A new use of PT-1 or its active ingredients in pathogenic microorganism detection and a method for processing pathogenic microorganism detection samples

Abstract

The present invention discloses a new use of PT-1 and its effective components in pathogenic microorganism detection and a method for processing detection samples, and relates to the field of biological detection. Among them, the sample processing method for pathogenic microorganism antigen detection is: put the sample into an antigen extracting solution for 15 seconds, inactivate the sample and complete the extraction; 1L of antigen extracting solution contains 1-100ml of PT-1 with a concentration of 0.5%-5%; the sample processing method for pathogenic microorganism extraction-free nucleic acid detection is: put the sample into a lysate for 15 seconds, inactivate the sample and complete the extraction; 1L of lysate contains 1-100ml of PT-1 with a concentration of 0.5%-3%; the present invention finds that PT-1 can inactivate pathogenic microorganisms but does not affect the detection results of pathogenic microorganisms. The sample processing method realizes that the sensitivity and accuracy of pathogenic microorganism detection are not affected while inactivating the sample.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, in particular to a new use of PT-1 or its effective components in pathogenic microorganism detection and a method for processing pathogenic microorganism detection samples. Background Art

[0002] The common steps for rapid detection of pathogenic microorganisms using immunochromatographic methods represented by colloidal gold method are: sample collection, sample processing, sample testing and infectious waste treatment. The processing of rapid antigen test samples usually uses antigen extraction solution to extract pathogenic microorganisms in the sample.

[0003] Common disinfectants inactivate pathogenic microorganisms by causing lethal structural changes in the proteins, enzymes, and nucleic acids of pathogenic microorganisms. In the process of rapid detection of pathogenic microorganisms using immunochromatographic methods represented by the colloidal gold method, in order to ensure the sensitivity and accuracy of the detection, it is usually required that the conformation of pathogen-related antigens cannot be changed, and the activity of proteins involved in the reaction is not affected. Therefore, commonly used antigen extracts only have the function of extracting antigens, but do not have the function of inactivating pathogenic microorganisms. As a result, there is still a risk of contamination and infection in the subsequent sample inspection and waste disposal process.

[0004] The same is true for extraction-free nucleic acid testing. The common steps for extraction-free nucleic acid testing of pathogenic microorganisms are: sample collection, sample processing, sample testing, and infectious waste treatment. The processing of samples for extraction-free nucleic acid testing usually uses nucleic acid lysis solution to treat pathogenic microorganisms in the sample.

[0005] In the process of rapid detection of pathogenic microorganisms by avoiding the extraction of nucleic acids, in order to ensure the sensitivity and accuracy of the detection, it is usually required that the nucleic acids related to the pathogenic microorganisms cannot be changed, and the activities of proteins and enzymes involved in the reaction are not affected. Therefore, the commonly used nucleic acid lysis only has the effect of nucleic acid lysis, but does not have the effect of inactivating pathogenic microorganisms. Therefore, there is still a risk of contamination and infection in the subsequent sample inspection and waste disposal process.

[0006] PT-1 is a product and material developed from the extract of Rutaceae fruit / pulp / seed, which has been used in Nashild disinfectant, but it is only known to have the effect of inactivating pathogenic microorganisms.

[0007] The prior art has not yet found a sample processing method that can inactivate pathogenic microorganisms without changing the detection results of pathogenic microorganisms. The present invention solves this problem by discovering new uses of PT-1 and a sample processing method. Summary of the invention

[0008] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a new use of PT-1 or its active ingredients in pathogenic microorganism detection and a method for processing samples for pathogenic microorganism detection. The present invention finds a sample processing method in which PT-1 can inactivate pathogenic microorganisms but does not change the detection results of pathogenic microorganisms, thereby achieving inactivation of samples without affecting the sensitivity and accuracy of pathogenic microorganism detection.

[0009] In order to achieve the above object, the present invention adopts the following technical solution:

[0010] A new use of PT-1 or its active ingredients in pathogenic microorganism detection, which is used for processing nucleic acid samples that inactivate pathogenic microorganisms but do not change the antigenic structure of pathogenic microorganisms.

[0011] The aforementioned new use of PT-1 or its active ingredients in pathogenic microorganism detection, PT-1 is a mixture of citron fruit extract CAS#92346-89-9 and citrus pulp CAS#68514-76-1.

[0012] The aforementioned new use of PT-1 or its active ingredients in the detection of pathogenic microorganisms, wherein PT-1 is derived from the fruit / pulp / seed extract of Rutaceae.

[0013] The aforementioned new use of PT-1 or its active ingredients in the detection of pathogenic microorganisms, PT-1 is derived from Nashild disinfectant.

[0014] The invention discloses a method for processing samples for pathogenic microorganism detection, including: a method for processing samples for pathogenic microorganism antigen detection and a method for processing samples for pathogenic microorganism extraction-free nucleic acid detection. The method for processing samples for pathogenic microorganism antigen detection is as follows: placing the sample in an antigen extraction solution for 1-60 seconds, inactivating the sample and completing the extraction; 1L of the antigen extraction solution contains 1-100ml of PT-1 with a concentration of 0.5%-5%; the method for processing samples for pathogenic microorganism extraction-free nucleic acid detection is as follows: placing the sample in a lysis solution for 1-60 seconds, inactivating the sample and completing the extraction; 1L of the lysis solution contains 1-100ml of PT-1 with a concentration of 0.5%-5%.

[0015] The aforementioned method for processing samples for pathogenic microorganism detection includes: a method for processing samples for pathogenic microorganism antigen detection and a method for processing samples for pathogenic microorganism nucleic acid detection without extraction. The method for processing samples for pathogenic microorganism antigen detection includes: adding equal volumes of Nashild disinfectant and corresponding antigen extracting solution, 150ul each, to the sample extraction tube, and mixing them evenly; placing the sample in the mixed extracting solution for 15 seconds to inactivate the sample and complete the extraction; the Nashild disinfectant contains 3% PT-1; the antigen extracting solution contains: 1.21-2.42g of tris(hydroxymethyl)aminomethane with a concentration of 0.01%-0.10%, 20g of sucrose with a concentration of 0.2%-3.00%, 5ml of Tween 20 with a concentration of 0.2%-0.8%, 10g of trisodium citrate with a concentration of 0.03%-0.10%, and 10g of casein sodium salt with a concentration of 0.2%-2.10%.

[0016] The aforementioned method for processing samples for pathogenic microorganism detection includes: a method for processing samples for pathogenic microorganism antigen detection and a method for processing samples for pathogenic microorganism non-extraction nucleic acid detection. The method for processing samples for pathogenic microorganism non-extraction nucleic acid detection is: adding equal volumes of Nashild disinfectant and corresponding nucleic acid lysis solution, 150ul each, to the sample lysis tube, and mixing them evenly; placing the sample in the mixed lysis solution for 15 seconds to inactivate the sample and complete lysis; the Nashild disinfectant contains 3% concentration of PT-1; the nucleic acid lysis solution contains: sodium chloride 0.7-1.0g / 100ml, EDTA 1-1.5g / 100ml.

[0017] The present invention is beneficial in that:

[0018] The present invention has found a sample processing method in which PT-1 can inactivate pathogenic microorganisms but will not affect the detection results of pathogenic microorganisms, thereby eliminating the risk of contamination while not affecting the results of pathogenic microorganism detection;

[0019] The present invention found that PT-1 cannot achieve the virus inactivation effect when the concentration of the antigen extract is lower than 0.5%; the commonly used concentration is 0.5-5%;

[0020] The present invention finds that when the concentration of PT-1 in the lysate is lower than 0.5%, the virus inactivation effect cannot be achieved; the commonly used concentration is 0.5%-5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the experimental result figure of Experiment 1 of the present invention;

[0022] Figure 2 It is the experimental result diagram of Experiment 3 of the present invention. DETAILED DESCRIPTION

[0023] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] A new use of PT-1 or its active ingredients in pathogenic microorganism detection, a sample processing method capable of inactivating pathogenic microorganisms but not affecting the detection results of pathogenic microorganisms.

[0025] PT-1 is a mixture of citron fruit extract CAS#92346-89-9 and citrus pulp CAS#68514-76-1; PT-1 is derived from Rutaceae fruit / pulp / seed extract; PT-1 is derived from Natshield disinfectant. All ingredients of Natshield disinfectant are recognized as safe by the FDA, listed in the United States Pharmacopeia (USP), and are also approved as a "no-rinse" disinfectant by New Zealand (EPA).

[0026] The processing method of pathogenic microorganism detection samples includes: sample processing for pathogenic microorganism antigen detection and sample processing for extraction-free nucleic acid detection.

[0027] As an embodiment, the sample processing method for pathogenic microorganism antigen detection is: placing the sample in an antigen extracting solution for 15 seconds, inactivating the sample and completing the extraction; 1L of antigen extracting solution contains 1-100ml of PT-1 with a concentration of 0.5%-5%; as a preferred embodiment, 1L of antigen extracting solution contains 1-100ml of PT-1 with a concentration of 0.5%-1.0%, and as a further preferred embodiment, 1L of antigen extracting solution contains: 1-100ml of PT-1 with a concentration of 0.5%-5%, 1.21-2.42g of tris(hydroxymethyl)aminomethane with a concentration of 0.01%-0.10%, 20g of sucrose with a concentration of 0.2%-3.00%, 5ml of Tween 20 with a concentration of 0.2%-0.8%, 10g of trisodium citrate with a concentration of 0.03%-0.10%, and 10g of casein sodium salt with a concentration of 0.2%-2.10%.

[0028] As an embodiment, the sample processing method for the extraction-free nucleic acid detection of pathogenic microorganisms is: put the sample into the lysis solution for 15 seconds, inactivate the sample and complete the extraction; 1L of lysis solution contains 1-100ml of PT-1 with a concentration of 0.5%-3%. As a preferred embodiment, 1L of lysis solution contains 1-100ml of PT-1 with a concentration of 0.5%-1%; as a further preferred embodiment, 1L of lysis solution contains 1-100ml of PT-1 with a concentration of 0.5%-1%, sodium chloride 0.7-1.0g / 100ml, EDTA 1-1.5g / 100ml, and pH 7-7.5.

[0029] As another embodiment, the sample processing method for pathogenic microorganism antigen detection is: add equal volumes of Nashild disinfectant and corresponding antigen extracting solution, 150ul each, to the sample extraction tube and mix them evenly; place the sample in the mixed extracting solution for 15 seconds to inactivate the sample and complete the extraction; the Nashild disinfectant contains 3% PT-1; the antigen extracting solution contains: 1.21-2.42g of tris(hydroxymethyl)aminomethane with a concentration of 0.01%-0.10%, 20g of sucrose with a concentration of 0.2%-3.00%, 5ml of Tween 20 with a concentration of 0.2%-0.8%, 10g of trisodium citrate with a concentration of 0.03%-0.10%, and 10g of casein sodium salt with a concentration of 0.2%-2.10%.

[0030] As another embodiment, a sample processing method for nucleic acid detection without extraction of pathogenic microorganisms is as follows: equal volumes of Nashild disinfectant and corresponding nucleic acid lysis solution, 150ul each, are added to the sample lysis tube and mixed evenly; the sample is placed in the mixed lysis solution for 15 seconds to inactivate the sample and complete lysis; the Nashild disinfectant contains 3% concentration of PT-1; the nucleic acid lysis solution contains: sodium chloride 0.7-1.0g / 100ml, EDTA 1-1.5g / 100ml.

[0031] Experiment 1: The following experiment verifies that PT-1 has excellent inactivation performance at concentrations of 0.5% and 1%:

[0032] 20uL of virus sample stock solution was mixed with different nucleic acid lysates (PBS, saline and 3 different nucleic acid lysates to be tested) so that the concentration of the mixed and post-liquid solution was 0.5% and 1.0%. Samples were taken after mixing for 15 seconds, and 2uL was taken from each sample to infect HEK293 cells. Fluorescence microscopy analysis was performed 44 hours after infection. If there is a fluorescent signal under the fluorescence microscope, it means that the virus has infected the cells and the virus has not been inactivated; if there is no fluorescent signal, it proves that the virus has been completely inactivated and has no ability to infect HEK293 cells.

[0033] Virus sample: Lentivirus (RNA virus, enveloped), virus titer 6x108 TCID50 / mL

[0034]

[0035] The results are as follows Figure 1 shown.

[0036] Result analysis: Lysis buffer A (0.5%, 1.0% PT-1), lysis buffer B (0.5%, 1.0% PT-1), and lysis buffer C (0.5%, 1.0% PT-1) can all inactivate the lentivirus samples within 15 seconds, and HEK293 cells show no fluorescent signal.

[0037] Experiment 2: Verify that PT-1 does not affect rapid nucleic acid extraction-free and direct amplification detection experiments:

[0038] Two nucleic acid lysis solutions with PT-1 concentrations of 0.5% and 1.0%, as well as a lysis solution without PT-1 (the three lysis solutions only differed in PT-1 concentration, and other components were the same) were lysed with positive and negative reference substances of influenza A virus and then tested. Each reference substance was tested 3 times with different lysis solutions, and the results of influenza nucleic acid detection reagents (PCR) were recorded to determine whether the test results were consistent.

[0039] A positive test result is indicated by "+". A negative test result is indicated by "-".

[0040] Virus samples:

[0041] Influenza A virus positive and negative references.

[0042] Among them, S1: titer is not higher than 8.0×10 3 copies / mL, the test result should be positive for influenza A virus; S2: the titer is not higher than 1.2×10 4 copies / mL, the test result should be positive for influenza A virus; S3: the titer is not higher than 1.8×10 3 copies / mL, the test result should be positive for influenza A virus.

[0043] Among them, NC1-10 is a negative reference, and the test result should be negative.

[0044] The test results are shown in Table 1 below:

[0045] Table 1

[0046]

[0047] From the above experiments, we can know that adding or not adding PT-1 can inactivate pathogenic microorganisms without affecting normal test results, and consistent results can be obtained in the verification of positive reference products and negative reference products. This shows that PT-1 does not affect rapid nucleic acid extraction-free and direct amplification, and the added test reagents can produce correct and accurate consistent results without any influence.

[0048] Experiment 3: The following experiment verifies that PT-1 has excellent inactivation performance at concentrations of 0.5% and 1%:

[0049] 20uL of virus sample stock solution was mixed with different antigen extracts (PBS, saline and 3 different antigen extracts to be tested) so that the concentration of the mixed and post-mixed liquids was 0.5% and 1.0%. Samples were taken after mixing for 15 seconds, and 2uL was taken from each sample to infect HEK293 cells. Fluorescence microscopy analysis was performed 44 hours after infection. If there is a fluorescent signal under the fluorescence microscope, it means that the virus has infected the cells and the virus has not been inactivated; if there is no fluorescent signal, it proves that the virus has been completely inactivated and has no ability to infect HEK293 cells.

[0050] Virus sample: Lentivirus (RNA virus, enveloped), virus titer 6x10 8 TCID 50 / mL

[0051]

[0052] The results are as follows Figure 2 shown.

[0053] in conclusion:

[0054] Extract A (0.5%, 1.0% PT-1), extract B (0.5%, 1.0% PT-1), and extract C (0.5%, 1.0% PT-1) can all inactivate the lentivirus samples within 15 seconds, and HEK293 cells show no fluorescent signal.

[0055] Experiment 4: Experiment to verify that PT-1 does not affect the sensitivity and accuracy of detection:

[0056] Two antigen extracts with PT-1 concentrations of 0.5% and 1.0%, and an extract without PT-1 (the three extracts differed only in PT-1 concentration, and other components were the same) were extracted with positive and negative reference materials of influenza A virus and then tested. Each reference material was tested 3 times with different extracts, and the color development of the influenza detection reagent (colloidal gold method) was observed to determine whether the test results were consistent.

[0057] A positive test result is indicated by "+++", "+" or "+" according to the color intensity. A negative result is indicated by "-".

[0058] Virus samples:

[0059] Influenza A virus positive and negative references.

[0060] Among them, S1: titer is not higher than 1.22×10 4 TCID 50 / L, the test result should be positive for influenza A virus; S2: the titer is not higher than 3.25×10 4 TCID50 / L, the test result should be positive for influenza A virus; S3: the titer is not higher than 1.25×10 3 TCID 50 / L, the test result should be positive for influenza A virus.

[0061] Among them, NC1-10 is a negative reference, and the test result should be negative.

[0062] The test results are shown in Table 2 below:

[0063] Table 2

[0064]

[0065]

[0066] From the above experiments, we can know that adding or not adding PT-1 can inactivate pathogenic microorganisms without affecting normal test results. Consistent results can be obtained in the verification of positive reference products and negative reference products, indicating that PT-1 does not affect the test of rapid antigens, and the added test reagents can produce correct and accurate consistent results without any influence.

[0067] In summary: the present invention has discovered a sample processing method in which PT-1 can inactivate pathogenic microorganisms but will not affect the detection results of pathogenic microorganisms, thereby eliminating the risk of contamination and infection while not affecting the sensitivity and accuracy of pathogenic microorganism detection, and obtaining accurate detection results;

[0068] The concentration of PT-1 in the antigen extract must be higher than 0.5% to achieve the effect of inactivating the virus;

[0069] The concentration of PT-1 in the lysate must be higher than 0.5% to achieve the effect of inactivating the virus.

[0070] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A new use of Nashild disinfectant in pathogenic microorganism detection, characterized in that: Used for inactivation of pathogenic microorganisms in samples for pathogenic microorganism detection.

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