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Real-time fluorescent nucleic acid isothermal amplification detection kit for bordetella pertussis as well as special primer and probe thereof

A technology of constant temperature amplification detection and real-time fluorescence, which is applied in the field of biomedical detection, can solve the problems of insufficient detection sensitivity, and achieve the effects of easy automation, improved detection sensitivity, and shortened time required

Pending Publication Date: 2021-10-19
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the problems faced by the application of SAT technology in the detection of different types of pathogens are different, and it is necessary to specifically analyze the characteristics of pathogens for special design.
[0005] The applicant's previous patent document CN111378724A relates to an RNA amplification detection method. The application of this method to the detection of B. pertussis is disclosed in the examples. The disclosed method needs to introduce a probe A for identifying the RAN to be detected in its detection system ( Its 3' end is the sequence that specifically binds to the RNA to be tested, and the 5' end is the first primer sequence), and a sequence that is not homologous to the RNA to be tested is introduced into the first primer sequence as a common primer, and the detection sensitivity can be Reaching 100copies / reaction, but still unable to meet the requirements of higher detection sensitivity

Method used

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  • Real-time fluorescent nucleic acid isothermal amplification detection kit for bordetella pertussis as well as special primer and probe thereof
  • Real-time fluorescent nucleic acid isothermal amplification detection kit for bordetella pertussis as well as special primer and probe thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Design of special primers and probes for real-time fluorescent nucleic acid constant temperature amplification to detect the ptxa gene of B. pertussis

[0047] According to the published nucleic acid sequences of B. pertussis in the Genbank database, the present inventors selected and determined a highly conserved sequence on the ptxa gene of B. pertussis and was quite different from other similar pathogens as the detection sequence (the nucleotide sequence corresponding to DNA such as SEQ ID Shown in NO: 1), primers and probes were designed according to the principles of primers and probes design, so as to perform real-time fluorescent nucleic acid constant temperature amplification detection of B. pertussis.

[0048] This embodiment designs multiple sets of primers and probes altogether, wherein selects following two groups of primers and probes (group 1 and group 2) to pertussis positive control (detailed below) and negative control (do not contain the syst...

Embodiment 2

[0066] Example 2: Real-time fluorescent nucleic acid constant temperature amplification detection kit for Bacillus pertussis ptxa gene

[0067] The kit for detecting B. pertussis nucleic acid provided in this example is a kit for detecting B. pertussis ptxa gene based on the principle of RNA nucleic acid constant temperature simultaneous amplification detection, and specifically includes the following components:

[0068] (1) Viral nucleic acid extraction solution: used to extract and purify Bacillus pertussis nucleic acid in the sample, which contains 250-800mM HEPES, 4-10% lithium dodecyl sulfate (LLS), and 1-50 μM specific capture probe (SEQ ID NO:2), 50-500mg / L magnetic beads, 25-150pmol / mL first primer (SEQ ID NO:3);

[0069] (2) Detection solution a: it contains 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl 2 , 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol / mL of the second primer (SEQ ID NO:4);

[0070] (3) Detection solution b: it contains 10-50mM Tris, 5-40mM KCl, 10...

Embodiment 3

[0077] Example 3: A method for real-time fluorescent nucleic acid constant temperature amplification to detect the Bacillus pertussis ptxa gene

[0078] The method of this example detects the ptxa gene of B. pertussis based on the principle of RNA constant temperature synchronous amplification detection. It uses the kit provided in the above-mentioned Example 2 to check whether the oropharyngeal swab / sputum sample obtained from the subject contains B. pertussis nucleic acid. Detection, the specific operation steps are:

[0079] 3.1. Sample preparation

[0080]Take 250 μL pertussis positive control (as described in Example 1), 250 μL negative control and 250 μL samples to be tested (oropharyngeal swab sample / sputum sample) and place them in sample processing tubes for later use;

[0081] 3.2. Nucleic acid extraction

[0082] (1) Add 100 μL to 800 μL of viral nucleic acid extraction solution (HEPES 500 mM, LLS 8%, specific capture probe (SEQ ID NO: 2) 15 μM, magnetic beads 150...

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Abstract

The invention discloses a real-time fluorescent nucleic acid isothermal amplification detection kit for bordetella pertussis as well as a special primer and a probe thereof, and belongs to the technical field of biomedical detection. The kit comprises a nucleic acid extracting solution, a detection solution a, a detection solution b and an SAT enzyme solution, primers and probes which are more suitable for bordetella pertussis detection are optimally designed, all components are added step by step in the detection process for step-by-step reaction, quick and accurate detection of bordetella pertussis can be achieved, and the kit is good in specificity, high in sensitivity, and the amplification product RNA is easy to degrade and does not cause sample cross contamination and environmental pollution.

Description

technical field [0001] The invention belongs to the technical field of biomedical detection, in particular to a real-time fluorescent nucleic acid constant temperature amplification detection kit for B. Primers, probes and related kits used in the real-time fluorescent nucleic acid constant temperature amplification detection of bordetella pertussis (BP) combined with SAT and SAT). Background technique [0002] Pertussis is mainly a severe acute respiratory infectious disease caused by Bacillus pertussis. The detection of Bacillus pertussis is not only meaningful for identifying pathogens, but also for environments that may be polluted by pathogens, such as river water, sewage, surface attachments, etc., and drinking water The detection of pathogenic bacteria in samples such as , food and so on is meaningful. [0003] At present, pertussis bacilli mainly rely on PCR detection of nasopharyngeal swabs and serum pertussis toxin IgG detection. The product of PCR detection is DN...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/6844C12Q2565/519C12Q2521/107C12Q2521/119C12Q2545/113C12Q2523/308C12Q2563/107Y02A50/30
Inventor 居金良崔振玲耿玥
Owner SHANGHAI RENDU BIOTECH
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