Kit for ultrasensitive extraction of hepatitis C virus RNA using magnetic beads and its extraction method

Abstract

The present invention provides a kit for ultrasensitive extraction of hepatitis C virus RNA by magnetic bead method and its extraction method. The kit includes an extraction reagent group for ultrasensitive extraction of hepatitis C virus RNA; the extraction reagent group consists of magnetic bead lysate, washing solution I, washing solution II and eluent; the magnetic bead lysate consists of sodium citrate, sodium lauroyl sarcosinate, sodium acetate, sodium metabisulfite, guanidine thiocyanate, ethanol and magnetic bead suspension; washing solution I consists of guanidine thiocyanate, sodium chloride, Tween-20, sodium acetate and ethanol aqueous solution. The present invention obtains the optimal ratio and extraction conditions for extracting hepatitis C virus nucleic acid by rationally compounding each raw material. While ensuring that the extracted hepatitis C virus RNA has a high detection rate, good repeatability and low error after fluorescent quantitative PCR, ultrasensitive detection of hepatitis C virus RNA is achieved. The extraction method can detect 100% of virus samples with a concentration of 10 IU / mL.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, in particular to a kit for ultra-sensitive extraction of hepatitis C virus RNA using a magnetic bead method and an extraction method thereof. Background Art

[0002] The goal of hepatitis C virus (HCV) treatment is to control liver inflammation and fibrosis, prevent or reduce complications caused by HCV infection, and cut off the basis for HCV transmission. HCV-RNA is a test method for confirming active hepatitis C infection. Patients should receive antiviral treatment as long as they test positive for HCV-RNA. Highly sensitive HCV-RNA detection has become an important basis for the diagnosis and pre-treatment evaluation of chronic hepatitis C. Highly sensitive HCV-RNA detection methods can not only be used to determine HCV infection and analyze viral load during antiviral treatment, but can also be used to evaluate the response of hepatitis C patients during and after antiviral treatment, as well as monitor them during dialysis treatment. At the same time, HCV-RNA testing has clear clinical significance in preoperative examinations and other aspects.

[0003] The 2018 European Association for the Study of the Liver Guidelines and the 2019 Chinese Hepatitis C Prevention and Control Guidelines both require that the sensitivity of HCV-RNA detection in hepatitis C patients be ≤15IU / mL. Currently available magnetic bead-based HCV detection kits on the market typically utilize nanomagnetic beads to specifically identify and efficiently bind to RNA molecules, without the need for heating or boiling. They incorporate an efficient amplification system and can achieve ultra-sensitive, wide linear range, multi-genotype, repeatable, and anti-interference-capable quantitative traceability of HCV-RNA. However, due to the influence of the efficacy of the kit's extraction reagents, the commonly used commercial magnetic bead-based HCV detection kits have poor sensitivity, and the extracted viral RNA is easily degraded, resulting in a high probability of missed detection, making it impossible to guarantee the accuracy of the test. Therefore, it is very necessary to develop an ultra-sensitive HCV-RNA detection kit with a high detection rate, small repeatability error, and high sensitivity.

[0004] Patent application number CN201710492564.5 discloses a kit for extracting hepatitis C virus RNA. The kit includes a lysis buffer, wash buffer I, wash buffer II, magnetic beads, proteinase K, mineral oil, an eluent, and a biological preservative. The lysis buffer contains 40-70 g / 100 mL guanidine hydrochloride, 2-20% (v / v) Triton X-100, 0-2 g / L citric acid, and 0-15 g / L sodium citrate, with a pH of 4.0-7.0. The concentration of the magnetic beads is 25 mg / mL, the proteinase K concentration is 20 mg / mL, and the volume of the mineral oil is 70-100% (v / v).

[0005] Patent application number CN201710407670.9 discloses a kit for rapid detection of hepatitis C virus nucleic acid. The kit includes a nucleic acid extract, an RT-qPCR reaction solution, an enzyme mixture, a negative control, a positive control, and a positive reference. The RT-qPCR reaction solution includes a reaction buffer, a pair of specific primers for detecting hepatitis C virus nucleic acid, a specific probe for detecting hepatitis C virus nucleic acid, a pair of specific primers for detecting human globin, and a specific probe for detecting human globin.

[0006] However, the above-mentioned test kits all have disadvantages such as low detection rate or low sensitivity, and cannot provide effective guarantees for clinical diagnosis and disease analysis.

[0007] In view of this, it is necessary to design an improved magnetic bead method for ultrasensitive extraction of hepatitis C virus RNA and its extraction method to solve the above problems. Summary of the Invention

[0008] The present invention aims to provide a kit for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method and an extraction method thereof. By compounding various substances in a magnetic bead lysis solution and compounding the magnetic bead lysis solution with a washing solution I, a synergistic effect is exerted between the various raw materials, so that the raw material system has excellent lysis and stable binding properties to hepatitis C virus RNA, while achieving ultrasensitive detection of hepatitis C virus RNA, with a detection rate of up to 100% for hepatitis C virus RNA at 9 to 12 IU / mL.

[0009] To achieve the above-mentioned object of the invention, the present invention provides a kit for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method, comprising an extraction reagent set for ultrasensitive extraction of hepatitis C virus RNA; the extraction reagent set comprises a magnetic bead lysis solution, a washing solution I, a washing solution II, and an eluent; the magnetic bead lysis solution comprises sodium citrate sodium salt, sodium lauroyl sarcosinate, sodium acetate, sodium metabisulfite, guanidine isothiocyanate, ethanol, and a magnetic bead suspension.

[0010] As a further improvement of the present invention, in the magnetic bead lysate, the concentration of the sodium citrate sodium salt is 10-100 mM, the concentration of the sodium lauroyl sarcosinate is 10-100 mM, the concentration of the sodium acetate is 0.1-10 M, the content of the sodium metabisulfite is 0.2-2 wt %, the concentration of the guanidine isothioate is 2-6 M, and the volume of the ethanol accounts for 40-60% of the total volume of the magnetic bead lysate.

[0011] As a further improvement of the present invention, the surfaces of the magnetic beads in the magnetic bead suspension are modified with hydroxyl groups or carboxyl groups; and the particle size of the magnetic beads is 0.2 to 2 μm.

[0012] As a further improvement of the present invention, the magnetic bead suspension is prepared by mixing magnetic beads and sterilized water in a mass ratio of 1:(35-65); the pH value of the magnetic bead lysate is 6.5-7.5.

[0013] As a further improvement of the present invention, the washing solution I is a mixed aqueous solution comprising 1-5M guanidine thiocyanate, 0.1-1M sodium chloride, 2-5% by volume of Tween-20, 0.1-1M sodium acetate and 40-60% by volume of ethanol.

[0014] As a further improvement of the present invention, the washing liquid II is an isopropyl alcohol aqueous solution or an ethanol aqueous solution with a volume fraction of 75-85%.

[0015] As a further improvement of the present invention, the eluent is one of distilled water, deionized water, and ultrapure water.

[0016] To achieve the above-mentioned object of the invention, the present invention also provides a method for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method, wherein the method uses the above-mentioned kit for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method, and comprises the following steps:

[0017] S1. Soak the swab sample in saline, shake thoroughly, let it sit for 2–5 minutes, and then centrifuge at 800–12,000 rpm to prepare a diluted HCV sample with a concentration of 9–12 IU / mL.

[0018] S2. Take 200-400 μL of the diluted HCV sample prepared in step S1, add 1-2 times the volume of the magnetic bead lysis solution to the diluted HCV sample, mix at 50-100°C, place on a magnetic stand, let stand, and after the magnetic beads are completely adsorbed, discard all the liquid to obtain the adsorbed magnetic beads;

[0019] S3. Add 500-1000 μL of washing solution I to the magnetic beads obtained in step S2, shake and mix at room temperature, place on a magnetic rack, let it stand until the magnetic beads are completely adsorbed, and discard all the liquid to ensure that no liquid remains; then add 500-1000 μL of washing solution II, shake and mix at room temperature, place on a magnetic rack, let it stand until the magnetic beads are completely adsorbed, and discard all the liquid to ensure that no liquid remains;

[0020] S4. Add 50-100 μL of eluent to the magnetic beads obtained in step S3, shake and mix at 50-100°C, and place on a magnetic rack. After the magnetic beads are adsorbed, collect the eluent to obtain a nucleic acid solution.

[0021] As a further improvement of the present invention, extraction is performed using a 96-channel automatic nucleic acid extractor, comprising the following steps:

[0022] S10. Take a 96-well deep-well plate that has been irradiated and sterilized, and add 400-800 μL of magnetic bead lysis buffer and 200-400 μL of the diluted HCV sample at a concentration of 9-12 IU / mL to the first and seventh columns of the deep-well plate, respectively; set the extraction instrument to: extraction time 300-600 s, extraction temperature 50-100°C, and magnetic absorption time 120-300 s;

[0023] S20. Add 500-1000 μL of wash solution I to columns 2 and 8 of the deep-well plate, respectively. Set the extraction instrument to: extraction time 30-180 s, extraction temperature at room temperature, and magnetic absorption time 30-180 s.

[0024] S30. Add 500-1000 μL of Wash Solution II to columns 3 and 9 of the deep-well plate, respectively. Set the extraction instrument to: extraction time 30-180 s, extraction temperature at room temperature, and magnetic absorption time 30-180 s.

[0025] S40. Add 50-100 μL of eluent to columns 5 and 11 of the deep-well plate, respectively. Set the extraction instrument to: extraction time 300-600 s, extraction temperature 50-100°C, and magnetic absorption time 120-300 s.

[0026] The remaining wells of the 96-well deep-well plate are blank; after the nucleic acid extraction is completed, the 5th and 11th columns of the deep-well plate contain the extracted nucleic acid solution.

[0027] As a further improvement of the present invention, the extracted nucleic acid solution is subjected to fluorescent quantitative PCR detection, and the detection rate of hepatitis C virus RNA at a concentration of 9 to 12 IU / mL is as high as 100%.

[0028] The beneficial effects of the present invention are:

[0029] (1) The kit for ultrasensitive extraction of hepatitis C virus RNA by magnetic bead method provided by the present invention adopts sodium citrate, sodium lauroyl sarcosinate, sodium acetate, sodium metabisulfite, guanidine isothiocyanate, ethanol and magnetic bead suspension to mix, and the obtained magnetic bead lysis solution has excellent lysis, degradation resistance and binding stability to hepatitis C virus RNA. At the same time, combined with the synergistic effect of washing solution I, the hepatitis C virus RNA extracted by the magnetic bead lysis solution has a high detection rate, small repeatability error, high sensitivity, is not easy to degrade, and has much better stability than the same type of commercial kits. Virus samples with a concentration of 10 IU / mL can be detected 100%, which is better than the 12 IU / mL concentration of the commercial kit, and has significant advantages in clinical testing; at the same time, the reagents used in the magnetic bead lysis solution and the washing solution are non-toxic, have little pollution to the environment, and can be stored at room temperature for a long time, and can be used to replace most similar products on the market.

[0030] (2) The kit for ultrasensitive extraction of HCV RNA by magnetic beads provided by the present invention, during the treatment of magnetic bead lysate, as components such as proteins, nucleases and nucleic acids in HCV are continuously released, firstly, guanidine isothiocyanate can lyse RNA in viral cells to the greatest extent, and at the same time, guanidine isothiocyanate can denature proteins, which is helpful for the subsequent purification of nucleic acids; it can also inhibit the degradation of nucleic acids by nucleases, thereby improving the stability of nucleic acids. Secondly, the pH value of the magnetic bead lysate is controlled to 6.5-7.5. In the presence of sodium citrate and sodium acetate, high concentrations of inorganic salt cations can neutralize the negative charge on RNA molecules, thereby reducing the repulsive force between negatively charged RNA molecules and promoting RNA aggregation and precipitation. At the same time, citrate and acetate can be adsorbed on the surface of magnetic beads, effectively protecting the activity of silanol or carboxyl groups on the surface of magnetic beads. Highly active magnetic beads are more conducive to the firm adsorption of bacterial genomic RNA on the surface of magnetic beads. In addition, ethanol in the lysate can also cause nucleic acid aggregation and precipitation, reducing the loss of nucleic acid during the extraction process. Thirdly, magnetic beads modified with hydroxyl or carboxyl groups on their surface can utilize the negative charge of their surface to form an "anion-cation-anion" salt bridge structure with negatively charged nucleic acids in the presence of salt ions, thereby specifically extracting nucleic acids. At the same time, the appropriate magnetic bead particle size of 0.2 to 2 μm can effectively ensure the magnetic separation of nucleic acids to achieve the purpose of separation and purification.

[0031] During the viral cell lysis process, sodium metabisulfite, as a strong reducing agent and inherently non-degradable, possesses strong reducing properties and storage stability. This can, to a certain extent, prevent oxidation of magnetic beads, which could weaken their magnetism. It can also effectively prevent nucleic acids from oxidizing and denaturing. This is particularly true for single-stranded RNA, which is susceptible to denaturation or degradation due to environmental influences. The strong reducing properties of sodium metabisulfite further prevent oxidation. Furthermore, the presence of sodium lauroyl sarcosinate provides a stable salt-containing extraction environment for the nucleic acid extraction system, preventing the acidity of the sodium metabisulfite solution from affecting the stability of the extraction reagents, further enhancing lysis.

[0032] The present invention is to obtain the optimal ratio of the composite system of magnetic bead lysate and washing solution I by rationally compounding the above-mentioned raw material components, so that the synergistic effect is exerted between the raw materials, so that the raw material system has excellent cleavage and stable binding to hepatitis C virus RNA while realizing ultrasensitive detection of hepatitis C virus RNA. The synergistic mechanism of this ultrasensitive detection is as follows: after the hepatitis C virus is lysed by the magnetic bead lysate, components such as protein, nuclease and nucleic acid in the virus are released together, and the high concentration of guanidine isothiocyanate in the magnetic bead lysate can lyse the RNA in the viral cells to the greatest extent, thereby greatly increasing the amount of RNA bound to the magnetic beads. At the same time, guanidine isothiocyanate can also denature proteins and nucleases, inhibit the degradation of nucleic acids by nucleases, and effectively ensure the RNA content in the solution; on the other hand, the strong reducing property of sodium metabisulfite in the magnetic bead lysate can effectively prevent the oxidative denaturation of nucleic acids, ensure the effective RNA content in the solution, enable the magnetic beads to effectively bind to more nucleic acids, and reduce the detection limit.

[0033] The nucleic acids obtained during the lysis step will, to a certain extent, be adsorbed with some proteins and nucleases, which can inhibit the subsequent PCR amplification process, thereby increasing the detection limit. The high concentration of guanidine isothiocyanate in Washing Solution 1 can further denature the adsorbed proteins and inhibit the activity of nucleases, thereby lowering the detection limit of this method. Furthermore, the high concentration of sodium salt in Washing Solution 1 can adjust the pH of the system, providing sufficient sodium ions to ensure effective binding of the magnetic beads and nucleic acids, avoiding nucleic acid loss, and further reducing the detection limit of nucleic acids. Furthermore, Tween-20 in Washing Solution 1 can prevent the denaturation and loss of low-load viral RNA during the washing process, thereby increasing RNA content. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The detection rate of hepatitis C virus RNA nucleic acid at a concentration of 10 IU / mL was obtained using the kit for ultrasensitive extraction of hepatitis C virus RNA using magnetic beads provided in Example 1 of the present invention.

[0035] Figure 2 The kit provided for comparative example 1 extracts the hepatitis C virus RNA nucleic acid detection rate result at a concentration of 10 IU / mL. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] It should also be noted here that, in order to avoid obscuring the present invention due to unnecessary details, only structures and / or processing steps closely related to the solutions of the present invention are shown in the drawings, while other details that are not closely related to the present invention are omitted.

[0038] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0039] The invention provides a kit for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method. The kit comprises an extraction reagent group for ultrasensitive extraction of hepatitis C virus RNA, wherein the extraction reagent group comprises a magnetic bead lysis solution, a washing solution I, a washing solution II and an eluent. RNA in the hepatitis C virus is released by the magnetic bead lysis solution, and the RNA is further completely separated from other components in the reaction system, such as proteins, salts and other impurities, by the washing solution I, the washing solution II and the eluent.

[0040] The magnetic bead lysis solution is composed of sodium citrate, sodium lauroyl sarcosinate, sodium acetate, sodium metabisulfite, guanidine isothiocyanate, ethanol and magnetic bead suspension. Among them, the concentration of sodium citrate is 10-100mM, the concentration of sodium lauroyl sarcosinate is 10-100mM, the concentration of sodium acetate is 0.1-10M, the content of sodium metabisulfite is 0.2-2wt%, the concentration of guanidine isothiocyanate is 2-6M, and the volume of ethanol accounts for 40-60% of the total volume of the magnetic bead lysis solution; the surface of the magnetic beads in the magnetic bead suspension is modified with hydroxyl or carboxyl groups; the particle size of the magnetic beads is 0.2-2μm. Specifically, the magnetic bead suspension is a mixture of magnetic beads and sterilized water in a mass ratio of 1: (35-65); the pH value of the magnetic bead lysis solution is 6.5-7.5.

[0041] The washing solution I is a mixed aqueous solution comprising 1-5M guanidine thiocyanate, 0.1-1M sodium chloride, 2-5% by volume of Tween-20, 0.1-1M sodium acetate and 40-60% by volume of ethanol.

[0042] The washing liquid II is an isopropyl alcohol aqueous solution or an ethanol aqueous solution with a volume fraction of 75 to 85%.

[0043] The eluent is one of distilled water, deionized water, and ultrapure water.

[0044] The present invention also provides a method for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method, wherein the method uses the above-mentioned kit for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method, and comprises the following steps:

[0045] S1. Soak the swab sample in saline, shake thoroughly, let it sit for 2–5 minutes, and then centrifuge at 800–12,000 rpm to prepare a diluted HCV sample with a concentration of 9–12 IU / mL.

[0046] S2. Take 200-400 μL of the diluted HCV sample prepared in step S1, add 1-2 times the volume of magnetic bead lysis buffer to the diluted HCV sample, mix at 50-100°C, place on a magnetic stand, and let it stand until the magnetic beads are completely adsorbed. Discard all the liquid to obtain the adsorbed magnetic beads;

[0047] S3. Add 500-1000 μL of washing solution I to the magnetic beads obtained in step S2, shake and mix at room temperature, place on a magnetic rack, let it stand until the magnetic beads are completely adsorbed, and discard all the liquid to ensure that no liquid remains; then add 500-1000 μL of washing solution II, shake and mix at room temperature, place on a magnetic rack, let it stand until the magnetic beads are completely adsorbed, and discard all the liquid to ensure that no liquid remains;

[0048] S4. Add 50-100 μL of eluent to the magnetic beads obtained in step S3, shake and mix at 50-100°C, and place on a magnetic rack. After the magnetic beads are adsorbed, collect the eluent to obtain a nucleic acid solution.

[0049] According to a preferred embodiment of the present invention, extraction is performed using a 96-channel automatic nucleic acid extractor, comprising the following steps:

[0050] S10. Take an irradiated 96-well deep-well plate and add 400-800 μL of magnetic bead lysis buffer and 200-400 μL of diluted HCV sample at a concentration of 9-12 IU / mL to columns 1 and 7, respectively. Set the extraction instrument to: extraction time 300-600 s, extraction temperature 50-100°C, and magnetic absorption time 120-300 s.

[0051] S20. Add 500-1000 μL of wash solution I to columns 2 and 8 of the deep-well plate, respectively. Set the extraction instrument to: extraction time 30-180 s, extraction temperature at room temperature, and magnetic absorption time 30-180 s.

[0052] S30. Add 500-1000 μL of Wash Solution II to columns 3 and 9 of the deep-well plate, respectively. Set the extraction instrument to: extraction time 30-180 s, extraction temperature at room temperature, and magnetic absorption time 30-180 s.

[0053] S40. Add 50-100 μL of eluent to columns 5 and 11 of the deep-well plate, respectively. Set the extraction instrument to: extraction time 300-600 s, extraction temperature 50-100°C, and magnetic absorption time 120-300 s.

[0054] The remaining wells of the 96-well deep-well plate were left blank. After nucleic acid extraction, columns 5 and 11 of the deep-well plate contained the extracted nucleic acid solution. Fluorescence quantitative PCR analysis of the extracted nucleic acid solution revealed a 100% detection rate for HCV RNA at concentrations of 9 to 12 IU / mL.

[0055] The present invention is described in detail below through a number of embodiments:

[0056] Example 1

[0057] The invention discloses a method for ultrasensitively extracting hepatitis C virus RNA using a magnetic bead method. The method adopts a kit for ultrasensitively extracting hepatitis C virus RNA using a magnetic bead method provided by the invention for extraction. The kit comprises an extraction reagent group for ultrasensitively extracting hepatitis C virus RNA; the extraction reagent group comprises a magnetic bead lysate, a washing solution I, a washing solution II, and an eluent; the magnetic bead lysate comprises sodium citrate, sodium lauroyl sarcosinate, sodium acetate, a reducing agent sodium metabisulfite, guanidine isothiocyanate, ethanol, and a magnetic bead suspension; the washing solution I is a mixed aqueous solution comprising guanidine isothiocyanate, sodium chloride, Tween-20, sodium acetate, and ethanol; the washing solution II is an ethanol aqueous solution with a volume fraction of 70%; and the eluent is distilled water.

[0058] Among them, the magnetic bead lysate is a mixed magnetic bead solution comprising 30mM sodium citrate, 20mM sodium lauroyl sarcosinate, 0.5M sodium acetate, 0.8wt% sodium metabisulfite, 4M guanidine isosulfate, 45% ethanol (that is, the volume of ethanol accounts for 45% of the total volume of the magnetic bead lysate) and 3mL of a 0.5μm-sized magnetic bead suspension modified with carboxyl groups on the surface, wherein the magnetic bead suspension is a mixture of magnetic beads and sterilized water in a mass ratio of 1:40, and the pH value of the magnetic bead lysate is 7.0.

[0059] Washing solution I is a mixed aqueous solution containing 3M guanidine thiocyanate, 0.5M sodium chloride, 3% Tween-20 by volume, 0.5M sodium acetate, and 50% ethanol.

[0060] Follow the steps below to extract HCV RNA: (Each kit is tested with 8 replicate wells)

[0061] S10. Take a 96-well deep-well plate that has been sterilized by irradiation, and add 600 μL of magnetic bead lysis buffer and 300 μL of the diluted HCV sample at a concentration of 10 IU / mL to columns 1 and 7, respectively. Set the extraction instrument to: extraction time 450 s, extraction temperature 75°C, and magnetic absorption time 200 s.

[0062] S20. Add 800 μL of wash solution I to columns 2 and 8 of the deep-well plate, respectively. Set the extraction instrument to: extraction time 100 s, extraction temperature at room temperature, and magnetic absorption time 100 s.

[0063] S30. Add 800 μL of wash solution II to columns 3 and 9 of the deep-well plate, respectively. Set the extraction instrument to: extraction time 100 s, extraction temperature at room temperature, and magnetic absorption time 100 s.

[0064] S40. Add 80 μL of eluent to columns 5 and 11 of the deep-well plate, respectively. The extraction instrument settings are: extraction time 450 s, extraction temperature 75°C, and magnetic absorption time 200 s.

[0065] The remaining wells of the 96-well deep-well plate are blank; after the nucleic acid extraction is completed, the 5th and 11th columns of the deep-well plate contain the extracted nucleic acid solution.

[0066] The nucleic acid solution extracted in Example 1 was subjected to fluorescence quantitative PCR detection, and the results were as follows: Figure 1 As shown by Figure 1 It can be seen that the detection rate of hepatitis C virus RNA extracted by the kit provided in Example 1 is 100%, and the repeatability error is small.

[0067] Examples 2-6

[0068] A method for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method is provided, using the kit for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method provided by the present invention. The kits for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method provided in Examples 2-6 differ from those in Example 1 in that the concentrations of sodium lauroyl sarcosinate and sodium metabisulfite in the magnetic bead lysis buffer are different, as shown in Table 1. Other details are generally the same as in Example 1 and are not further described here.

[0069] The nucleic acid solutions extracted from Examples 1-6 were subjected to fluorescent quantitative PCR detection, and the results are shown in Table 1. (The detection concentration was 10 IU / mL, the detection channel was FAM, and 8 replicate wells were made for each kit)

[0070] Table 1 Detection rate of nucleic acid solutions extracted from Examples 1-6

[0071]

[0072] As shown in Table 1, the CT value of Example 1 is small and the stability is good, indicating that the concentration of nucleic acid extracted by the magnetic bead lysate of Example 1 is large, and the detection rate of hepatitis C virus RNA is 100%, the repeatability error is 1.95%, and the repeatability error is relatively small (this is consistent with the Figure 1 The results are consistent).

[0073] As can be seen from Examples 1-4, when the content of sodium lauroyl sarcosinate in the magnetic bead lysate is in the range of 10-50mM, the change in the CT value is small; as the content of sodium lauroyl sarcosinate in the magnetic bead lysate further increases, the CT value continues to increase, indicating that the nucleic acid concentration extracted gradually decreases, that is, the degree of cracking of hepatitis C virus RNA by the magnetic bead lysate decreases, and the detection time increases therewith. If the content of sodium lauroyl sarcosinate is too low (below 10mM), the CT value will also be larger. This shows that the presence of sodium lauroyl sarcosinate in the magnetic bead lysate can prevent the acidity of the sodium metabisulfite aqueous solution from affecting the stability of the extraction reagent in the nucleic acid extraction system. However, if the content of sodium lauroyl sarcosinate is too little or too much, the pH of the magnetic bead lysate will be affected, thereby destroying the stability of the extraction reagent in the magnetic bead lysate, and then affecting the extraction effect.

[0074] The data from Examples 1, 5, and 6 show that the CT value increases when the sodium metabisulfite content in the magnetic bead lysis solution is low or high. This is primarily because, when the sodium metabisulfite content in the magnetic bead lysis solution is low, the reducing properties of the system are weak, the magnetic beads are easily oxidized, and the magnetism is weakened. At the same time, the nucleic acids are easily oxidized and denatured, resulting in a low nucleic acid concentration and an increased CT value. When the sodium metabisulfite content is high, the acidity of the sodium metabisulfite aqueous solution affects the stability of the extraction reagents in the nucleic acid extraction system, thereby affecting the extraction effect.

[0075] Examples 7-9

[0076] A method for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method is provided, using the kit for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method provided by the present invention. The kits for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method provided in Examples 7-9 differ from those in Example 1 in that the concentration of guanidine isothiocyanate in the magnetic bead lysis buffer and the particle size of the magnetic beads are different. The specific parameters are shown in Table 2. Other parameters are substantially the same as in Example 1 and are not further described here.

[0077] The nucleic acid solutions extracted from Examples 7-9 were subjected to fluorescent quantitative PCR detection, and the results are shown in Table 2. (The detection concentration was 10 IU / mL, the detection channel was FAM, and 8 replicate wells were made for each kit)

[0078] Table 2 Detection rate of nucleic acid solutions extracted from Examples 7-9

[0079]

[0080]

[0081] As shown in Table 2, as the guanidine isothiocyanate content in the magnetic bead lysis buffer increases, the CT value first decreases and then increases (but the increase trend is small). This is mainly because when the guanidine isothiocyanate content is low, the degree of RNA cleavage in the viral cells is less, resulting in a lower nucleic acid concentration and a larger CT value.

[0082] As the particle size of the magnetic beads in the magnetic bead lysis solution increases, the CT value does not change much, but missed detections occur.

[0083] Examples 10-12

[0084] A method for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method is provided, using the kit for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method provided by the present invention. The kits for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method provided in Examples 10-12 differ from those in Example 1 in that the concentrations of guanidine isothiocyanate and Tween-20 in Washing Solution I are different, as shown in Table 3. Other details are generally the same as in Example 1 and are not further described here.

[0085] The nucleic acid solutions extracted from Examples 10-12 were subjected to fluorescent quantitative PCR detection, and the results are shown in Table 3. (The detection concentration was 10 IU / mL, the detection channel was FAM, and 8 replicate wells were made for each kit)

[0086] Table 3 Detection rate of nucleic acid solutions extracted from Examples 10-12

[0087]

[0088] As shown in Table 3, when the content of guanidine thiocyanate in the washing solution I is low, the CT value will be slightly larger. However, when the content of guanidine thiocyanate increases or the content of Tween-20 increases, the CT value does not change much, and the repeatability error does not change much either, and the overall extraction effect is better.

[0089] Comparative Example 1

[0090] The invention discloses a method for ultrasensitively extracting HCV RNA by magnetic bead method, wherein the HCV RNA is extracted by using a commercially available HCV nucleic acid detection kit produced by Shengxiang Biotechnology Co., Ltd.

[0091] Comparative Example 2

[0092] A method for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method is disclosed. The kit used for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method is different from that in Example 1, in that sodium metabisulfite is not added to the magnetic bead lysate. Other details are substantially the same as those in Example 1 and are not further described herein.

[0093] Comparative Example 3

[0094] A method for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method is disclosed. The kit used for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method is different from that in Example 1, the antioxidant added to the magnetic bead lysate is dithiothreitol instead of sodium metabisulfite. Other details are the same as those in Example 1 and are not further described herein.

[0095] Comparative Example 4

[0096] A method for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method is disclosed. The kit for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method is different from that in Example 1, except that Tween-20 is not added to the washing solution I. Other details are substantially the same as those in Example 1 and are not further described herein.

[0097] The nucleic acid solutions extracted from Comparative Examples 1-4 were subjected to fluorescent quantitative PCR detection, and the results are shown in Table 4. (The detection concentration was 10 IU / mL, the detection channel was FAM, and 8 replicate wells were made for each kit)

[0098] Table 4 Detection rate of nucleic acid solution extracted from comparative examples 1-4

[0099]

[0100]

[0101] As shown in Table 4, the detection rate of the nucleic acid solution extracted by the kit of Comparative Example 1 is significantly lower than that of Example 1. Comparative Example 1 has missed detections, and the repeatability error is also higher than that of Example 1.

[0102] In Comparative Example 2, when the magnetic bead lysis buffer does not contain sodium metabisulfite, not only is the CT value larger, but there is also a high probability of missed detection. This further illustrates that sodium metabisulfite can prevent the magnetic beads from being oxidized to a certain extent, thereby weakening their magnetism. It can also effectively prevent nucleic acids from being oxidized and denatured, thereby improving the extraction effect.

[0103] In Comparative Example 3, after replacing the sodium metabisulfite in the magnetic bead lysis solution with dithiothreitol, although there was no missed detection, the CT value was larger, indicating that the concentration of the extracted nucleic acid was low.

[0104] In Comparative Example 4, when Tween-20 was not added to the washing solution I, the CT value increased significantly and the repeatability error value was also large, indicating that Tween-20 can prevent the denaturation loss of viral RNA during the washing process.

[0105] In summary, the kit for ultrasensitive extraction of hepatitis C virus RNA by magnetic bead method and the extraction method provided by the present invention adopt sodium citrate, sodium lauroyl sarcosinate, sodium acetate, sodium metabisulfite, guanidine isothiocyanate, and ethanol to mix with a magnetic bead suspension, and the obtained magnetic bead lysis solution has excellent lysis, degradation resistance and binding stability for hepatitis C virus RNA. The hepatitis C virus RNA extracted by the magnetic bead lysis solution has a high detection rate, small repeatability error, high sensitivity, is not easy to degrade, and has much better stability than commercially available kits of the same type. Virus samples with a concentration of 10 IU / mL can be detected 100%, which is better than the 12 IU / mL concentration of commercially available kits, and has significant advantages in clinical testing. At the same time, the reagents are non-toxic, have little pollution to the environment, and can be stored at room temperature for a long time, and can be used to replace most similar products on the market.

[0106] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A kit for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method, characterized in that: The invention comprises an extraction reagent set for ultrasensitive extraction of hepatitis C virus RNA; the extraction reagent set comprises a magnetic bead lysis solution, a washing solution I, a washing solution II and an eluent; the magnetic bead lysis solution comprises sodium citrate, sodium lauroyl sarcosinate, sodium acetate, sodium metabisulfite, guanidine isothiocyanate, ethanol and a magnetic bead suspension; In the magnetic bead lysis solution, the concentration of sodium lauroyl sarcosinate is 10 to 100 mM, the content of sodium metabisulfite is 0.2 to 2 wt%, and the concentration of guanidine isothiocyanate is 2 to 6 M; the washing solution I is a mixed aqueous solution containing 1 to 5 M guanidine isothiocyanate and 3 to 5% by volume of Tween-20; the particle size of the magnetic beads in the magnetic bead suspension is 0.5 to 2 μm.

2. The kit for ultrasensitive extraction of hepatitis C virus RNA by magnetic bead method according to claim 1, characterized in that: In the magnetic bead lysate, the concentration of the sodium citrate is 10-100 mM, the concentration of the sodium acetate is 0.1-10 M, and the volume of the ethanol accounts for 40-60% of the total volume of the magnetic bead lysate.

3. The kit for ultrasensitive extraction of hepatitis C virus RNA by magnetic bead method according to claim 1, characterized in that: The surfaces of the magnetic beads in the magnetic bead suspension are modified with hydroxyl groups or carboxyl groups.

4. The kit for ultrasensitive extraction of hepatitis C virus RNA by magnetic bead method according to claim 1, characterized in that: The magnetic bead suspension is prepared by mixing magnetic beads and sterilized water in a mass ratio of 1:(35-65); the pH value of the magnetic bead lysate is 6.5-7.

5.

5. The kit for ultrasensitive extraction of hepatitis C virus RNA by magnetic bead method according to claim 1, characterized in that: The washing solution I further comprises 0.1-1M sodium chloride, 0.1-1M sodium acetate and 40-60% by volume ethanol.

6. The kit for ultrasensitive extraction of hepatitis C virus RNA by magnetic bead method according to claim 1, characterized in that: The washing liquid II is an isopropyl alcohol aqueous solution or an ethanol aqueous solution with a volume fraction of 75-85%.

7. The kit for ultrasensitive extraction of hepatitis C virus RNA by magnetic bead method according to claim 1, characterized in that: The eluent is one of distilled water, deionized water and ultrapure water.

8. A method for ultrasensitive extraction of hepatitis C virus RNA using magnetic beads, characterized in that: Extraction is performed using the kit for ultrasensitive extraction of hepatitis C virus RNA using a magnetic bead method according to any one of claims 1 to 7, comprising the following steps: S1. Soak the swab sample in saline, shake thoroughly, let it sit for 2–5 minutes, and then centrifuge at 800–12,000 rpm to prepare a diluted HCV sample with a concentration of 9–12 IU / mL. S2. Take 200-400 μL of the diluted HCV sample prepared in step S1, add 1-2 times the volume of the magnetic bead lysis solution to the diluted HCV sample, mix at 50-100°C, place on a magnetic stand, let stand, and after the magnetic beads are completely adsorbed, discard all the liquid to obtain the adsorbed magnetic beads; S3. Add 500-1000 μL of washing solution I to the magnetic beads obtained in step S2, shake and mix at room temperature, place on a magnetic rack, let it stand until the magnetic beads are completely adsorbed, and discard all the liquid to ensure that no liquid remains; then add 500-1000 μL of washing solution II, shake and mix at room temperature, place on a magnetic rack, let it stand until the magnetic beads are completely adsorbed, and discard all the liquid to ensure that no liquid remains; S4. Add 50-100 μL of eluent to the magnetic beads obtained in step S3, shake and mix at 50-100°C, and place on a magnetic rack. After the magnetic beads are adsorbed, collect the eluent to obtain a nucleic acid solution.

9. The method for ultrasensitive extraction of hepatitis C virus RNA by magnetic bead method according to claim 8, characterized in that: Extraction was performed using a 96-channel automatic nucleic acid extractor, including the following steps: S10. Take a 96-well deep-well plate that has been irradiated and sterilized, and add 400-800 μL of magnetic bead lysis buffer and 200-400 μL of the diluted HCV sample at a concentration of 9-12 IU / mL to the first and seventh columns of the deep-well plate, respectively; set the extraction instrument to: extraction time 300-600 s, extraction temperature 50-100°C, and magnetic absorption time 120-300 s; S20. Add 500-1000 μL of wash solution I to columns 2 and 8 of the deep-well plate, respectively. Set the extraction instrument to: extraction time 30-180 s, extraction temperature at room temperature, and magnetic absorption time 30-180 s. S30. Add 500-1000 μL of Wash Solution II to columns 3 and 9 of the deep-well plate, respectively. Set the extraction instrument to: extraction time 30-180 s, extraction temperature at room temperature, and magnetic absorption time 30-180 s. S40. Add 50-100 μL of eluent to columns 5 and 11 of the deep-well plate, respectively. Set the extraction instrument to: extraction time 300-600 s, extraction temperature 50-100°C, and magnetic absorption time 120-300 s. The remaining wells of the 96-well deep-well plate are blank; after the nucleic acid extraction is completed, the 5th and 11th columns of the deep-well plate contain the extracted nucleic acid solution.

10. The method for ultrasensitive extraction of hepatitis C virus RNA by magnetic beads according to claim 9, characterized in that: The extracted nucleic acid solution was subjected to fluorescent quantitative PCR detection, and the detection rate of hepatitis C virus RNA at a concentration of 9-12 IU / mL was as high as 100%.

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