An extractable nucleic acid-protection liquid for a protozoa

Abstract

The application discloses an extractable nucleic acid trichomonad activity protection solution and relates to the technical field of biotechnology, which comprises two component reagents A and B, the reagent A comprises 0.8-1.0% NaCl, 0.1-5% Tris-Cl buffer, 0.1-1.5% SDS, 1.5-5% glucose, 5-15% serum, 0.5-5% betaine, and the balance is sterile deionized water, and the reagent B is a proteinase K solution. The application can not only dilute secretion, preserve trichomonas and culture trichomonas, but also be used for extracting trichomonad nucleic acid in PCR molecular experiment, so that the application can realize double application of trichomonad detection. The application not only helps to solve the health problems of women and improve fertility protection, but also is the first domestic invention to provide an example for new technology transformation service to the society.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a protective solution for the activity of Trichomonas vaginalis from which nucleic acids can be extracted. Background Technology

[0002] Trichomoniasis is an inflammatory disease of the female reproductive tract caused by Trichomonas vaginalis. It mainly infects the squamous epithelium of the urogenital tract. Infection can also lead to cervicitis, urethritis, premature birth, miscarriage, premature rupture of membranes, low birth weight and other adverse pregnancy symptoms. It seriously affects women's urinary and reproductive systems and makes them more susceptible to inflammation during pregnancy, which is a major factor leading to adverse pregnancy and miscarriage.

[0003] Clinical diagnosis of Trichomonas vaginalis infection in women's lower genital tract relies on morphological and chemical testing. However, morphological and immunological diagnostic methods are prone to false negatives, with a sensitivity and specificity of only about 50-60%. Molecular diagnostic methods are gradually being promoted and applied in the field of pathogen detection. Currently, there are no commercially available nucleic acid detection reagents for Trichomonas vaginalis in China. Therefore, the development of molecular biological diagnostic reagents for Trichomonas vaginalis not only helps address women's health issues and improve fertility protection but also serves as a pioneering example in China for the commercialization of new technologies. (This approach addresses women's health issues, improves fertility protection, and pioneers the commercialization of new technologies in China). Currently, there is a certain gap between molecular biological techniques and traditional methods, with subjective biases and low accuracy leading to numerous misdiagnoses. Misdiagnosis results in low cure rates and high recurrence rates. Therefore, sequencing is needed to establish a research foundation and serve as a gold standard for molecular biological and morphological comparisons. The specific pathogenic species and proportions in the lower genital tract flora and mixed infections such as mycoplasma, chlamydia, and viruses in women with genital tract infections are still unclear. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies by providing a Trichomonas vaginalis activity preservation solution for nucleic acid extraction. Its advantages include: it can both dilute and preserve cultured Trichomonas vaginalis and extract the Trichomonas vaginalis genome for molecular experiments such as PCR.

[0005] To achieve the above objectives, the technical solution adopted in this application is as follows: a protective solution for the activity of Trichomonas vaginalis that can extract nucleic acids, comprising two components, reagent A and reagent B. Reagent A comprises 0.8%-1.0% NaCl, 0.1%-5% Tris-Cl buffer, 0.1%-1.5% SDS, 1.5%-5% glucose, 5%-15% serum, 0.5%-5% betaine, with the remainder being sterile deionized water. Reagent B is a proteinase K solution.

[0006] Preferably, the pH of the Tris-Cl buffer in reagent A is 7.5-8.5 and the concentration is 10-100 mmol / L.

[0007] Preferably, the proportions of the components in reagent A are as follows: NaCl 0.9%, Tris-Cl buffer 0.6%, SDS 0.5%, glucose 2%, serum 10%, betaine 1%, and the remainder is sterile deionized water.

[0008] Preferably, the concentration of proteinase K in reagent B is 10-20 mg / mL.

[0009] Preferably, the serum is bovine serum albumin.

[0010] Preferably, the serum is fetal bovine serum.

[0011] Preferably, the method for preparing the preservation solution includes the following steps: S1: Dissolve NaCl, Tris-Cl, SDS, glucose, and betaine in sterile deionized water in sequence according to the ratio, and stir to dissolve; S2: Add serum, mix well, adjust pH, and prepare reagent A; S3: Prepare reagent B from proteinase K; S4: Mix 0.1 mL of reagent B with 10 mL of reagent A and store at -20℃.

[0012] Compared with the prior art, the beneficial effects of this application are as follows: (1) This application can both dilute and preserve the culture of Trichomonas vaginalis and extract the genome of Trichomonas vaginalis for PCR amplification, sequencing and other detection, realizing the dual application of Trichomonas vaginalis detection. It can solve women's health problems, improve fertility protection and create a new quality technology in China to serve the society. Detailed Implementation

[0013] The present application will be further described below with reference to specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0014] It should be noted that the terms "first," "second," etc., in the specification and claims of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence.

[0015] This application discloses a protective solution for the activity of Trichomonas vaginalis that can be used to extract nucleic acids, comprising two components, reagent A and reagent B. Reagent A comprises 0.8%-1.0% NaCl, 0.1%-5% Tris-Cl buffer, 0.1%-1.5% SDS, 1.5%-5% glucose, 5%-15% serum, 0.5-5% betaine, and the balance being sterile deionized water. Reagent B is a proteinase K solution. The pH value of the Tris-Cl buffer in reagent A is 7.5-8.5, and the concentration is 10-100 mmol / L. The proportions of the components in reagent A are as follows: 0.9% NaCl, 0.6% Tris-Cl buffer, 0.5% SDS, 2% glucose, 10% serum, 1% betaine, and the balance being sterile deionized water. The concentration of proteinase K in reagent B is 10-20 mg / mL. The serum is bovine serum albumin or fetal bovine serum.

[0016] At room temperature, Trichomonas vaginalis can survive stably in reagent A, and may proliferate at 37 degrees Celsius. Reagent B can enzymatically digest the proteins in Trichomonas vaginalis at a temperature of 60-70 degrees Celsius, allowing the genome to be released into the preservation solution.

[0017] Among them, NaCl is used to maintain cell osmotic pressure; Tris-Cl buffer is used to adjust pH; SDS is a surfactant that inhibits bacterial growth; glucose is used to adjust osmotic pressure and provide basic energy for Trichomonas vaginalis; serum is used as nutrients for Trichomonas vaginalis to grow; and low temperature conditions inhibit ice crystal formation and maintain cell morphology. Highly efficient lysis and inactivation: SDS, as a strong anionic detergent, effectively lyses cell and nuclear membranes and denatures proteins; proteinase K rapidly digests the nucleases released during lysis, protecting the integrity of nucleic acids. The synergistic effect of these two components results in excellent lysis performance on various tissues, cells, and whole blood samples.

[0018] Excellent low-temperature stability: By storing reagent A, which contains SDS, salt, and sugar, separately from proteinase K reagent B, the long-term instability of proteinase K under high-concentration denaturant conditions is avoided. Glucose and betaine, as natural cryoprotectants and co-diffusion agents, effectively protect the physicochemical stability of the reagents under -20°C freezing conditions, preventing SDS crystallization or enzyme activity reduction caused by repeated freeze-thaw cycles.

[0019] Excellent nucleic acid preservation: The high-salt environment (NaCl) and the presence of serum albumin can effectively inhibit the degradation of nucleic acids and reduce the non-specific adsorption of nucleic acids on the tube wall, making it especially suitable for long-distance transportation and long-term preservation of micro-samples.

[0020] Example 1 The formula for 100 mL of reagent A is: NaCl 0.9 g; 5 mL of Tris-Cl buffer (50 mmol / L, pH 8.0); SDS 0.5 g; 2 g of glucose; 10 mL of fetal bovine serum; 1 g of betaine; Add sterile deionized water to a final volume of 100 mL.

[0021] Reagent B is: 20 mg / mL proteinase K solution.

[0022] Preparation method: S1: Dissolve NaCl, Tris-Cl, SDS, glucose, and betaine in sterile deionized water in sequence according to the ratio, and stir to dissolve; S2: Add serum, mix well, adjust pH, and prepare reagent A; S3: Prepare reagent B from proteinase K; S4: Mix 0.1 mL of reagent B with 10 mL of reagent A and store at -20℃.

[0023] Example 2 The formula for 100 mL of reagent A is: 1g of NaCl; 10 mL of Tris-Cl buffer (20 mmol / L, pH 7.5); SDS 0.5 g; 2 g of glucose; 5 mL of bovine serum albumin; 1 g of betaine; Add sterile deionized water to a final volume of 100 mL.

[0024] Reagent B is: 10 mg / mL proteinase K solution.

[0025] Preparation method: S1: Dissolve NaCl, Tris-Cl, SDS, glucose, and betaine in sterile deionized water in sequence according to the ratio, and stir to dissolve; S2: Add serum, mix well, adjust pH, and prepare reagent A; S3: Prepare reagent B from proteinase K; S4: Mix 0.1 mL of reagent B with 10 mL of reagent A and store at -20℃.

[0026] Nucleic acid extraction and detection methods: 1. Sample collection: Collect cervical specimens using a disposable cervical brush: Pinch the brush handle with your thumb and forefinger, insert the tip of the cervical brush into the cervical opening and rotate it in one direction 3-5 times, then pull out the cervical brush, remove the brush head and place it in a vial containing the protective solution of this application. 2. Nucleic acid extraction: Pretreatment: Centrifuge the cervical preservation solution at 1000 rpm for 10 minutes, discard the supernatant, and retain approximately 1 ml of precipitate.

[0027] 2.1 Add 300 µL of cervical cells to a 1.5 mL centrifuge tube.

[0028] 2.2 Add 180 µL of Lysis to the centrifuge tube, vortex for 5 seconds to mix thoroughly, then incubate the centrifuge tube in a 25°C constant temperature mixer for 4 minutes, or incubate the centrifuge tube in a 25°C water bath for 4 minutes, vortexing for 10 seconds every minute during the incubation period.

[0029] 2.3 After briefly centrifuging the centrifuge tube, remove it and let it stand for 5 minutes to reach room temperature. Add 10 µL of thoroughly mixed LMJbeads H and 320 µL of isopropanol (if additional agents are needed, 10 µL of nucleic acid precipitation aid can be added at this time), vortex to mix for 5 seconds, then place the centrifuge tube on a 25°C constant temperature mixer and vortex to mix for 5 minutes, or continuously invert the centrifuge tube to mix for 10 minutes.

[0030] 2.4 Place the centrifuge tube on the magnetic rack and let it stand for 1 minute. Once the magnetic beads are completely adsorbed onto the side wall of the centrifuge tube, discard the solution completely (keep the centrifuge tube fixed on the magnetic rack).

[0031] 2.5 Remove the centrifuge tube from the magnetic rack, add 600 µL of Washing A (check that ethanol has been added before use), vortex for 1 minute or vortex for 5 seconds, then place it on a 25°C constant-temperature mixer and mix for 30 seconds. Afterward, place the centrifuge tube on the magnetic rack and let it stand for 30 seconds until the magnetic beads are completely adhered to the side wall of the centrifuge tube. Then gently invert the magnetic rack to wash away any impurities from the centrifuge tube cap and discard the solution completely (keeping the centrifuge tube fixed on the magnetic rack). Note: If subsequent tests require high sample purity, step 5 can be repeated once.

[0032] 2.6 Remove the centrifuge tube from the magnetic rack, add 600 µL of Washing B (check that ethanol has been added before use), vortex for 1 minute or vortex for 5 seconds, then place it on a 25°C constant-temperature mixer and mix for 30 seconds. Afterward, place the centrifuge tube on the magnetic rack and let it stand for 30 seconds until the magnetic beads are completely adhered to the side wall of the centrifuge tube. Gently invert the magnetic rack to wash away any impurities from the centrifuge tube cap, then discard the solution completely (keeping the centrifuge tube fixed on the magnetic rack). Note: If subsequent tests require high sample purity, step 6 can be repeated once.

[0033] 2.7 Keep the centrifuge tubes fixed on the magnetic rack. Use a pipette to further remove the solution from the bottom and cap of the centrifuge tubes, then let them stand at room temperature for 5-10 minutes to allow the ethanol to evaporate completely. Note: If there are droplets on the side of the centrifuge tubes, add 600 µL of anhydrous ethanol to the tubes. After capping, invert the centrifuge tubes (keeping them fixed on the magnetic rack), and then completely discard the anhydrous ethanol.

[0034] 2.8 Remove the centrifuge tube from the magnetic rack, add 50 µL of Elution, vortex to completely suspend the magnetic beads in the eluent, and then place it on a 65°C constant temperature mixer to elute for 3 minutes, or place the centrifuge tube in a 65°C water bath to incubate for 3 minutes, vortexing for 10 seconds every minute during this period.

[0035] 2.9 Place the centrifuge tube on the magnetic rack and let it stand for 2 minutes until the magnetic beads are completely adsorbed onto the side wall of the centrifuge tube. Then use a pipette to transfer the eluent to a new centrifuge tube for later use or store at -20°C.

[0036] 3. Nucleic acid testing: 3.1 Reagent preparation: 3.1.1 Prepare the reaction system as shown in Table 1. The amounts of negative control and positive control need to be prepared, and possible losses during dispensing should be considered.

[0037] Table 1: After the above-prepared mixture is thoroughly mixed, it is instantly centrifuged and dispensed into 10 μl per well in an eight-well tube or a 96-well plate.

[0038] 3.1.2 Add 10 μl of sample or negative or positive control to the eight-tube strip or 96-well plate containing the reaction system from the previous step (the final system is 20 μl).

[0039] 3.1.3 Tightly close the tube cap or seal with film. Centrifuge briefly.

[0040] 3.2 Fluorescent RT-PCR amplification (sample amplification area): 3.2.1 Place the reaction tube into a fluorescent PCR amplification instrument for amplification and detection.

[0041] 3.2.2 As shown in Table 2, set the loop parameters; Table 2: As shown in Table 3, fluorescence selection FAM Table 3: 4. Result determination: 4.1 Quality Control: 1. Negative control: No typical S-type amplification curve or no CT; 2. Positive control: showing a typical S-shaped amplification curve and a CT value ≤32; 3. The experiment is valid if conditions 1 and 2 are met simultaneously; otherwise, it is invalid.

[0042] 4.2 Result Interpretation: a) If the sample shows S-type amplification in the detection channel and the Ct value is ≤37, the determination shall be made according to the following table based on the fluorescence channel corresponding to the detected pathogen; b) If the sample shows S-type amplification in the detection channel and 37 < Ct value ≤ 40, it is considered an uncertain sample and needs to be re-extracted for nucleic acid testing; if the retested sample still shows S-type amplification in the detection channel and Ct value ≤ 40, it should be judged according to the following table based on the fluorescence channel corresponding to the pathogen being detected; otherwise, it is considered negative. c) If the sample does not show a clear S-shaped amplification curve in the detection channel, it is judged as negative.

[0043] The basic principles, main features, and advantages of this application have been described above. Those skilled in the art should understand that this application is not limited to the above embodiments. The embodiments and descriptions in the specification are merely the principles of this application. Various changes and modifications can be made to this application without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claims. The scope of protection claimed by this application is defined by the appended claims and their equivalents.

Claims

1. A protective solution for the activity of Trichomonas vaginalis from which nucleic acids can be extracted, characterized in that, The reagent comprises two components, reagent A and reagent B. Reagent A includes 0.8%-1.0% NaCl, 0.1%-5% Tris-Cl buffer, 0.1%-1.5% SDS, 1.5%-5% glucose, 5%-15% serum, 0.5%-5% betaine, and the balance being sterile deionized water. Reagent B is a proteinase K solution.

2. The trichomonas activity protective solution from which nucleic acids can be extracted as described in claim 1, characterized in that, The pH of the Tris-Cl buffer in reagent A is 7.5–8.5, and the concentration is 10–100 mmol / L.

3. The trichomonas activity protective solution from which nucleic acids can be extracted as described in claim 1, characterized in that, The proportions of each component in reagent A are as follows: NaCl 0.9%, Tris-Cl buffer 0.6%, SDS 0.5%, glucose 2%, serum 10%, betaine 1%, and the remainder is sterile deionized water.

4. The trichomonas activity protective solution from which nucleic acids can be extracted as described in claim 1, characterized in that, The concentration of proteinase K in reagent B is 10-20 mg / mL.

5. The trichomonas activity protective solution from which nucleic acids can be extracted as described in claim 1, characterized in that, The serum in question is bovine serum albumin.

6. The trichomonas activity protective solution from which nucleic acids can be extracted as described in claim 1, characterized in that, The serum in question is fetal bovine serum.

7. A method for preparing a protective solution for extracting nucleic acids from Trichomonas vaginalis as described in any one of claims 1-6, characterized in that, Includes the following steps: S1: Dissolve NaCl, Tris-Cl, SDS, glucose, and betaine in sterile deionized water in sequence according to the ratio, and stir to dissolve; S2: Add serum, mix well, adjust pH, and prepare reagent A; S3: Prepare reagent B from proteinase K; S4: Mix 0.1 mL of reagent B with 10 mL of reagent A and store at -20℃.

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