CN103033613A - Quick mouth mucosa exudate detection method - Google Patents

Abstract

The invention discloses a quick mouth mucosa exudate detection method and belongs to the field of biotechnology. The method comprises the following steps: lightly reversing the bottom for about three times to mix the sample buffer solution in the bottle; distributing the mouth mucosa exudate in a dropper into a test tube; taking a clean swab, immediately placing the swab into the tube with the sample buffer solution; repeatedly inserting the swab into the sample buffer solution tube for 6-8 times, and rubbing two sides of the swap by leaning against the tube wall; taking out the swap from the tube; and preparing the mouth mucosa exudate for detection; transferring 200 microliters of mouth mucosa exudate to an empty test tube; taking out a determination test strip from a jar, and immediately covering the jar; avoiding touching the membrane surface at the center of the test strip by a hand; placing the determination test strip into the tube with 200 microliters of diluted sample, making the arrow on the determination test strip downward; recording the time of the determination test strip added into the sample; reading the detection result after 20 minutes; and throwing the test strip, the tube and the swap into a biological dangerous cargo container.

Description

A kind of oralmucosal transudate fluid method for quick

Technical field

The present invention relates to a kind of oralmucosal transudate fluid method for quick, belong to biological technical field.

Background technology

People have developed the analytical approach of various analytes in many qualitative or quantitative detection of biological somas and the fluid.Reagent such as a kind of diagnosing oral diseases, selecting rose-bengal is coloring agent, itself and DNA of tumor cell polymerase have affinity, its painted diagnosis to carcinoma of mouth has specificity, its painted weight can be differentiated carcinoma of mouth and Precancerous lesion, preparing rose-bengal carcinoma of mouth diagnostic reagent is made of following raw material: sodium bicarbonate, formaldehyde or ethanol, rose-bengal, distilled water, all by a certain percentage preparation.

The detection method of streptococcus mutans and streptococcus sobrinus in a kind of people's saliva at first is the design primer, and the nested polymerase chain reaction that provides comprises secondary PCR, and 4 pairs of primers, streptococcus mutans two align anti-primer and streptococcus sobrinus two aligns anti-primer; Next is that bacterial chromosomal dna and salivary chromosome DNA extract; The 3rd is PCR for the first time, adds successively two pairs of primers, four kinds of dNTP potpourris and template DNA and Taq enzyme; The 4th is the PCR second time, and for the first time PCR product dilution adds other two pairs of primers, increases; The 5th is electrophoresis.

Collect saliva of buccal cavity for detection of bringing relatively less privacy to invade, it is comparatively safe, and can relatively easily finish fast.

Although the analytic approach by above-mentioned detection strip has advantages of directly, needs complex steps fast and not, for detected object, it is inconvenient all wanting interlock to detect strip in whole testing process.

Summary of the invention

The purpose of this invention is to provide a kind of oralmucosal transudate fluid method for quick, a kind of simple, convenient and cost-efficient oralmucosal transudate fluid collection method with the side direction immunity-chromatography test strip, it is suitable for sensitive and detects rapidly analyte in the oralmucosal transudate fluid.

A kind of oralmucosal transudate fluid method for quick contains following steps;

At first, approximately come biased sample damping fluid (with 0.15M sodium chloride, 0.l% Triton X-100,15% hot deactivation chicken serum, 30 μ g/ml affinities element, 0.2% Tween 80,0.2% Tetronic T-904 and the 0.0285%(active component of pH7.2+/-0.2 potassium phosphate buffering) ProClin 950 for 3 times by inverted bottle lightly);

Take off bottle cap and damping fluid is filled to the dropper to groove from bottle;

The oralmucosal transudate fluid of dropper is dispensed in the test tube; Then a clean swab that is provided by bag is provided;

Catch the swab handle, avoid touching the fabric end of swab;

Subsequently, apply suitable pressure and gently with swab fabric end gums line in the wiping to and fro.One jiao at mouth begins softly and lentamente wiping until arrive another angle of mouth, then returns starting point along the wiping of upper gums line, and the time is 5-6 second;

Then rotate swab with gums under the opposite side wiping of using swab;

The opposite side that uses swab is gums line under the wiping back and forth softly and lentamente;

One jiao at mouth begins, and finishes at another angle of mouth, then returns the place of setting out along the wiping of lower gums line, and the time is 5-6 second;

To wipe away immediately to give and be put in the pipe that fills sample buffer;

Hold the swab handle, swab is back and forth inserted in the sample buffer pipe 6-8 time, be close to the both sides of pipe friction swab; From pipe, take out swab;

Oralmucosal transudate fluid has been ready for detection now;

Transferase 12 00 this oralmucosal transudate fluid of μ l is to empty test tube;

To use following mensuration strip to detect this sample;

If oralmucosal transudate fluid carried out the detection that detects strip more than one, a plurality of 200 μ l samples can be transferred to single empty test tube;

Open the tank of measuring strip is housed; Taking out one from tank measures strip and again builds at once tank;

Keep away the film surface that manual-free touches strip central authorities;

Place the pipe that contains 200 μ l dilution sample with measuring strip, make under the arrow points of measuring on the strip;

Set the timer to 20 minutes, or the time that strip adds to sample will be measured in record;

Read later on testing result in 20 minutes;

Then strip, pipe and swab are abandoned in bio-hazard product waste canister.

Advantage of the present invention is the diagnosis that can easily be used for fast many illnesss, and these illnesss need to be carried out immune detection to the analyte in the saliva, especially easily uses concept of the present invention to be used for sexually transmitted disease and detects, such as c-hepatitis antibody and hbv antibody.

Embodiment

Obviously, the many modifications and variations done based on aim of the present invention of those skilled in the art belong to protection scope of the present invention.

Embodiment 1:

Use the methods known to those skilled in the art, be applicable to mensuration strip of the present invention can comprise from any source natural, chemosynthesis or recombinant production and obtain.For example, the peptide moiety of preferred SEQ ID No.1 to 5 can use the chemosynthesis of solid-phase peptide synthetic technology, or by operability will encode the nucleic acid of expectation peptide be connected to express in the carrying agent and in suitable host the described nucleic acid of expression carry out recombinant production.In case separate, can use known technology with described peptide biotinylation.

Can use any the methods known to those skilled in the art that suitable antigen is fixed to matrix, described method is not destroyed antigen to the specific binding of target antibody.

For water absorptivity matrix, use the technology that well known to a person skilled in the art, usually before sealing, the streptavidin of compound is coupled to matrix.

Preferably, in containing the streptavidin binding site equivalent of 4:1 ratio at least and contrast the solution of each biotin structure, realize coupling, in although other ratio is also included within such as (mark) ratio in the middle of 0.5:1,1:1,2:1,3:1 and 5:1 and all, as a part of the present invention.

For water absorptivity matrix, resulting composite can be applied to host material and dry simply, subsequently with sealing with suitable sealer.

The suitable antigen that has enough high molecular and can directly be combined with matrix needn't adhere to by the method for biotin/streptavidin connexon such as a lot of recombinant proteins.

After inserting oralmucosal transudate fluid, described mensuration strip can be observed the label signal between 15 to 60 minutes, more preferably between 15 to 50 minutes, most preferably between 20 to 45 minutes.

After detecting beginning early than 15 minutes or be later than 50 minutes and read testing result may be to the result who makes mistake; The signal that produces by aforesaid coloured label can directly detect from detecting strip without further processing usually; Fluorescent marker may need photofluorometer to detect.

In method well-known to those skilled in the art, use silver salt solution can strengthen the signal that is produced by the metal-sol label; Similarly, when using enzyme, label must contact with the substrate of enzyme labeling thing, can detect product to produce.

Therefore, these Enhancement Method have departed from the conventional single stage method that adopts coloured particle label and colloidal sol to carry out to be measured, because matrix must contact with imaging liquid (silver salt or substrate solution) before the certification mark thing.

Illustrate the program that oralmucosal transudate fluid fast immune chromatographic according to a preferred embodiment of the invention detects, for testing, with the up and down gums of polyester swab wiping object, then described swab is placed approximately 1000 μ l saliva sample damping fluids (0.15M sodium chloride, 0.1%Triton X-100,15% hot deactivation chicken serum .30 μ g/ml affinity element, 0.2%Tween 80,0.2%Tetronic T-904 and the 0.0285%(active component of pH 7.2+/-0.2 potassium phosphate buffering) ProClin 950 of test tube) and mix at test tube.

Fluid in the extrusion swab also discards, and this sample potpourri of 200 μ l is transferred to clean tube to measure; Put into vertically the test tube that contains 200 μ I sample potpourris with measuring strip.

When the sample of dilution when measuring strip and upwards move, it makes blush Protein G-colloid gold reagent rehydration on the strip, and IgG is combined with Protein G-colloid gold particle and is formed IgG/ conjugate compound in the sample.

Described IgG/ conjugate compound continues along upwards migration of strip, at first arrives the detection zone of measuring strip (described detection zone contain can with the HIV antigen that anti-HIV antibody is combined) and is fixed on the antigen line of detection zone and the erythroid colo(u)r streak that has occurs; This Indicator Reaction or positive findings; The intensity of line not with sample in the antibody amount that exists proportional.

Lacking in the detection zone has the colo(u)r streak indicateing arm originally not contain anti-HIV antibody; IgG/ conjugate compound continues upwards to move until it arrives the check plot along measuring strip; The check plot is contained to be fixed in and is measured the goat anti-human F of strip in reaching the standard grade (ab ') ₂The IgG fragment antibody.

Remaining IgG/ conjugate compound and immobilization F (ab ') ₂The fragment combination, and the erythroid colo(u)r streak that has appears; The appearance of control line is the evidence that detects normal operation and contain IgG; No matter sample is reactive or non-reacted to the antibody of HIV-1 or HIV-2, and the blush control line all will occur in the check plot between all effective detection periods carrying out.

Sample continues migration and enters final absorption pad through the check plot, and described absorption pad helps pulling IgG/ conjugate compound by strip and removes any background color.

Introduced in the dilution sample 15 minutes but after being no more than 60 minutes, the result is made an explanation will measuring strip; After detecting beginning early than 15 minutes or be later than 60 minutes and read testing result may be to the result who makes mistake; Remaining dilute sample can be used for other detection, tests such as authenticity.

As mentioned above, embodiments of the invention are explained, but as long as not breaking away from fact inventive point of the present invention and effect can have a lot of distortion, this will be readily apparent to persons skilled in the art.Therefore, such variation also all is included within protection scope of the present invention.

Claims (1)

1. an oralmucosal transudate fluid method for quick is characterized in that containing following steps;

At first, approximately take the biased sample damping fluid 3 times by inverted bottle lightly, with 0.15M sodium chloride, 0.l% Triton X-100,15% hot deactivation chicken serum, 30 μ g/ml affinities element, 0.2% Tween 80,0.2% Tetronic T-904 and 0.0285% active component of pH7.2+/-0.2 potassium phosphate buffering, ProClin 950;

Take off bottle cap and damping fluid is filled to the dropper to groove from bottle;

The oralmucosal transudate fluid of dropper is dispensed in the test tube; Then a clean swab that is provided by bag is provided;

Catch the swab handle, avoid touching the fabric end of swab;

Subsequently, apply suitable pressure and gently with swab fabric end gums line in the wiping to and fro; One jiao at mouth begins softly and lentamente wiping until arrive another angle of mouth, then returns starting point along the wiping of upper gums line, and the time is 5-6 second;

Then rotate swab with gums under the opposite side wiping of using swab;

The opposite side that uses swab is gums line under the wiping back and forth softly and lentamente;

One jiao at mouth begins, and finishes at another angle of mouth, then returns the place of setting out along the wiping of lower gums line, and the time is 5-6 second;

To wipe away immediately to give and be put in the pipe that fills sample buffer;

Hold the swab handle, swab is back and forth inserted in the sample buffer pipe 6-8 time, be close to the both sides of pipe friction swab; From pipe, take out swab;

Oralmucosal transudate fluid has been ready for detection now;

Transferase 12 00 this oralmucosal transudate fluid of μ l is to empty test tube;

To use following mensuration strip to detect this sample;

If oralmucosal transudate fluid carried out the detection that detects strip more than one, a plurality of 200 μ l samples can be transferred to single empty test tube;

Open the tank of measuring strip is housed; Taking out one from tank measures strip and again builds at once tank;

Keep away the film surface that manual-free touches strip central authorities;

Place the pipe that contains 200 μ l dilution sample with measuring strip, make under the arrow points of measuring on the strip;

Set the timer to 20 minutes, or the time that strip adds to sample will be measured in record;

Read later on testing result in 20 minutes;

Then strip, pipe and swab are abandoned in bio-hazard product waste canister.