CN103088154A - Molecular motor biosensor kit for detecting norovirus - Google Patents

Abstract

The invention relates to a molecular motor biosensor kit for detecting food-borne norovirus, belonging to the field of quick detection of food-borne viruses. The invention mainly solves the problems of overlong time, low sensitivity and low specificity 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. A norovirus nucleic acid 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 norovirus in a sample to be detected at high flux.

Description

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

Technical field

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

Background technology

Norovirus is the important pathogen that causes that human virus's property gastroenteritis is broken out.Norovirus all occupies critical role in Sporadic cases or in epidemic outbreaks, from both at home and abroad in recent years to the situation that detects of food norovirus, food-borne virus is more serious to the threat of food safety.The method that detects at present norovirus mainly contains the methods such as electron microscopy, round pcr, elisa technique, gene chip.Round pcr is the main method that present norovirus detects, but needs the steps such as evaluation of viral enrichment, RNA extraction, design of primers, PCR reaction, amplified production, and whole process required time is long.And the economic level present according to China, electron microscopy, round pcr, biochip technology is used for still difficult popularization of medical institutions' conventional sense.Although the ELISA method is simple to operation, do not need valuable equipment, it has certain deficiency on sensitivity and specificity.Many norovirus the infecteds are arranged in the outpatient clinic case, and the frontier port also becomes the main path of norovirus infectious diseases input.Therefore, investigation and confirmation method can avoid epidemic situation to spread on a large scale fast and efficiently, and development fast as early as possible, high, the highly sensitive food source property norovirus detection technique of specificity makes a definite diagnosis Biosafety, the medical clinic of guarantee frontier port and have very important significance.

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 1h, and detection sensitivity can reach 0.005ng/mL, satisfies existing Microbiological detection of foods scope.

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 NV probe, and 5 is norovirus RNA.

Accompanying drawing 2 is chroNV to the detected result of food-borne virus of the same race not, and ordinate zou represents fluorescent value, and X-coordinate represents the sample that detects, and wherein 1 is water, and 2 is hepatitis A virus (HAV), and 3 is rotavirus.

Accompanying drawing 3 adds norovirus food samples, 2 detected results that do not contain the norovirus food samples for chro NV to 13, 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 one section high conservative region gene design specific dna probe 5 '-AGGAGATYGCGATCYCCTGTCCAYA between norovirus ORF1 and ORF2,5 ' of its middle probe carries out mark with vitamin H.This probe is connected to above molecular motor by biotin antibody, and note is Chro NV.Utilize the molecular motor biosensor can the RNA of sample to be tested be detected, when sample was norovirus 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 the Salmonellas in food easily and fast.

Test kit forms:

Numbering	The component title	Quantity	Preservation condition
					1	Chro NV	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 the 1.5mLEP pipe, add sample to be tested 10 μ L.

2. get 2 μ L chroNV, 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 room temperature 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 NV has good specificity, and to water, rotavirus, hepatitis A virus (HAV), norovirus, the fluorescent value of norovirus is apparently higher than the fluorescent value of water and other contrast bacterium with chro NV, concrete data see the following form, and the results are shown in accompanying drawing 2.

Sample (0.7ng/mL)	Fluorescent value
		Water	195554
Norovirus	232606
		Hepatitis A virus (HAV)	196456
Rotavirus	195808

Add norovirus food samples, 2 with chro NV to 13 and detect through the ELISA method and do not contain norovirus food samples (No. 3, No. 8) and detect, detected result is consistent with the ELISA detected result, and specifically data see the following form, and the results are shown in accompanying drawing 3.

Sample number	Fluorescent value	Sample number	Fluorescent value
				Water	199611	8	201274
1	232076	9	233679
				2	228769	10	227417
3	198809	11	222508
				4	236870	12	224056
5	222963	13	232949
				6	232544	14	222036
7	202697	15	235579

Experimental result shows, this test kit is 1～1.5h to the detection time of norovirus RNA, detects and is limited to 0.005ng/ml.To 15 strain food inspection sample bacterial strains to detect the result that result and ELISA method detect consistent.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 (3)

1. 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 norovirus nucleic acid probe on the epsilon subunit of-atp synthase.
2. 2. the reaction conditions of test kit detection system: room temperature incubation 10min.
3. 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.

Images