, the hybridized product was incubated with SAPE, and then detected by the ABC BioCode3000 system (the hybridization detection principle is as follows figure 2 shown), so as to determine the type of pathogen contained in the sample.

[0073] The kit specifically includes a first reaction solution, a second rea...

Embodiment 2

[0083] In this embodiment, it is known that sample 1 contains influenza A virus H1N1, and the kit described in Example 1 is used for detection. The specific steps of the detection method are as follows:

[0084] a) The total nucleic acid in sample 1 is automatically extracted using a Pre-Nat instrument.

[0085] b) PCR reaction uses the extracted sample nucleic acid as a template, and uses the specific primers of SEQ ID NO.1 to SEQ ID NO.48 and the general primer shown in SEQ ID NO.73 to carry out the experiment, and the same template is added to two tubes for detection , add 10uL template to each tube. Among them, add 8uL of the first reaction solution and 2uL of mixed enzyme in the first tube; add 8uL of the second reaction solution and 2uL of mixed enzyme in the second group, and then put it into the BioCode3000 instrument for PCR reaction to obtain the PCR product . The reaction conditions are: 37°C for 2min; 50°C for 10min; 95°C for 10min; 95°C for 15s, 59°C for 30s, 72...

Embodiment 3

[0091] In this embodiment, it is known that samples 2 / 3 / 4 / 5 / 6 contain M.p, H7N9, C.p, HKU1 and H5 respectively, and are detected according to the detection method of the present invention. The specific steps are the same as in the second embodiment. The test results are as follows:

[0092] a) Determine the result based on the fluorescence signal value. When the fluorescent signal value of the calibration magnetic beads is 1000, and the fluorescent signal value of the internal standard reference product is >1000. At the same time, the magnetic bead detection fluorescence signal value of Mycoplasma pneumoniae (M.p) is 4475, and the other 23 targets have no non-specific signal >1000, confirming that the sample contains Mycoplasma pneumoniae (M.p);

[0093] b) Determine the result based on the fluorescence signal value. When the fluorescent signal value of the calibration magnetic beads is 1000, and the fluorescent signal value of the internal standard reference product is >10...

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Abstract

The invention relates to a composition for detecting 23 respiratory pathogens. The composition comprises a primer composition consisting of the sequences shown by SEQ ID NO. 1-SEQ ID NO. 48 and a probe composition consisting of the sequences shown by SEQ ID NO. 49-SEQ ID NO. 72. The invention also relates to a kit for detecting 23 respiratory pathogens and a detection method of the kit. The kit comprises a first reaction solution, a second reaction solution, a first hybridization solution and a second hybridization solution containing the primers and probes respectively; the kit also comprises a mixed enzyme, a streptavidin-phycoerythrin solution, a negative reference, a positive reference and an interior label. The kit and the detection method provided by the invention integrate the functions of RT-PCR, full-automatic liquid-phase chip hybridization detection, data analysis and the like, and also have the advantages of high throughput, multiple detection target spots, high degree of automation, easiness in operation, short time consumption among the inventions of the same kind of liquid-phase chip technologies, etc.; moreover, the kit and the detection method provide important information for addressing the respiratory tract infection epidemic in time, and are of great significance to the prevention and control of respiratory pathogens.

Description

technical field [0001] The invention relates to the technical field of nucleic acid detection, in particular to a composition, a kit and a detection method for detecting 23 kinds of respiratory pathogens. Background technique [0002] The current clinical methods for virus detection mainly include: cultivation and isolation of pathogens, immunological detection and nucleic acid detection. Nucleic acid detection method is a method for detecting pathogen-specific nucleic acid sequences through molecular biological methods, which mainly includes ordinary PCR, real-time fluorescent quantitative PCR, nucleic acid sequencing and gene chips and other methods. The technology closest to the present invention is the gene chip method. Nucleic acid detection method is a method for detecting pathogen-specific nucleic acid sequences by molecular biological methods. [0003] Among the nucleic acid detection methods, the ordinary PCR method generally requires gel electrophoresis of the am...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12Q1/04C12N15/11C12R1/93C12R1/35C12R1/01
CPCC12Q1/6813C12Q1/689C12Q1/701C12Q2600/16C12Q2531/113C12Q2537/143Y02A50/30
Inventor 董万强何晓辉张平林敏
Owner SUZHOU SYM BIO LIFESCI CO LTD
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