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Micro-fluidic chip reagent kit for detecting ten respiratory tract infection pathogens and use method of reagent kit

A microfluidic chip, respiratory technology, applied in the field of molecular biology, can solve the problems of low sensitivity, inconvenient operation, poor culture effect, etc., and achieve the effect of strong specificity

Active Publication Date: 2018-01-19
NANJING LANSION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although ordinary fluorescent PCR detection reagents have good sensitivity and specificity, most of them are aimed at single or several pathogens. If you want to detect multiple pathogens, you need to use multiple sets of different detection kits, which is inconvenient to operate and expensive; Although the immunoassay detection reagent can detect multiple pathogens at the same time, because the immunoassay detects the specific antibodies produced by the organism caused by the pathogens in the sample, there is a long window period and the sensitivity is relatively low; and for bacteria For the detection of pathogen-like pathogens, although the bacterial culture method has high accuracy, it takes a long time and the culture effect is poor. Many bacteria cannot be cultured, which delays the best time for treatment

Method used

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  • Micro-fluidic chip reagent kit for detecting ten respiratory tract infection pathogens and use method of reagent kit
  • Micro-fluidic chip reagent kit for detecting ten respiratory tract infection pathogens and use method of reagent kit
  • Micro-fluidic chip reagent kit for detecting ten respiratory tract infection pathogens and use method of reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Microfluidic chip detection kit for detecting 10 kinds of respiratory infection pathogens, the kit is composed of primer-probe mixture, RT-PCR buffer, mixed enzyme solution, positive quality control and RNase-free water; RT-PCR buffer includes PCR buffer, dATP, dUTP, dCTP, dGTP, and MgCl 2 ; The mixed enzyme solution includes HotStart Taq enzyme, reverse transcriptase and UNG enzyme; the positive quality control contains plasmids corresponding to the amplified gene sequences of the above 10 respiratory pathogens.

[0131] The final concentration of the primer in the amplification system is 100-1000nM; the final concentration of the probe in the amplification system is 50-500nM.

[0132] Kit operation and result determination

[0133] (1) Dilute 50 μL of sample DNA / RNA co-extraction template (extracted from human sputum, throat swab, etc.) with RNase-free water to 500 μL into the sample well of the chip, and mix with the pre-loaded mixed enzyme in the sample well solut...

Embodiment 2

[0140] 15 samples were tested with a microfluidic chip, 5 negative samples were numbered 1 to 5, and 10 positive samples were numbered 6 to 15 containing the nucleic acids of 10 positive strains of pathogens respectively. Sample No. 6 contained Mycoplasma pneumoniae nucleic acid, and Sample No. 7 contained pneumonia Chlamydia nucleic acid, sample No. 8 contained Legionella pneumophila nucleic acid, sample No. 9 contained Bacillus pertussis nucleic acid, sample No. 10 contained rhinovirus nucleic acid, sample No. 11 contained respiratory adenovirus nucleic acid, sample No. 12 contained influenza A virus nucleic acid, and No. 13 Samples contained influenza B virus nucleic acid, sample No. 14 contained respiratory syncytial virus nucleic acid, and sample No. 15 contained parainfluenza virus nucleic acid. The detection operation was carried out according to the same method as in Example 1, and the detection results are shown in Table 1.

[0141] Table 1

[0142]

[0143]

...

Embodiment 3

[0146] Use the microfluidic chip to detect ten kinds of pathogenic plasmid mixed samples respectively, according to the dilution concentration of 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 Sensitivity test was carried out with five gradients of copies / μL and 10copies / μL. Detection was carried out in the same manner as in Example 1.

[0147] The test results show that the kit of the present invention has good sensitivity in the microfluidic chip, and the Ct value changes in a gradient as the concentration decreases. The results are shown in Table 2.

[0148] Table 2

[0149]

[0150]

[0151] The test results show that the kit is effective against respiratory pathogens Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Bordetella pertussis, rhinovirus, influenza A virus, influenza B virus, respiratory adenovirus, syncytial virus, The diagnosis of parainfluenza virus is highly sensitive, and the detection sensitivity of each item can reach ...

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Abstract

The invention provides a micro-fluidic chip reagent kit for detecting ten respiratory tract infection pathogens and a use method of the reagent kit. The reagent kit adopts a combination of a Taqman probe fluorescent PCR (polymerase chain reaction) technology and a micro-fluidic chip, detects the ten common respiratory tract infection pathogens, can obtain a detection result within 2h, and is highin specificity, and the sensitivity can reach 100 copies / microliter. The kit comprises a sample introduction chamber, at least ten reaction chambers and a micro-fluidic flow channel, wherein the reaction chambers are mutually independent; each reaction chamber is provided with a reagent dry powder for amplifying one respiratory tract pathogen in advance; each reagent dry powder comprises a primerfor amplifying the corresponding respiratory tract pathogen and a TaqMan probe; the reagent dry powder arranged in each reaction chamber in advance can amplify any one of the ten respiratory tract pathogens; the respiratory tract pathogens amplified by the reagent dry powder in all the reaction chambers can include the ten respiratory tract pathogens.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a microfluidic chip detection kit for detecting 10 kinds of respiratory tract infection pathogens and a use method thereof. Background technique [0002] Respiratory tract infection is one of the most common diseases in the world, and its incidence rate plays a major role in the overall structure of the morbidity rate of residents in various countries. About 10% of the residents suffer from respiratory tract infection at the peak of the epidemic each year. Respiratory tract infection can lead to symptoms such as rhinitis, runny nose, nasal congestion, cough, mild pharyngitis, general fever, etc., and severe cases can lead to dyspnea and even death. Acute respiratory infection has become one of the important infectious diseases of children at home and abroad, and its incidence rate ranks first among all kinds of diseases in children. The pathogens that cause respiratory tract inf...

Claims

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Application Information

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IPC IPC(8): C12M1/34C12M1/00C12Q1/70C12Q1/686C12Q1/04
CPCY02A50/30
Inventor 许行尚杰弗瑞·陈于沛朱滔张蓉蓉
Owner NANJING LANSION BIOTECH CO LTD
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