WO2022088511A1

WO2022088511A1 - Reagent for lysing virus sample

Info

Publication number: WO2022088511A1
Application number: PCT/CN2021/071117
Authority: WO
Inventors: 张敏; 杨蓉; 张权峰; 宋学文; 叶凯璐; 周康
Original assignee: 杭州微策生物技术股份有限公司
Priority date: 2020-10-29
Filing date: 2021-01-11
Publication date: 2022-05-05
Also published as: CN113281503B; CN113281503A

Abstract

The present invention provides a reagent for lysing a virus sample. The reagent contains nano titanium dioxide, such that a lysing effect on a component to be tested in the sample can be obviously improved, thereby greatly improving the virus test sensitivity, especially the lysing of SARS-CoV-2.

Description

A reagent for splitting virus samples

technical field

The invention belongs to the field of disease diagnosis, and relates to a reagent for splitting virus samples, in particular to a reagent for splitting novel coronavirus samples.

Background technique

Immunochromatographic Assay (ICA) is an analytical method developed at the end of the 20th century that combines immunological and chromatographic techniques. , food safety and other important areas. The general immunochromatographic detection test paper includes a support layer, a sample pad, a marker pad, a reaction area, and an absorbent paper. The sample pad, the marker pad, the reaction area, and the absorbent paper are overlapped on the support layer in turn, and the reaction area includes the quality control line (C line ) and the detection line (T line), when a certain amount of the sample to be tested is applied to the sample pad, due to capillary action, after being evenly dispersed through the sample pad, after the marking of the marking pad, in the reaction area, it is respectively connected with the C line and the C line of the reaction area. The antigen or antibody on the T line reacts, and the remaining liquid after the reaction is absorbed by the absorbent paper. When the C line signal value tends to be stable, the result of the reaction with the antigen or antibody shows the strength of the T line signal value.

When conducting virus detection, such as new coronavirus detection, it is necessary to lyse the collected samples on the nasal swab or throat swab to obtain the sample to be tested, and then apply it to the fluorescent immunochromatography test paper for reaction, lysis The lysis effect of the solution on the component to be tested (virus) in the sample is directly reflected in the strength of the T-line signal value, so the formulation of the lysis solution is particularly important. Due to the poor lysis effect of the existing virus lysate, the detection sensitivity of the virus is affected, especially when the virus content is particularly low, it is very easy to cause false detection and missed detection.

SUMMARY OF THE INVENTION

Aiming at the deficiencies of the prior art, the present invention provides a new lysis solution formulation, which contains nano-titanium dioxide, which can significantly improve the lysis effect of the components to be tested in the sample, thereby greatly improving the virus detection sensitivity, especially for novel The lysis of the coronavirus.

The present invention provides a virus-splitting reagent, comprising titanium dioxide, buffer, surfactant and preservative.

Further, the titanium dioxide is nano titanium dioxide.

Further, the content of the nano titanium dioxide is 0.01%-0.1%.

Further, the buffer solution includes sodium chloride, phosphate and sodium caseinate.

Further, the buffer solution includes sodium chloride 0.15-0.45 mol/L, phosphate 1.15-2.9 g/L, and sodium caseinate content of 0.5%.

Further, the surfactant is Triton X-100.

Further, the preservative is Proclin 300.

On the other hand, the present invention also provides a fluorescence immunochromatography detection kit, which is characterized in that it includes the virus splitting reagent according to any one of claims 1 to 7, and an immune layer for detecting virus antigens Analysis test paper.

In one aspect, the present invention provides the use of nano-titanium dioxide for preparing a reagent for splitting virus samples.

Further, the virus sample is a novel coronavirus sample.

The beneficial effect of the present invention is that the virus can be better split through the splitting of the present invention, and more antigens or antigenic sites can be exposed, thereby improving the detection sensitivity. When the amount of virus in the sample is particularly low, it is hoped that a positive result can be obtained, and more antigen fragments or virus fragments are expected to be obtained. Using the lysis solution provided by the present invention to lyse the sample can significantly increase the virus antigen concentration in the sample. Therefore, the immunofluorescence test strip is used to increase the minimum threshold of detection and prevent missed detection.

Description of drawings

Fig. 1 is the T-line mean value level comparison diagram obtained for lysate formulations 1-6 in Example 8

Detailed ways

The present invention will be described in further detail below with reference to the examples. It should be noted that the following examples are intended to facilitate the understanding of the present invention, but do not have any limiting effect on it. The reagents not specified in this example are all known products, obtained by purchasing commercially available products.

The positive samples in the following examples are virus cultures with a virus titer of 1.51×10 ⁶ TCID ₅₀ /mL, diluted to 15,000 times, 10,000 times, 5,000 times, and 1,000 times, respectively, as samples to be tested. For the lysis effect of the lysis solution formula, after the dilution ratio in the existing lysis solution system is greater than 10,000 times, the T line signal value is similar to the background value, and it is judged as negative.

Explanation of terms: The background value refers to the signal value detected when no sample is injected.

Example 1: Fluorescence immunochromatography detection test paper

The preparation method of the fluorescence immunochromatography detection test paper used in the present invention is as follows, and the fluorescence immunochromatography detection test paper used in the present invention is the test paper produced in the same batch:

Step 1: Fluorescent microspheres are labeled with monoclonal antibody against NP protein of novel coronavirus.

Mix the fluorescent microspheres with the monoclonal antibody of the novel coronavirus NP protein to obtain the antibody marker, which is reconstituted with the reconstituting solution.

Step 2: Sample Pad Processing

(1) Configure the sample pad treatment solution: 0.01M pH 7.4 phosphate buffered saline solution, fixed mass fraction of BSA, surfactant, PVP K30.

(2) Disperse the treatment solution evenly on the glass fiber, dry it in a drying oven at 37°C overnight, take it out and place it in an aluminum foil bag and seal it for later use;

Step 3: Bonding Pad Treatment

(1) Configure the binding pad treatment solution: 0.02M pH 7.4 phosphate buffer solution, fixed mass fraction of BSA, sucrose, and preservatives.

Step 4: Dilute and spray the antibody marker

(1) The novel coronavirus NP protein monoclonal antibody labeled with fluorescent microspheres in step 1 was sprayed on the glass fiber treated in step 3 at a speed of 3.0uL/cm with a gold sprayer, and placed in a drying oven at 37°C overnight. Dry, take out and place in an aluminum foil bag and seal it for later use;

Step 5: Scribing the nitrocellulose membrane

(1) Dilute the novel coronavirus NP protein monoclonal antibody to a fixed concentration with 0.015M pH 7.4PBS as a T-line solution;

(2) Dilute the goat anti-mouse polyclonal antibody to a fixed concentration with 0.015M pH 7.4PBS as a C-line solution;

(3) Scribe the film at a speed of 1.0uL/cm with a film-scribing gold sprayer, place it in a drying oven at 37°C to dry overnight, take it out and place it in an aluminum foil bag and seal it for later use;

Step 6: Assembly Cut and Bag

(1) Cut the sample pad processed in step 2, the binding pad of the antibody marker by spraying in step 4, the nitrocellulose membrane processed in step 5, and the absorbent paper to an appropriate width, and stick them on the bottom plate in turn. 1-2mm between them.

(2) Cut the glued large card into 4mm wide test cards with a chopping machine, and put them into the detection device.

(3) Put it in an aluminum foil bag and keep it sealed.

Embodiment 2 Lysis solution formula 1

Lysis solution 1 contains the following components by mass fraction: 0.15mol/L NaCl, 1.15g Na2HPO4, 0.12g KH2PO4, 0.5% Casein Na, 1% Triton X-100, 0.05% proclin300. The pH of the lysate was 8.0.

Use this formula lysate to treat the new coronavirus-positive cultures with concentrations of 1/15000, 1/10000, 1/5000, and 1/1000, respectively, to obtain the sample to be tested, and apply 50 microliters of the sample to the fluorescence immunochromatography test paper. , 15 minutes later, the T-line signal was detected in the fluorescence meter, the average value was recorded and calculated, and the test was repeated 5 times. The test strip used was the test strip prepared in Example 1.

Table 1 Test results of formula 1

As can be seen from the above table, when the new coronavirus positive culture concentration is 1/15000 of the original concentration, although the T line has a signal, it is almost similar to the line background signal, that is, the T line signal when the culture concentration is When the result is negative. On the other hand, when the culture concentration was 1/10000, 1/5000 and 1/5000 of the original concentration, it was judged as a positive result.

Embodiment 3 Lysis solution formula 2

Lysis solution 2 contains the following components in mass fractions: 0.15mol/L NaCl, 1.15g Na2HPO4, 0.5% Casein Na, 1% Triton X-100, 0.05% proclin300. The pH of the lysate was 8.0.

Use the formula lysate to treat the new coronavirus cultures with concentrations of 1/15000, 1/10000, 1/5000, and 1/1000, respectively, to obtain the sample to be tested, and apply the sample on the fluorescence immunochromatography detection test paper. After 15 minutes T-line signal was detected, recorded and averaged.

Table 2 formula 2 test results

As can be seen from the above table, although the lysing liquid has changed, for the same dilution, when the culture concentration is 1/15000 of the original concentration, the fluorescent signal cannot be detected, and the result is still negative.

Embodiment 4 Lysis solution formula 3

Lysis solution 3 contains the following components in mass fractions: 0.15mol/L NaCl, 1.15g Na2HPO4, 0.12g KH2PO4, 0.5% Casein Na, 1% Triton X-100, 0.05% proclin300, 0.01% Nano Titanium Dioxide. The pH of the lysate was 8.0.

The detection result of table 3 formula 3

It can be seen from the above table that the addition of nano-titanium dioxide can increase the minimum detection threshold. When the culture concentration is 1/15000 of the original concentration, the presence of viral antigens can still be detected, which shows that this substance has improved detection sensitivity.

Embodiment 5 Lysis solution formula 4

Lysis solution 4 contains the following components in mass fractions: 0.3mol/L NaCL, 2.9g Na2HPO4, 0.5% Casein Na, 1% Triton X-100, 0.05% proclin300, 0.02% nano-titanium dioxide. The pH of the lysate was 8.0.

The detection result of table 4 formula 4

Embodiment 6 Lysis solution formula 5

Lysis solution 5 contains the following components in mass fractions: 0.3mol/L NaCL, 2.9g Na2HPO4, 0.5% Casein Na, 1% Triton X-100, 0.05% proclin300, 0.03% nano-titanium dioxide. The pH of the lysate was 8.0.

The detection result of table 5 formula 5

Embodiment 7 Lysis solution formula 6

Lysis solution 6 contains the following components by mass fraction: 0.3mol/L NaCL, 2.9g Na2HPO4, 0.5% Casein Na, 1% Triton X-100, 0.05% proclin300, 0.05% nano-titanium dioxide. The pH of the lysate was 8.0.

The detection result of table 6 formula 6

Example 8

According to the results of Examples 2-7, the average value of the T-line signal was compared, and the statistics are shown in Table 7 and FIG. 1 . It can be clearly seen that formula 1 and formula 2, and formula 3 to formula 6, the difference in the average value of the T-line signal, the addition of nano-titanium dioxide significantly improves the detection sensitivity.

Table 7 Summary table of T-line signal average values for recipes 1-6

培养物浓度/配方Culture concentration/recipe	配方1Recipe 1	配方2Recipe 2	配方3Recipe 3	配方4Recipe 4	配方5Recipe 5	配方6Recipe 6
00	3333	3232	2828	1919	24twenty four	2727
1/150001/15000	3131	3434	154154	179179	169169	163163
1/100001/10000	315315	321321	451451	469469	460460	432432
1/50001/5000	602602	640640	874874	899899	881881	825825
1/10001/1000	27092709	27392739	42144214	43974397	42574257	40754075

All patents and publications mentioned in the specification of the present invention indicate that they are disclosed technology in the art and can be used in the present invention. All patents and publications cited herein are also incorporated by reference as if each publication was specifically and individually incorporated by reference. The inventions described herein may be practiced in the absence of any element or elements, limitation or limitations, such limitation not specifically stated herein. For example, the terms "comprising", "consisting essentially of" and "consisting of" in each instance herein may be replaced by either of the remaining two terms. The so-called "one" here only means "one", and it does not exclude that only one is included, and it may also mean that two or more are included. The terms and expressions used herein are by way of description, not limitation, and there is no intention here to indicate that these terms and interpretations described in this book exclude any equivalent features, but it is understood that the present invention and the rights make any suitable changes or modifications to the extent required. It can be understood that the embodiments described in the present invention are all preferred embodiments and features, and any person skilled in the art can make some changes and changes according to the essence of the description of the present invention, and these changes and changes are also considered to belong to The scope of the invention is limited by the independent and dependent claims.

Claims

A reagent for splitting virus, characterized in that it comprises titanium dioxide, buffer solution, surfactant and preservative.
The reagent according to claim 1, wherein the titanium dioxide is nano titanium dioxide.
The reagent according to claim 2, wherein the content of the nano titanium dioxide is 0.01%-0.1%.
The reagent according to any one of claims 3, wherein the buffer solution comprises sodium chloride, phosphate and sodium caseinate.
The reagent according to claim 4, wherein the buffer solution comprises sodium chloride 0.15-0.45 mol/L, phosphate 1.15-2.9 g/L, and sodium caseinate content of 0.5%.
The reagent of claim 5, wherein the surfactant is Triton X-100.
The reagent of claim 6, wherein the preservative is Proclin 300.
A fluorescence immunochromatography detection kit, characterized in that it comprises the virus splitting reagent according to any one of claims 1 to 7, and an immunochromatographic test paper for detecting virus antigens.
Use of nano-titanium dioxide for preparing reagents for splitting virus samples.
The use according to claim 9, wherein the virus sample is a novel coronavirus sample.