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Compositions, kits and methods for detecting pathogens

A composition and kit technology, which is applied in the fields of biochemical equipment and methods, recombinant DNA technology, and the determination/inspection of microorganisms to achieve the effects of high sensitivity, good specificity, simple and efficient detection methods

Active Publication Date: 2020-06-12
SANSURE (SHANGHAI) GENE TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When performing multiplex PCR amplification, it is also necessary to face the challenge of optimizing the PCR reaction system with each additional pair of primers

Method used

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  • Compositions, kits and methods for detecting pathogens
  • Compositions, kits and methods for detecting pathogens
  • Compositions, kits and methods for detecting pathogens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] sequence

[0054] 30 primer sequences and 2 internal standard sequences: SEQ. ID NO: 01-SEQ. ID NO: 32;

[0055] Wherein SEQ. ID NO: 08 and SEQ. ID NO: 24 are internal standard primer pairs;

[0056] Specific detection of Haemophilus influenzae (HI), Streptococcus pneumoniae (SP), Neisseria meningitidis (NM), Listeria monocytogenes (LM), Escherichia coli (ECO), Japanese encephalitis virus (JEV) , enterovirus (EV), human herpesvirus type 6 (HHV6), varicella-zoster virus (VZV), Nipah virus (NVD), Powassen virus (POWV), West Nile virus (WNV), SEQ ID NO: 1~7, 9~23 and 25~32 primer sequences of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), Cryptococcus neoformans (CN);

[0057]

[0058]

Embodiment 2

[0060] DNA / RNA extraction

[0061] DNA / RNA is extracted by the magnetic bead method, and the following operations are performed in the sample processing room:

[0062] Take an appropriate amount of 1.5 mL sterilized centrifuge tubes, label the negative control, positive control and test samples respectively, and add 300 μL DNA / RNA extraction solution 1 to each tube;

[0063] Add 200 μL of the sample to be tested or negative control or positive control to each tube; cover the tube cap, shake and mix for 10 seconds, and centrifuge briefly;

[0064] Add 100 μL of DNA / RNA Extraction Solution 2-mix to each tube, mix well and pipette, shake and mix for 10 seconds, then let stand at room temperature for 10 minutes;

[0065] After centrifuging briefly, place the centrifuge tube on the separator, and slowly suck out the solution after 3 minutes (be careful not to touch the brown substance adsorbed on the tube wall);

[0066] Add 600 μL DNA / RNA Extraction Solution 3 and 200 μL DNA / R...

Embodiment 3

[0069] PCR reaction

[0070] Add 50 μL PCR-mix (Mg 2+ , dNTPs, MMLV, Taq enzyme, primers, PCR buffer), use a tip to draw PCR-mix to elute the brown residue adsorbed on the wall of the centrifuge tube, repeat several times to make it completely eluted, and then wash the eluted Transfer all the brown mixture to a 0.2mL PCR reaction tube, cover the tube cap, and transfer to the amplification detection area.

[0071] Use Hongshi SLAN-96P automatic medical PCR analysis system for the following analysis.

[0072] Wherein, the fluorescent PCR reaction system is shown in Table 1. The PCR amplification program is shown in Table 2.

[0073]

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Abstract

The present invention provides a composition, especially for detecting various pathogens of encephalitis / meningitis / respiratory tract, wherein said composition comprises SEQ ID NO: 1~7, 9~23 and 25~32 primer sequences for pathogens , wherein SEQ ID NO:1~7 and 9~16 have fluorescent reporter groups. In addition, the present invention also relates to the use of the above composition in the preparation of kits, and related kits and methods of use thereof. The invention realizes single-channel multi-target detection on a conventional fluorescent PCR instrument, thereby increasing the detection throughput of the existing fluorescent PCR by 4 times, and is carried out under single-tube closed conditions, greatly reducing the pollution of molecular detection.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and in particular relates to a composition for detecting encephalitis / meningitis / respiratory tract multiple pathogens by using single-channel multiplex real-time fluorescent PCR technology. And the present invention also relates to a related kit and a method for using the kit. Background technique [0002] Encephalitis (encephalitis) and meningitis (brain fever; meningitis) are diseases caused by the infection of the brain by pathogens. infection with pathogens. They are often accompanied by complications of bacterial or viral infections, such as ear, sinus, or upper respiratory tract infections. Among them, the pathogens causing encephalitis and / or meningitis are various, and the scope and degree of lesions vary greatly, and the clinical manifestations are very complicated. [0003] At present, there are many clinical methods for detecting encephalitis and / or meningitis pathogens, s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/689C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/701C12Q1/705C12Q1/689C12Q1/686C12Q2600/16C12Q2561/113C12Q2563/107C12Q2537/143C12Q2527/107C12Q1/6883Y02A50/30
Inventor 谭德勇任小梅孙青芝程星邓中平刘佳戴立忠
Owner SANSURE (SHANGHAI) GENE TECH LTD