CN103088155A

CN103088155A - Molecular motor biosensor kit for detecting rotavirus

Info

Publication number: CN103088155A
Application number: CN2012104060731A
Authority: CN
Inventors: 张捷; 杨丽君; 张惠媛; 卢晓宇; 许美玲; 顾德周; 刘岩; 汪琦; 张昕; 王佩荣; 陈广全; 乐加昌
Original assignee: YANTAI ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU; Beijing Entry Exit Inspection and Quarantine Bureau of Peoples Republic of China
Current assignee: YANTAI ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU; Beijing Entry Exit Inspection and Quarantine Bureau of Peoples Republic of China
Priority date: 2012-10-23
Filing date: 2012-10-23
Publication date: 2013-05-08

Abstract

The invention relates to a molecular motor biosensor kit for detecting food-borne rotavirus, belonging to the field of quick detection of food-borne viruses. The invention mainly solves the problems of overlong period and expensive instrument and equipment in the traditional detection. 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. A VP7 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 H-DHPE. The kit can be used for quickly and sensitively detecting food-borne rotavirus in a sample to be detected at high flux, and can effectively shorten the detection period and enhance the work efficiency.

Description

A kind of molecular motor biosensor test kit for detection of rotavirus

Technical field

The invention relates to a kind of of food-borne virus Fast Detection Technique.

Background technology

Rotavirus is the infantile diarrhea main pathogens.The classical the Methods of Detection of Pathogens of rotavirus comprises electron microscopy and separation and Culture.Electron microscopy requires to comprise in every milliliter of sample approximately 10 ⁶Individual virion, and virion is easy to degraded aligns and makes a definite diagnosis stopping pregnancy and give birth to impact.The detection sensitivity of separation and Culture detection technique is lower, needs the specific culture condition, and is limited to fundamental research and the diagnostic value of rotavirus.Lower take Detection of antigen as detection method specificity and the sensitivity on basis, and can not effectively carry out detection by quantitative.Round pcr has higher sensitivity, do not need cell cultures, but testing cost is high, and complex operation step, needs the time long.In the rotavirus detection method of exploitation, improve sensitivity, the specificity of detection method at present, the test kit of exploiting economy practicality is still the main direction of rotavirus detection technique.Development fast, efficient rotavirus detects new detection method to the breaking out of effective prevention rotavirus epidemic situation, and identifies the pollution situation of rotavirus, and Evaluation Environment sanitary condition etc. will play a significant role.

Summary of the invention

Take controlled molecular motor technology as core, 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 the ATP enzyme as carrier, target compound to be detected to have highly sensitive, high specificity, characteristics fast.At F ₀F ₁-ATPase molecular motor connects special nucleic acid probe, can realize to the specific food products microorganism fast, specificity, high-throughout detection.The present invention can foreshorten to detection time 1～1.5h, and detection sensitivity can reach 0.005ng/mL, satisfies existing food-borne virus sensing range.

Description of drawings

Accompanying drawing 1 is molecular motor biosensor mode chart (wherein a, b, c, α, β, δ, γ, ε are the atp synthase subunit), 1 is epsilon subunit antibody, and 2 is Streptavidin (Strptavidin), and 3 is N-biotin, 4 is the VP7 probe, and 5 is the rotavirus single stranded RNA.

Accompanying drawing 2 is chro VP7 to the detected result of bacterial strain of the same race not, and ordinate zou represents fluorescent value, and X-coordinate represents the sample that detects, and wherein 1 is water, 2 rotaviruss, and 3 is hepatitis A virus (HAV), 4 is norovirus.

Accompanying drawing 3 is chro VP7 to 15 detected results of adding the rotavirus food samples, and ordinate zou represents fluorescent value, and X-coordinate represents the sample that detects, and 1～15 is the food inspection sample number.

Embodiment

According to rotavirus vp7 gene design specific dna probe 5 '-AGCACAACATTATAATCACAGTATAGTTTGGGTCGA-3 ', 5 ' of its middle probe carries out mark with vitamin H.This probe is connected to above molecular motor by biotin antibody, utilize the molecular motor biosensor can the RNA of sample to be tested be detected, when sample was rotavirus RNA, obvious variation can occur in the fluorescent value of detection system, got final product judgement sample by this variation positive.For this reason, we have designed a test kit, can detect food source property rotavirus easily and fast.

Test kit forms:

Numbering	The component title	Quantity	Preservation condition
					1	Chro VP7	20μl	-20℃
2	Synthetic buffer	10ml	Room temperature
					3	ADP(1.6mol/l)	1ml	-20℃
4	1×PBS	25ml	Room temperature
					5	Luciferase/luciferin	100 units	-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. get 2 μ l chro VP7, be diluted to certain multiple with synthetic buffer.The chro10 μ l that gets after dilution adds above-mentioned EP pipe.

3. separately get 1 EP pipe and add 10 μ l sterilized waters, then add the synthetic buffer of 10 μ l to contrast as background, short term oscillation makes the reaction system mixing.

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

5. above-mentioned reaction system is placed in incubation 10min.

6. the EP pipe is taken out from shaking table, add respectively 200 μ l PBS damping fluids, vibration makes the system mixing.

7. 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 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 to each well, cover bottle stopper, repeatedly put upside down mixing several times, can not vibrate.Mixed solution should be placed 1h in room temperature before using), repeatedly blow and beat with rifle and make several times the system mixing.

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

By experiment, molecular motor biosensor chro VP7 has good specificity, with chro VP7, water, rotavirus, hepatitis A virus (HAV), norovirus are detected, the fluorescent value of rotavirus 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 (0.6ng/mL)	Fluorescent value
		Water	351774
Rotavirus	419008
		Hepatitis A virus (HAV)	356623
Norovirus	350977

With chro VP7, the shellfish sample of 14 interpolation rotaviruss, 1 shellfish sample that does not contain rotavirus are carried out blind sample and detect (No. 5 samples), No. 5 sample fluorescence values and water fluorescent value difference are not remarkable, be judged to and do not detect rotavirus, and other samples and water fluorescent value obvious difference are judged to and detect rotavirus.Detected result is consistent with the interpolation result, can detect the sample standard deviation that adds, and concrete data see the following form, and the results are shown in accompanying drawing 3.

Sample number	Fluorescent value	Sample number	Fluorescent value
				Water	389386	8	455971
1	419739	9	443609
				2	445509	10	447321
3	457229	11	435403
				4	400312	12	447404
5	391020	13	446144
				6	434933	14	442853
7	451273	15	439719

Experimental result shows, this test kit is 1h to the detection time of rotavirus RNA, detects and is limited to 0.005ng/ml.To 15 strain food inspection sample bacterial strains to detect result consistent with the result of RT-PCR technology for detection.Have good specificity and higher sensitivity by this molecular motor biosensor of a large amount of experimental verifications, and can the hole Chemiluminescent plate is high-throughout that sample is detected by 96.

Claims

The present invention claimed in have:

1. specific dna probe and F ₀F ₁The mode that-atp synthase connects: at F ₀F ₁Connect successively epsilon subunit antibody, vitamin H, Streptavidin, biotin labeled rotavirus nucleic acid probe on the epsilon subunit of-atp synthase.
2. the reaction conditions of test kit detection system: room temperature incubation 10min.
3. the formula that synthesizes buffer, startup buffer, PBS.Synthetic buffer: glycerine 20%, magnesium chloride 5mM, Tricine50mM, dipotassium hydrogen phosphate 5mM; Start buffer:ADP (1.6mM): above-mentioned synthetic buffer=1: 3 (v/v); PBS:137mM sodium-chlor, 2.7mM Repone K, 10mM Sodium phosphate dibasic, 2mM potassium primary phosphate.