Hydroxyl magnetic bead extraction and capture probe of group of GBS RNA, kit and extraction method

Abstract

The invention discloses a group of hydroxyl magnetic bead extraction and capture probes for GBSRNA, a kit and an extraction method, and relates to the technical field of nucleic acid extraction. The capture probe provided by the invention comprises five nucleotide sequences, the five nucleotide sequences are loaded on a hydroxyl magnetic bead and are used for preparing the kit for extracting the GBSRNA, the extraction efficiency of the RNA is greatly improved by rapidly splitting bacteria and releasing the RNA and specifically capturing the GBSRNA by utilizing the probe on the magnetic bead, and finally, DNA impurities are removed by utilizing DNase, so that the high-purity RNA is obtained. The technical problem that the extraction efficiency of RNA is relatively low due to relatively high content of impurities in an extract caused by inaccurate extraction is solved.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid extraction technology, specifically to a set of GBS RNA hydroxyl magnetic bead extraction and capture probes, kits, and extraction methods. Background Technology

[0002] Group B Streptococcus (GBS), also known as agalactiae, is an opportunistic pathogen and part of the normal flora in the human body, normally not causing disease. The carrier rate in the lower digestive tract of normal individuals is approximately 15%–35%. GBS can invade the urogenital tract and coexist with Candida. The carrier rate varies depending on age, region, and ethnicity; for example, women in groups with multiple sexual partners have a significantly higher carrier rate than the general population. In addition, diabetic patients, newborns, and pregnant women are all high-risk groups. In pregnant women, GBS infection of the reproductive tract can easily lead to premature rupture of membranes, amniocentesis (chorioamnionitis), premature birth, puerperal infection, bacteremia, amniotic membrane infection, endometrial infection, wound infection, and pneumonia. It can also cause fetal distress, asphyxia, jaundice, and other adverse pregnancy outcomes. Infection in newborns can cause neonatal sepsis, pneumonia, and meningitis, and in severe cases, death. Newborns who survive infection may suffer from severe neurological sequelae, such as hydrocephalus, intellectual disability, microcephaly, and deafness. Most neonatal infections with Group B Streptococcus are closely related to mother-to-child vertical transmission and are associated with childbirth. If the mother is infected with Group B Streptococcus, the newborn may inhale infected amniotic fluid at birth or become infected while passing through the birth canal. Even a cesarean section cannot completely prevent neonatal Group B Streptococcus infection.

[0003] Rapid and accurate bacterial detection is a crucial prerequisite for effective treatment, disease monitoring, and control of disease spread. With the development of detection technologies, nucleic acid detection, due to its high sensitivity and specificity, has become one of the important methods for bacterial detection. RNA, one of the most important basic substances in biomolecules, has also become a research object in this field, making nucleic acid (RNA) extraction particularly important in experimental processes. For the extraction of bacterial RNA, such as RNA from Group B Streptococcus, classic methods include the Trizol method, as well as commercial kits such as column extraction and magnetic bead extraction. Among these, magnetic bead extraction has become a commonly used extraction method in the molecular field. Furthermore, different extraction methods, due to differences in the concentration and purity of the extracted products and the degree of RNA protection, will, to some extent, affect the detection of nucleic acids, especially in borderline positive samples. Compared to other methods, the magnetic bead method is simpler, more efficient, and produces higher quality results. The magnetic bead method for nucleic acid extraction and purification utilizes nanotechnology to modify and enhance the surface of superparamagnetic nanoparticles, creating superparamagnetic nanobeads. These nanobeads serve as a solid-phase carrier, allowing nucleic acids released from the sample to specifically bind to them. An external magnetic field is then applied to separate and purify the nucleic acids. A washing solution is used to remove proteins, polysaccharides, and salt ions from the sample solution. High-purity RNA solutions are obtained under heating conditions. This method has been widely adopted in recent years due to its applicability to various sample types, low sample requirements, and the ability to be integrated with automated nucleic acid extractors for automated and high-throughput extraction. For example, patent applications CN112501162A and CN108192892A both mention techniques for extracting GBS RNA using hydroxyl-containing magnetic nanobead suspensions combined with RNA. However, these methods suffer from imprecise extraction, resulting in high impurities in the extract and consequently, low RNA extraction efficiency. Summary of the Invention

[0004] To address the aforementioned problems in the prior art, this invention provides a set of GBS RNA hydroxyl magnetic bead extraction and capture probes, kits, and extraction methods to solve the technical problem of low RNA extraction efficiency due to high impurities in the extract caused by insufficient extraction precision in the prior art.

[0005] The technical solution adopted in this invention is as follows:

[0006] A set of GBS RNA hydroxyl magnetic bead extraction and capture probes, comprising 5 nucleotide sequences, as shown in SEQ ID NO:1 to SEQ ID NO:5 respectively:

[0007] SEQ ID NO:1 TACGATCCGAAAACCTTCTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA;

[0008] SEQ ID NO:2 CGTTCTTCTCTAACAACAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA;

[0009] SEQ ID NO:3 TTCTGGTTAGTTACCGTCACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA;

[0010] SEQ ID NO:4 ATCGTTTACGGCGTGGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA;

[0011] SEQ ID NO:5 CTACCTTGTTACGACTTCACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA.

[0012] The GBS RNA hydroxy magnetic bead extraction kit includes lysis buffer, binding buffer, washing buffer, magnetic bead suspension modified with capture probe, DNase, and DNase reaction solution, wherein the capture probe is one of the above 5 nucleotide sequences.

[0013] Preferably, the lysis buffer consists of lysozyme, RNA inhibitor, and buffer solution, wherein the concentration of lysozyme is 0.1-100 mg / mL; the RNA inhibitor is selected from one or more of guanidine hydrochloride, urea, guanidine isothiocyanate, and vanadate ribonucleoside complex; and the buffer solution is one or more of Tris-HCl, EDTA, KCl, NaCl, and MgCl2.

[0014] Preferably, the binding solution is composed of one or more of Tris-HCl, EDTA, NaCl, NaOH, MgCl2 and SDS.

[0015] More preferably, the binding solution consists of 20–40 mM Tris-HCl with pH 7.0–8.0, 0.5–5.0 M NaCl, 1–20 mM MgCl2 and 10–20 mM EDTA.

[0016] Preferably, the washing solution consists of washing solution I and washing solution II. Washing solution I is composed of one or more of Tris-HCl, EDTA, NaCl, NaOH, MgCl2 and SDS, and washing solution II is one of DEPC water, RNase-free water or double-distilled water.

[0017] More preferably, the washing solution I is composed of 20-40 mM Tris-HCl with pH 7.0-8.0, 0.1-1 M NaCl, 0.1-1 mM MgCl2 and 10-20 mM EDTA.

[0018] Preferably, the treatment process of the magnetic bead suspension modified with the capture probe is as follows: the magnetic beads modified with streptavidin are soaked in 1% diethyl pyrocarbonate solution for 5-10 hours, washed 3-5 times with PBS buffer at pH 7.0, and then biotin-modified capture probe is added and conditioned at 37°C for half an hour, and then stored in a refrigerator at 4°C for later use.

[0019] A method for extracting GBS RNA using any of the above-described kits, the method comprising the following steps:

[0020] (1) Take a certain amount of cultured GBS into a 2mL centrifuge tube, add 100μl of lysis buffer, and shake at 37℃ to mix for a period of time;

[0021] (2) Add 10 μl of magnetic bead suspension and 100 μl of binding solution to a test tube, and shake at 37°C to mix for a period of time.

[0022] (3) Place the incubated sample on a magnetic rack and let it stand until it clarifies, then discard the supernatant;

[0023] (4) Add 200 μl of washing solution 1, mix thoroughly, place on a magnetic rack and let stand until clear, then discard the supernatant;

[0024] (5) Add 100 μl of washing solution 2, mix thoroughly, place on a magnetic rack and let stand until clear, then discard the supernatant; add 20 μl of washing solution 2, mix thoroughly, and incubate at 65°C for a period of time.

[0025] (6) Quickly place the incubated sample on a magnetic rack to stand still. After clarification, immediately aspirate the supernatant into a new EP tube.

[0026] (7) Add 10U DNase and 10μl DNase reaction solution to the above solution, incubate at 60℃ for a period of time, and then incubate at 85℃ for 10min to inactivate the enzyme to obtain high-purity target nucleic acid.

[0027] Furthermore, the shaking and mixing time in step (1) is 1-15 minutes, the shaking and mixing time in step (2) is 1-15 minutes, the incubation time in step (5) is 1-15 minutes, and the incubation time in step (7) is 1-30 minutes.

[0028] In summary, compared with the prior art, the present invention has the following advantages and benefits:

[0029] 1. This invention rapidly lyses bacteria and releases RNA, then uses probes on magnetic beads to specifically capture GBS RNA, significantly improving RNA extraction efficiency. Finally, DNase is used to remove DNA impurities, resulting in high-purity RNA. This overcomes the technical problem of high impurities in the extract due to insufficient extraction precision, which leads to low RNA extraction efficiency.

[0030] 2. This invention provides a rapid RNA extraction kit for Group B Streptococcus based on magnetic beads. The extraction process is simple and can be performed manually. It can be integrated with various automated nucleic acid extractors, simplifying the steps to include sample addition and automatic nucleic acid extraction. This greatly simplifies experimental procedures and ensures the safety of laboratory personnel. It meets the needs of hospitals with large clinical sample volumes for high-speed and high-throughput nucleic acid extraction, as well as high-quality extraction. Furthermore, this kit features low toxicity, ease of operation, high sample throughput, and superior nucleic acid extraction results. Attached Figure Description

[0031] Figure 1 RT-qPCR results curves for different capture probes;

[0032] Figure 2 This is a graph showing the RT-qPCR results of GBS RNA extracted in Example 2;

[0033] Figure 3 This is a graph showing the RT-qPCR results of RNA extracted using four different methods.

[0034] Figure 4 This is a bar chart comparing the efficiency and stability of four methods for RNA extraction;

[0035] Figure 5 This is a graph showing the RT-qPCR results of RNA extracted with gradient concentrations of GBS using the method of this invention.

[0036] Figure 6 This is a linear graph showing the results of extracting RNA with gradient concentrations of GBS using the method of this invention. Detailed Implementation

[0037] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0038] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0039] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0040] The lysis buffer consists of lysozyme, an RNA inhibitor, and a buffer solution. The concentration of the lysozyme is 0.1-100 mg / mL. The RNA inhibitor is selected from one or more of guanidine hydrochloride, urea, guanidine isothiocyanate, and vanadate ribonucleoside complex. The buffer solution is one or more of Tris-HCl, EDTA, KCl, NaCl, and MgCl2. The binding buffer consists of 20-40 mM Tris-HCl at pH 7.0-8.0, 0.5-5.0 M NaCl, 1-20 mM MgCl2, and 10-20 mM EDTA. The washing buffer consists of Washing Buffer I and Washing Buffer II. Washing Buffer I consists of 20-40 mM Tris-HCl at pH 7.0-8.0, 0.1-1 M NaCl, 0.1-1 mM MgCl2, and 10-20 mM EDTA. Washing Buffer II is one of DEPC water, RNase-free water, or double-distilled water.

[0041] The magnetic bead suspension of this invention is obtained through the following treatment: streptavidin-modified magnetic beads are soaked in a 1% diethyl pyrocarbonate solution for 5-10 hours, washed 3-5 times with PBS buffer at pH 7.0, and then biotin-modified capture probes are added and incubated at 37°C for half an hour. The suspension is then stored at 4°C for later use. The streptavidin-modified magnetic beads have a diameter of 100 nm to 5 μm.

[0042] The present application will now be described in detail with reference to embodiments and experimental data.

[0043] Example 1

[0044] This embodiment verifies the extraction efficiency of different capture probes. Different capture probes were incubated with magnetic beads, and the following steps were followed. The extracted RNA was then detected using an RT-qPCR kit:

[0045] (1) Collect GBS and prepare a bacterial suspension. The specific procedure is as follows:

[0046] Remove GBS in the exponential growth phase from the 37°C incubator and, in a biosafety cabinet, use an inoculation loop to pick up a certain number of bacteria into a centrifuge tube containing 1×PBS.

[0047] Transfer 2 mL of bacterial culture to a new EP tube and centrifuge at 10,000 rpm for 1 minute to remove any possible culture medium. Resuspend the culture in 2 mL of 1×PBS.

[0048] (2) Bacterial lysis. Centrifuge at 10,000 rpm for 1 minute, add 100 μl of lysis buffer, and mix by shaking at 37°C for 10 minutes.

[0049] (3) RNA capture. Add 10 μl of magnetic bead suspension and 100 μl of binding solution to a test tube. Shake at 37°C for 10 minutes to mix.

[0050] Among them, the magnetic bead suspension includes magnetic bead suspensions with and without capture probes (MB), and the capture probes modified on the magnetic bead suspensions with capture probes are shown in Table 1.

[0051] Table 1 Sequences of the capture probes

[0052]

[0053] After incubation, place the sample on a magnetic rack and let it stand until it clarifies, then discard the supernatant.

[0054] Add 200 μl of washing solution 1, mix thoroughly, place on a magnetic rack and let stand until clear, then discard the supernatant.

[0055] Add 100 μl of washing solution 2, mix thoroughly, place on a magnetic rack and let stand until clear, then discard the supernatant.

[0056] Add 20 μl of washing buffer 2, mix thoroughly, and incubate at 65°C for 5 minutes.

[0057] Quickly place the incubated sample on a magnetic rack to stand until it clarifies, then immediately aspirate the supernatant into a new EP tube.

[0058] Add 10U DNase and 10μl DNase reaction solution, mix thoroughly, and incubate at 60℃ for 30 minutes and 85℃ for 10 minutes.

[0059] (4) RT-qPCR quantification. The Luna universal one-step RT-qPCR (NEB, #E3005) kit was used for detection. The reaction system is shown in Table 2.

[0060] Table 2 RT-PCR reaction system (20 μL)

[0061] Element Volume (μL) Mix 10 Upstream primer (10 μM) 1 Downstream primer (10 μM) 1 Taqman probe (10μM) 0.5 RT Enzyme 1 sample 1 RNase-free water Make up to 20 μL

[0062] The reaction program settings are as follows:

[0063] Reverse transcription: 55℃ for 10 minutes;

[0064] Denaturation: 95℃ for 1 minute;

[0065] Annealing, extension and fluorescence acquisition: 60℃ for 30 seconds, 45 cycles.

[0066] The results are as follows Figure 1 As shown, it is easy to see that magnetic bead suspensions with capture probes can be extracted more efficiently, with Capture-390-409 exhibiting the best capture efficiency.

[0067] Example 2

[0068] This embodiment further investigates the stability of Capture-390-409 by repeatedly performing the operations described in Embodiment 1, with the following results: Figure 2 As shown, the method provided by this invention can efficiently and stably extract GBS RNA.

[0069] Example 3

[0070] To demonstrate the high extraction efficiency and stability of the kit provided by this invention, this embodiment verifies the effectiveness of the kit provided in Example 2 in extracting RNA from bacteria and compares the effectiveness of this extraction method with other kit methods, including the UNlQ-10 column-based total RNA extraction kit (Shanghai Sangon Biotech, B511361), the cultured cell / bacterial total RNA extraction kit (Tiangen Biotech, DP430), and the bacterial total RNA rapid extraction kit - centrifuge column type (Zhuangmeng Biotechnology, ZP403). Using the same sample of bacteria, the procedures outlined in Example 1 and the instructions of the corresponding kits were followed, and the extracted RNA was detected using an RT-qPCR kit.

[0071] RT-qPCR results of RNA extracted by four methods are as follows Figure 3 As shown, the efficiency and stability of RNA extraction are as follows: Figure 4 As shown in the figure. The above results demonstrate that the kit provided by this invention has better RNA extraction efficiency and stability compared with other commercial kits.

[0072] Example 4

[0073] To verify the analytical performance of this kit in extracting bacterial RNA, the RNA extracted in Example 2 was diluted 10-fold, 100-fold, 1000-fold, 10000-fold, and 100000-fold, respectively. Real-time quantitative PCR was performed according to the method in Example 1. The linear regression equation was Y = -3.438X + 43.205, with a correlation coefficient of 0.993. Each sample was tested three times. Specific detection results are shown below. Figure 5 and Figure 6 As shown, the results exhibit a good linear relationship.

[0074] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. A set of GBSRNA hydroxyl magnetic bead extraction and capture probes, characterized in that, It includes 5 nucleotide sequences, as shown in SEQ ID NO:1 to SEQ ID NO:5 respectively.

2. A kit for extracting hydroxyl magnetic beads from GBS RNA, characterized in that, It includes a lysis buffer, a binding buffer, a washing buffer, a magnetic bead suspension modified with a capture probe, DNase, and a DNase reaction solution, wherein the capture probe is one of the five nucleotide sequences described in claim 1.

3. The GBSRNA hydroxyl magnetic bead extraction kit as described in claim 2, characterized in that, The lysis buffer consists of lysozyme, RNA inhibitor, and buffer solution. The concentration of the lysozyme is 0.1-100 mg / mL. The RNA inhibitor is selected from one or more of guanidine hydrochloride, urea, guanidine isothiocyanate, and vanadate ribonucleoside complex. The buffer solution is one or more of Tris-HCl, EDTA, KCl, NaCl, and MgCl2.

4. The GBSRNA hydroxyl magnetic bead extraction kit as described in claim 2, characterized in that, The binding solution consists of one or more of Tris-HCl, EDTA, NaCl, NaOH, MgCl2, and SDS.

5. The GBSRNA hydroxy magnetic bead extraction kit as described in claim 4, characterized in that, The binding solution consists of 20–40 mM Tris-HCl with pH 7.0–8.0, 0.5–5.0 M NaCl, 1–20 mM MgCl2, and 10–20 mM EDTA.

6. The GBSRNA hydroxyl magnetic bead extraction kit as described in claim 2, characterized in that, The washing solution consists of washing solution I and washing solution II. Washing solution I is composed of one or more of Tris-HCl, EDTA, NaCl, NaOH, MgCl2 and SDS, and washing solution II is one of DEPC water, RNase-free water or double-distilled water.

7. The GBSRNA hydroxyl magnetic bead extraction kit as described in claim 6, characterized in that, The washing solution I consists of 20–40 mM Tris-HCl with pH 7.0–8.0, 0.1–1 M NaCl, 0.1–1 mM MgCl2, and 10–20 mM EDTA.

8. The GBSRNA hydroxyl magnetic bead extraction kit as described in claim 2, characterized in that, The process for treating the magnetic bead suspension modified with the capture probe is as follows: the streptavidin-modified magnetic beads are soaked in 1% diethyl pyrocarbonate solution for 5-10 hours, washed 3-5 times with PBS buffer at pH 7.0, and then biotin-modified capture probes are added and incubated at 37°C for half an hour. The mixture is then stored in a 4°C refrigerator for later use.

9. A method for extracting GBSRNA using the kit described in any one of claims 2 to 8, characterized in that, The method includes the following steps: (1) Take a certain amount of cultured GBS into a 2mL centrifuge tube, add 100μl of lysis buffer, and shake at 37℃ to mix for a period of time; (2) Add 10 μl of magnetic bead suspension and 100 μl of binding solution to a test tube, and shake at 37°C to mix for a period of time. (3) Place the incubated sample on a magnetic rack and let it stand until it clarifies, then discard the supernatant; (4) Add 200 μl of washing solution 1, mix thoroughly, place on a magnetic rack and let stand until clear, then discard the supernatant; (5) Add 100 μl of washing solution 2, mix thoroughly, place on a magnetic rack and let stand until clear, then discard the supernatant; add 20 μl of washing solution 2, mix thoroughly, and incubate at 65°C for a period of time. (6) Quickly place the incubated sample on a magnetic rack to stand still. After clarification, immediately aspirate the supernatant into a new EP tube. (7) Add 10 μL of UDNase and 10 μL of DNase reaction solution to the above-obtained solution, incubate at 60°C for a period of time, and then incubate at 85°C for 10 min to inactivate the enzymes to obtain high-purity target nucleic acid.

10. The method as described in claim 9, characterized in that, The shaking and mixing time in step (1) is 1-15 minutes, the shaking and mixing time in step (2) is 1-15 minutes, the incubation time in step (5) is 1-15 minutes, and the incubation time in step (7) is 1-30 minutes.

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