Nucleic acid extraction-free RT-qPCR quantitative analysis method for LNP-mRNA in plasma

Abstract

The invention discloses a nucleic acid extraction-free RT-qPCR quantitative analysis method for LNP-mRNA in plasma, the method comprises four steps of chemical pretreatment, physical pretreatment, reverse transcription and qPCR, the chemical pretreatment uses PBS (named as PBST / I) containing 0.5% of Triton X-100 and 0.2% of IGEPAL CA-630 for splitting decomposition, and the physical pretreatment uses a 95 DEG C high temperature to treat a sample. According to the method provided by the invention, time-consuming and labor-consuming nucleic acid extraction operation is not needed, time and cost are saved, and more importantly, the loss of target mRNA in the nucleic acid extraction process is avoided by omitting the nucleic acid extraction and purification step; the condition that the loss degrees of different samples are greatly different due to instability and uncontrollability of the extraction efficiency is prevented, so that the detection result is more accurate and reliable.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for quantitatively analyzing LNP-mRNA in plasma by RT-qPCR without nucleic acid extraction. Background Art

[0002] In recent years, as a new type of drug, mRNA has great development potential in the research and development of innovative drugs for various diseases. The nanolipid particle (LNP) delivery system can encapsulate mRNA in the LNP structure to form a stable LNP-mRNA complex, protect mRNA from nuclease degradation, and prolong the half-life of mRNA. At the same time, LNP encapsulation can also improve the efficiency of mRNA molecules passing through the cell membrane and improve its absorption in cells. Targeted delivery can be achieved by modifying the ingredients and modifying the surface of LNP to increase antibodies. With the successful launch of SARS-CoV-2 vaccines using LNP-mRNA molecules, LNP-mRNA has been used in various types of biopharmaceutical products such as preventive and therapeutic vaccines, in vitro and in vivo gene editing drugs, etc. In the research and development and production of LNP-mRNA drugs, mRNA needs to be quantitatively detected and analyzed. Real-time fluorescence quantitative reverse transcription polymerase chain reaction RT-qPCR is a commonly used mRNA detection and analysis method.

[0003] The use of RT-qPCR technology to quantitatively analyze mRNA in plasma samples usually requires a pretreatment step for the biological matrix sample, first separating the mRNA from the LNP-mRNA polymer package, then enriching and purifying to obtain high-purity mRNA, and then using it as a PCR template for subsequent RT-qPCR amplification reactions. If pretreatment is not performed, it will be difficult for mRNA nucleic acid molecules to contact specific primers and probes, and thus it will be difficult to perform RT-qPCR reactions; in addition, due to the complex composition of biological matrices such as plasma, there are a large number of substances that affect the PCR reaction. If no treatment is performed, the RT-qPCR amplification reaction will be severely inhibited, resulting in the failure of PCR analysis. In addition, inappropriate pretreatment will also cause mRNA to be degraded by extracellular nucleases during the extraction process.

[0004] In the prior art, common mRNA pretreatment methods include: organic solvent extraction, boiling, surfactant direct demulsification, purification column, etc., and the extraction method is to extract mRNA by centrifugation, precipitation dissolution or chromatographic separation. However, these conventional methods will inevitably have the following problems in the nucleic acid extraction process: 1. Loss of target mRNA molecules; 2. Poor extraction stability; 3. Large differences in nucleic acid extraction recovery rates between batches, resulting in inaccurate mRNA quantitative analysis results; 4. The extraction process is too complicated, time-consuming and labor-intensive, resulting in increased costs.

[0005] Therefore, there is an urgent need to develop a highly specific and versatile RT-qPCR method that does not require nucleic acid extraction to quantitatively analyze mRNA in plasma, which can be well applied to monitoring the pharmacokinetic / toxicokinetic changes of the active ingredient mRNA of LNP-mRNA drugs in plasma. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for quantitatively analyzing LNP-mRNA in plasma by RT-qPCR without nucleic acid extraction, comprising the following steps:

[0007] 1) Chemical pretreatment: prepare lysis buffer, mix the lysis buffer and plasma sample thoroughly, and place them on a PCR thermal cycler for low-temperature incubation;

[0008] 2) Physical pretreatment: Add DEPC-treated water to the sample after incubation in step 1), mix well, place on a PCR thermal cycler for high-temperature incubation, then cool and store, centrifuge after incubation, and take the supernatant as a reverse transcription template;

[0009] 3) Reverse transcription: Design specific forward primers, reverse primers and probes for the mRNA to be tested, and reverse transcribe the reverse transcription template obtained in step 2). During reverse transcription, add the reverse primer to the reverse transcription system to obtain cDNA by reverse transcription;

[0010] 4) Fluorescence quantitative PCR amplification: Using the cDNA obtained in step 3) as a template, a qPCR reaction system is prepared using a forward primer, a reverse primer, a probe and a qPCR Mix to perform fluorescence quantitative PCR amplification to complete quantitative analysis of the sample.

[0011] Specifically, in step 1), the lysis solution includes 0.5% Triton X-100, 0.2% IGEPAL CA-630 and PBS.

[0012] Specifically, in step 1), the volume ratio of the lysate and the plasma sample is 1:1.

[0013] Specifically, in step 1), the low-temperature incubation refers to incubation at 37° C. for 10 min.

[0014] Specifically, in step 2), the volume of DEPC-treated water added is 3-5 times the volume of the plasma sample.

[0015] Specifically, in step 2), the high temperature incubation refers to incubation at 95° C. for 10 min.

[0016] Specifically, in step 2), the cooling refers to cooling to 16°C.

[0017] Specifically, in step 2), the centrifugation condition is 20000g at 4°C for 5 min.

[0018] Specifically, in step 3), the reverse transcription system also includes a reverse transcription template, a reverse transcription premix and DEPC-treated water.

[0019] Specifically, in step 3), in the reverse transcription system, the concentration of the reverse primer is 10 μM, and the amount of the reverse primer added is 1 μL.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The method provided by the present invention first uses chemical reagents to cleave LNP to release mRNA, then denatures substances in plasma that are unfavorable to PCR reaction at 95°C, and finally obtains supernatant as PCR template by centrifugation. Through chemical and physical pretreatment of plasma samples, the inhibitory effect of plasma matrix on PCR reaction can be minimized, so that the RT-qPCR quantification of mRNA can be carried out smoothly and accurate test results can be obtained.

[0022] 2. The method provided by the present invention has simple chemical pretreatment and physical pretreatment processes, which can be completed in only about 30 minutes. Compared with the conventional nucleic acid extraction operation that takes about 5 hours to complete, the method provided by the present application significantly shortens the sample pretreatment time and greatly improves the efficiency of RT-qPCR.

[0023] 3. In the method provided by the present invention, Triton X-100, IGEPAL CA-630 and PBS used in the chemical pretreatment are all conventional reagents. Compared with conventional nucleic acid extraction operations, the price is much lower, which significantly reduces the reagent cost; especially when facing the demand for high sample volume detection, the reagent cost of sample processing can be ignored when using the method provided by the present application for detection, while the cost of the extraction kit for ordinary nucleic acid extraction will increase linearly. Therefore, the method provided by the present application significantly reduces the cost of detection and has good economic value and practicality.

[0024] 4. Conventional nucleic acid extraction methods inevitably cause the loss of target mRNA molecules due to the presence of multiple steps of repeated elution during the nucleic acid purification process, and the degree of loss between each sample is technically uncontrollable, that is, the extraction efficiency is also correspondingly uncontrollable, therefore, the mRNA quantitative analysis results are unstable and the accuracy is poor. The method provided by the present invention does not require nucleic acid extraction operations, therefore, there is no extraction-related mRNA loss, and the amount of plasma samples is also reduced; in addition, the method provided by the present application has a unified and simple processing method for all samples, which reduces the uncontrollable errors generated in the technology, and can obtain more accurate, stable and reliable mRNA quantitative analysis results. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the process of sample pretreatment according to the method provided by the present invention.

[0026] Figure 2 It is a schematic diagram of the process of reverse transcription according to the method provided by the present invention.

[0027] Figure 3 It is a schematic diagram of the process of performing fluorescent quantitative PCR amplification according to the method provided by the present invention.

[0028] FIG4 is a qPCR quantitative analysis of LNP-Cas9 mRNA amplification curve and standard curve in plasma in Example 1 of the present invention, wherein: Figure 4A is the amplification curve, Figure 4B is the standard curve.

[0029] Figure 5 This is the qPCR amplification curve of LNP-Cas9 mRNA plasma samples with and without pretreatment in Comparative Example 1 of the present invention.

[0030] Figure 6 It is a comparison diagram of qPCR amplification curves with and without adding a specific reverse primer to the reverse transcription system in Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0031] The technical scheme of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The materials, instruments and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0032] Example 1 Detection of Cas9 mRNA encapsulated in LNP using the method provided by the present invention

[0033] 1. Prepare plasma samples containing LNP-Cas9 mRNA. Take out the plasma matrix from the -80℃ refrigerator, thaw the plasma at room temperature, and use the plasma as a diluent to prepare a series of standard plasma samples with known LNP-Cas9 mRNA gradient dilution concentrations.

[0034] 2. Prepare lysis buffer, which is PBS lysis buffer containing 0.5% Triton X-100 and 0.2% IGEPAL CA-630, named PBST / I.

[0035] 3.Reference Figure 1 The process diagram shown in the figure is used for pretreatment. Take 5 μL of each standard plasma sample, then add 5 μL of PBST / I lysis solution in equal proportion, shake and mix, place on the PCR thermal cycler, and set the program to incubate at 37°C for 10 minutes. After completion, take out the sample, add 40 μL of DEPC-treated water to each sample, shake and mix. Then place the sample on the PCR thermal cycler, set the program to incubate at 95°C for 10 minutes, and hold at 16°C. After the above treatment, take out the sample, centrifuge at 20,000g for 5 minutes, and take the plasma supernatant directly as a template for PCR.

[0036] 4. Design specific forward primers, reverse primers and TaqMan probes for the Cas9 mRNA sequence. The sequence information is shown in the following table:

[0037]

[0038] 5. Prepare 20 μL reverse transcription system: Figure 2 The reverse transcription process schematic diagram shown in the figure, 4μL LunaScript RT SuperMix (5X) (NEB reverse transcription kit, catalog number E3010L), 1μL reverse primer (10μM), 10μL DEPC treated water and 5μL plasma supernatant PCR template obtained after pretreatment are added to the PCR reaction tube in sequence, oscillated and mixed evenly, and then centrifuged instantaneously to collect the reaction solution to the bottom of the reaction tube. Then, it is placed in a PCR thermal cycler and first set to 25℃ for 2min to anneal so that the primer and template can be complementary to each other; then set to 55℃ for 10min to make the reverse transcriptase have the maximum enzyme activity, and the template paired with the reverse primer is reverse transcribed to synthesize cDNA in the 3' direction of the reverse primer; finally, it is set to 95℃ for 1min to heat inactivate the reverse transcriptase.

[0039] 6.Finally, perform qPCR amplification: refer to Figure 3As shown in the process diagram, 5 μL of TaqMan FastAdvanced Master Mix (Thermo fisher, catalog number 4444557) was added to a 384-well plate, followed by 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 0.2 μL of 10 μM TaqMan probe, and 1 μL of cDNA template. The mixture was made up to 10 μL with DEPC-treated water, and then the mixture was shaken and mixed, and then centrifuged instantaneously, and then fluorescent quantitative PCR amplification was performed in a qPCR instrument. The Ct value obtained by qPCR amplification is inversely proportional to the mRNA concentration, that is, the larger the Ct value, the lower the mRNA concentration. The standard curve is drawn with the Ct value of the known concentration of the gradient diluted LNP-Cas9 mRNA plasma standard sample as the ordinate and the logarithm of the concentration as the abscissa. The true concentration of the sample to be tested can be calculated by the Ct value of the sample to be tested. As shown in Figure 4, the results showed that the quantitative range of direct RT-qPCR analysis of LNP-Cas9 mRNA in plasma without nucleic acid extraction was 1.6 pg / mL to 100000 pg / mL, with a sensitivity of 1.6 pg / mL.

[0040] Comparative Example 1: Plasma pretreatment and plasma not pretreated but directly used as PCR template:

[0041] By using chemical reagents in Example 1 to treat at a specific temperature (37°C) for a specific time (10 minutes), the complete structure of LNP can be effectively destroyed, and mRNA can be released from the LNP encapsulation state, which is beneficial to the binding of PCR primers and enzymes to the template. In addition, the plasma is diluted with DEPC-treated water to a certain volume (5 times the volume), and then treated at high temperature (37°C) for a specific time (10 minutes). It can ensure that the high temperature will not cause the high-concentration plasma sample to coagulate, while ensuring that plasma proteins, lipids, etc. are denatured without degrading the target mRNA. Finally, the supernatant is taken by centrifugation (20000g centrifugation for 5 minutes), and the interference of the plasma matrix on PCR amplification is further reduced. In this comparative example 1, the plasma is not pretreated, and other operations are the same as in Example 1 for detection. Expected results: Due to the encapsulation of LNP, mRNA cannot come into contact with the PCR reagent. At the same time, the large amount of proteins, lipids, etc. present in the plasma will also seriously inhibit the PCR reaction. Actual results: As Figure 5 As shown, it can be seen that if the plasma is not pretreated, the Ct value will be too large, making it impossible to amplify effectively.

[0042] Comparative Example 2: Comparative experiment of adding reverse primer and not adding reverse primer in the reverse transcription system:

[0043] Adding a certain concentration (10 μM) and volume (1 μl) of reverse primer to the reverse transcription system in Example 1 can increase the binding of specific primers to target mRNA, and significantly enhance the efficiency of reverse transcriptase in synthesizing specific cDNA using target mRNA as a template. In this comparative example 2, no specific reverse primer was added to the reverse transcription system, but only random primers were used, and the remaining operations were the same as in Example 1 for detection. Expected results: Random primers will be used by all RNAs, and random reverse transcription will also be performed on the full-length sequence of mRNA, which will significantly reduce the efficiency of synthesizing specific cDNA fragments, and the amount of specifically synthesized cDNA will be less. The qPCR results show that the Ct value is too large, and the sensitivity of the analysis is lower than that of the reverse transcription system of the present invention with the addition of specific reverse primers. Actual results, such as Figure 6 As shown in the figure, adding specific reverse primers to the reverse transcription system resulted in better PCR amplification, earlier Ct values, and fewer cycles. However, samples without specific reverse primers showed poor PCR amplification and lower efficiency.

[0044] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for quantitatively analyzing LNP-mRNA in plasma by RT-qPCR without nucleic acid extraction, characterized in that: The following steps are involved: 1) Chemical pretreatment: prepare lysis buffer, mix the lysis buffer and plasma sample thoroughly, and place them on a PCR thermal cycler for low-temperature incubation; 2) Physical pretreatment: Add DEPC-treated water to the sample after incubation in step 1), mix well, place on a PCR thermal cycler for high-temperature incubation, then cool and store, centrifuge after incubation, and take the supernatant as a reverse transcription template; 3) Reverse transcription: Design specific forward primers, reverse primers and probes for the mRNA to be tested, and reverse transcribe the reverse transcription template obtained in step 2). During reverse transcription, add the reverse primer to the reverse transcription system to obtain cDNA by reverse transcription; 4) Fluorescence quantitative PCR amplification: Using the cDNA obtained in step 3) as a template, a qPCR reaction system is prepared using a forward primer, a reverse primer, a probe and a qPCR Mix to perform fluorescence quantitative PCR amplification to complete quantitative analysis of the sample.

2. The method for quantitatively analyzing LNP-mRNA in plasma by RT-qPCR without nucleic acid extraction according to claim 1, characterized in that: In step 1), the lysis solution includes 0.5% Triton X-100, 0.2% IGEPAL CA-630 and PBS.

3. The method for quantitatively analyzing LNP-mRNA in plasma by RT-qPCR without nucleic acid extraction according to claim 1, characterized in that: In step 1), the volume ratio of the lysate to the plasma sample is 1:

1.

4. The method for quantitatively analyzing LNP-mRNA in plasma by RT-qPCR without nucleic acid extraction according to claim 1, characterized in that: In step 1), the low-temperature incubation refers to incubation at 37° C. for 10 min.

5. The method for quantitatively analyzing LNP-mRNA in plasma by RT-qPCR without nucleic acid extraction according to claim 1, characterized in that: In step 2), the volume of DEPC-treated water added is 3-5 times the volume of the plasma sample.

6. The method for quantitatively analyzing LNP-mRNA in plasma by RT-qPCR without nucleic acid extraction according to claim 1, characterized in that: In step 2), the high temperature incubation refers to incubation at 95° C. for 10 min.

7. The method for quantitatively analyzing LNP-mRNA in plasma by RT-qPCR without nucleic acid extraction according to claim 1, characterized in that: In step 2), the cooling refers to cooling to 16°C.

8. The method for quantitatively analyzing LNP-mRNA in plasma by RT-qPCR without nucleic acid extraction according to claim 1, characterized in that: In step 2), the centrifugation condition is 20000g at 4°C for 5 min.

9. The method for quantitatively analyzing LNP-mRNA in plasma by RT-qPCR without nucleic acid extraction according to claim 1, characterized in that: In step 3), the reverse transcription system also includes a reverse transcription template, a reverse transcription premix and DEPC-treated water.

10. The method for quantitatively analyzing LNP-mRNA in plasma by RT-qPCR without nucleic acid extraction according to claim 1, characterized in that: In step 3), in the reverse transcription system, the concentration of the reverse primer is 10 μM, and the amount of the reverse primer added is 1 μL.

Images