CN103088118B

CN103088118B - Molecular motor biosensor kit for detecting Enterobacter sakazakii

Info

Publication number: CN103088118B
Application number: CN201210409160.2A
Authority: CN
Inventors: 张捷; 马贵平; 周琦; 杨向莹; 刘国传; 张惠媛; 李冰玲; 卢晓宇; 刘岩; 顾德周; 汪琦; 张昕; 王佩荣; 乐加昌; 陈广全
Original assignee: BEIJING TESTING QUARANTINE SCIENCE TECHNOLOGY RESEARCH INSTITUTE; Inspection and Quarantine Technology Center Beijing Entry-Exit Inspection and Q
Current assignee: BEIJING TESTING QUARANTINE SCIENCE TECHNOLOGY RESEARCH INSTITUTE; Inspection and Quarantine Technology Center Beijing Entry-Exit Inspection and Q
Priority date: 2012-10-23
Filing date: 2012-10-23
Publication date: 2014-10-22
Anticipated expiration: 2032-10-23
Also published as: CN103088118A

Abstract

The invention relates to a molecular motor biosensor kit for detecting Enterobacter sakazakii, belonging to the field of quick detection of animal-derived food microbes. The invention mainly solves the problem of overlong time (5-6 days) in the traditional detection method. The core technique is to utilize an F0F1-ATPase molecular motor biosensor on chromatophore; and since the F0F1-ATPase can quickly rotate, coupling between the catalytic site and the proton transfer is established through the rotation. An ITS probe is connected to an epsilon subunit of the ATPase; after a sample to be detected and a negative control are respectively combined with the biosensor, the ATP synthesis amounts under the catalytic action after 10 minutes are compared; and the ATP synthesis amount can be measured according to the amount of H<+> in the environment, and the amount of H<+> is acquired according to the fluorescence intensity embodied by the F-DHPE. The kit can be used for quickly and sensitively detecting Enterobacter sakazakii in a sample to be detected at high flux.

Description

A kind of molecular motor biosensor test kit for detection of Enterobacter sakazakii

Technical field

The invention relates to a kind of of animal derived food microbial rapid detection technology.

Background technology

Traditional detection of pathogens method requires each Interventions Requested to be carried out to the steps such as non-selective increasing bacterium, selective enrichment, separation, screening and identification, generally needs just can provide for 6 days examining report, has a strong impact on quality and the shelf-life of goods.Some new technology, as the detection methods such as round pcr, immunology detection technology, biochip technology still depend on traditional detecting step, need to increase bacterium, take time and effort, and completing one-time detection needs the time of several days conventionally.So not only increased the workload in laboratory, nor be beneficial to the monitoring of Production Flow Chart in foodstuffs industry, the quality control of finished product and government department to the management of food safety and control.Due to rolling up of import and export food, be therefore badly in need of reliable and effective method for quick, shorten detection time, promote the foreign trade of China's food.

Summary of the invention

The controlled molecular motor technology of take is core, and the technology such as the identification of integrated nucleic acid probe, fluorescent probe mark and detection have been set up the Fast Detection Technique system of a novel concept.Utilize ATP enzyme as carrier, target compound to be detected to there is highly sensitive, high specificity, feature fast.At F ₀f ₁-ATPase molecular motor connects special nucleic acid probe, can realize to specific food products microorganism fast, specificity, high-throughout detection.The present invention can foreshorten to detection time 2 days, and detection sensitivity can reach 10 ²cFU/ml, meets existing Microbiological detection of foods scope.

Accompanying drawing explanation

Accompanying drawing 1 is molecular motor biosensor mode chart (wherein a, b, c, α, β, δ, γ, ε are atp synthase subunit), 1 is epsilon subunit antibody, and 2 is Streptavidin (Strptavidin), and 3 is N-biotin, 4 is ITS probe, and 5 is Enterobacter sakazakii single stranded DNA.

Accompanying drawing 2 is chro ITS to the detected result of bacterial strain of the same race not, and ordinate zou represents fluorescent value, and X-coordinate represents the sample detecting, and wherein 1 is water, and 2 is Enterobacter sakazakii, and 3 is Vibrio parahemolyticus, and 4 is intestinal bacteria, and 5 is Salmonellas, and 6 is vibrio cholerae.

Embodiment

According to Enterobacter sakazakii ITS gene design specific dna probe 5 '-actctgacacaccgcgcattcctg, 5 ' of its middle probe carries out mark with vitamin H.This probe is connected to above molecular motor by biotin antibody, utilize molecular motor biosensor to detect the DNA of sample to be tested, when sample is Enterobacter sakazakii DNA, can there is obvious variation in the fluorescent value of detection system, get final product judgement sample positive by this variation.For this reason, we have designed a test kit, can to the Enterobacter sakazakii in food, detect easily and fast.

Test kit forms:

Numbering	Component title	Quantity	Preservation condition
				1	Chro?ITS	20μl	﹣20℃
2	Synthetic buffer	10ml	Room temperature
				3	ADP(1.6mol/l)	1ml	﹣20℃
4	1×PBS	25ml	Room temperature
				5	Luciferase/luciferin	100Ge unit	﹣20℃
6	Luciferase/luciferin reassembly buffer liquid	12ml	﹣20℃
				7	Sterilized water	5ml	Room temperature

Operation steps is as follows:

1. get 1.5ml EP pipe, add sample to be tested 10 μ l.

2. above-mentioned EP pipe is placed in to 95 ℃ of water-bath 5min, is then transferred to 50 ℃ of water-bath 1min.

3. get 2 μ l chro ITS, with synthetic buffer, be diluted to certain multiple.The chro 10 μ l that get after dilution add above-mentioned EP pipe.

4. separately get 1 EP pipe and add 10 μ l sterilized waters to be placed in 95 ℃ of water-bath 5min, be then transferred to 50 ℃ of water-bath 1min, then add the synthetic buffer of 10 μ l to contrast as background, short term oscillation mixes reaction system.

5. in 2 EP pipes, add respectively 30 μ l startup buffer (to start buffer by ADP (1.6mol/l) and synthetic buffer 1:3 preparation by volume again, certain amount preparation is got in each experiment on demand, matching while using), vibration mixes reaction system, then of short duration centrifugal to remove the globule on cap wall immediately.

6. above-mentioned reaction system is put into 37 ℃ of constant-temperature table incubation 10min.

7. EP pipe is taken out from shaking table, add respectively 200 μ l PBS damping fluids, vibration mixes system.

8. get 96 clean orifice plates, end reaction system in 2 pipes is added wherein, every individual system adds 3 holes, every hole application of sample 50 μ l, then to each well, add respectively the luciferase solution that 30 μ l have configured (luciferase/luciferin reassembly buffer liquid to be added in the Brown Glass Brown glass bottles and jars only that luciferase/luciferin is housed, cover bottle stopper, repeatedly put upside down several times and mix, can not vibrate.Before use, mixed solution should be placed to 1h in room temperature), with rifle, repeatedly blow and beat and make several times system mix.

9. by machine testing on 96 orifice plates, processing data, averages to each group data, then with sample numerical value, deducts the actual fluorescent value that background numerical value is sample.

By experiment, molecular motor biosensor chro ITS has good specificity, with chro ITS, water, Enterobacter sakazakii, Vibrio parahemolyticus, intestinal bacteria, Salmonellas, vibrio cholerae are detected, the fluorescent value of Enterobacter sakazakii is apparently higher than the fluorescent value of water and other contrast bacterium, concrete data see the following form, and the results are shown in accompanying drawing 2.

Sample (40ng/mL)	Fluorescent value
		Water	284994
Enterobacter sakazakii	391027
		Vibrio parahemolyticus	309041
Intestinal bacteria	328063
		Salmonellas	299080
Vibrio cholerae	348079

Experimental result shows, this test kit is 40min to the detection time of Enterobacter sakazakii DNA, detects and is limited to 10ng/ml.To Enterobacter sakazakii reference culture to detect the result that result and PCR detect consistent.By this molecular motor biosensor of a large amount of experimental verifications, there is good specificity and higher sensitivity, and can hole Chemiluminescent plate is high-throughout that sample is detected by 96.

Claims

1. for detection of a molecular motor biosensor test kit for Enterobacter sakazakii, comprise following component:

Wherein, described molecular motor biosensor is Chro-ITS, and its construction process is:

According to Enterobacter sakazakii ITS gene design specific dna probe 5 '-ACTCTGACACACCGCGCATTCCTG, 5 ' of its middle probe carries out mark with vitamin H, and this probe is connected to above molecular motor by biotin antibody: by biotin labeled epsilon subunit antibodies at F ₀f ₁on the epsilon subunit of-ATPase, then use the vitamin H of Streptavidin (Streptavidin) on epsilon subunit antibody to be combined, finally biotin labeled probe is combined with Streptavidin;

The step of applying described molecular motor biosensor test kit detection Enterobacter sakazakii comprises:

1) get 1.5mlEP pipe, add sample to be tested 10 μ l;

2) above-mentioned EP pipe is placed in to 95 ℃ of water-bath 5min, is then transferred to 50 ℃ of water-bath 1min;

3) get 2 μ lchroinvA, with synthetic buffer, be diluted to certain multiple, the chro10 μ l getting after dilution adds above-mentioned EP pipe;

4) separately get 1 EP pipe and add 10 μ l sterilized waters to be placed in 95 ℃ of water-bath 5min, be then transferred to 50 ℃ of water-bath 1min, then add the synthetic buffer of 10 μ l to contrast as background, short term oscillation mixes reaction system;

5) in 2 EP pipes, add respectively 30 μ l to start buffer again, start buffer by ADP and the synthetic buffer preparation in 1: 3 by volume of 1.6mol/l, certain amount preparation is got in each experiment on demand, matching while using, vibration mixes reaction system, then of short duration centrifugal to remove the globule on cap wall immediately;

6) above-mentioned reaction system is put into 37 ℃ of constant-temperature table incubation 10min;

7) EP pipe is taken out from shaking table, add respectively 200 μ lPBS damping fluids, vibration mixes system;

8) get 96 clean orifice plates, end reaction system in 2 pipes is added wherein, every individual system adds 3 holes, every hole application of sample 50 μ l, the luciferase solution that then adds respectively 30 μ l to configure to each well, described luciferase solution preparation method is: luciferase/luciferin reassembly buffer liquid is added in the Brown Glass Brown glass bottles and jars only that luciferase/luciferin is housed, cover bottle stopper, repeatedly put upside down several times and mix, can not vibrate, before use, mixed solution should be placed to 1h in room temperature; With rifle, repeatedly blow and beat and make several times system mix;

9), by machine testing on 96 orifice plates, processing data, averages to each group data, then with sample numerical value, deducts the actual fluorescent value that background numerical value is sample.