WO2024046097A1 - Method for measuring terminal transferase activity and kit - Google Patents

Abstract

The present invention provides a method for measuring terminal transferase activity and a kit, and relates to the technical field of molecular biology. With the measurement method provided, the terminal transferase activity of reverse transcriptase can be truly and accurately measured, so that the template conversion efficiency of a reverse transcription reaction can be reflected more truly and accurately, thereby providing a scientifically accurate evaluation for the efficiency of capturing cell mRNA. The method provided is suitable for adjusting and testing reverse transcription systems. The method is simple and cheap, and the measurement period is short; the method can meet the requirements of large-scale measurement.

Description

A method and kit for detecting terminal transferase activity

Cross-references to related applications

This disclosure claims the priority of the Chinese patent application submitted to the China Patent Office on August 29, 2022 with the application number "202211042763.3" and the invention title "A method and kit for detecting terminal transferase activity", all of which The contents are incorporated into this disclosure by reference.

Technical field

The present disclosure relates to the technical field of molecular biology, and relates to a method and a kit for detecting terminal transferase activity.

Background technique

The template switching effect of RNA reverse transcription reaction refers to that during the entire reverse transcription process, reverse transcriptase first uses RNA as a template to extend, and then uses its terminal deoxyribonucleotidyl transferase (Tdt) activity to switch the extension of RNA as a template. to DNA-templated extension. This process mainly utilizes the three major activities of reverse transcriptase: (1) DNA polymerization activity using RNA as template; (2) terminal deoxyribonucleotidyl transferase (Tdt) activity; (3) DNA using DNA as template polymerization activity. Among them, Tdt activity is the most critical factor affecting the template switching effect. Tdt activity refers to the ability to add several nucleotides to the 3′ end of a synthetic product without the need for a template. Under certain conditions, reverse transcriptase can exhibit Tdt activity. For example, M-MLV reverse transcriptase will preferentially add cytosine to the 3' end of the amplicon. In the reverse transcription reaction, the principle of the template switching effect of reverse transcriptase is: reverse transcriptase first uses mRNA as a template, and after extending its 5' end, it uses the Tdt activity of reverse transcriptase to add several peptides to the 3' end of the extension product. Cytidylic acid, these few cytidylic acid can be annealed to a template-switching oligo (TSO) with a corresponding number of guanylic acid at the 3' end, so that the extension product can be a TSO molecule. New DNA template implementations continue to be extended.

Currently, many single cell transcriptome sequencing (Single cell RNA-Seq, ScRNA-seq) solutions mostly use rapid amplification of cDNA ends (RACE) technology to construct cDNA samples, and the key step in the implementation of RACE relies on template conversion. Effect of reverse transcription reaction. RACE based on the template switching effect has the following advantages when applied to ScRNA-seq: (1) It can quickly construct and amplify cDNA. The fast here means that it does not need to use random primers or oligodT primers, but uses a specific primer GSP to amplify one strand of cDNA of the target mRNA. At this time, this one-strand cDNA will have a TSO-binding region at the 3' end and a GSP primer-binding region at the 5' end due to the template switching effect. In this way, primers can be designed for rapid amplification of cDNA using these two regions; (2) The TSO region can introduce unique molecular or cell tags (UMI, Cell barcode), which allows every cell or even every mRNA to be With a unique label, it is convenient to correct and trace the source of each read after sequencing. This method allows each cell to carry a unique identity tag, which facilitates the tracing of the sequencing information of each cell. Therefore, RACE based on the template switching effect helps reduce the difficulty of analysis based on high-throughput sequencing. .

It can be seen that the key to realizing RACE and applying it to ScRNA-seq is the template switching effect in the reverse transcription reaction. The main factor affecting the template switching effect is the Tdt activity of reverse transcriptase. The higher the Tdt activity, the higher the transcription efficiency of the reverse transcription template. Therefore, the higher the concentration of the one-strand cDNA that successfully converted the template (successfully carrying TSO), which means The more efficiently cellular mRNA is captured, the greater the abundance and accuracy obtained after sequencing. On the contrary, if the Tdt activity is low and the amount of cellular mRNA captured is low, then the final sequencing result will have a low degree of recovery of the sequence information of the cell sample. Therefore, accurate and simple measurement of the template conversion efficiency of the reverse transcription reaction is a key point to ensure the success of ScRNA-seq and even to apply this sequencing technology to industrial production.

At present, almost all methods for measuring template conversion efficiency are characterized based on cDNA with successful product-template conversion (successfully carrying TSO). However, these techniques cannot directly measure the concentration of cDNA with successful template conversion. This is because even if the Tdt activity of reverse transcriptase is high, the template conversion efficiency cannot completely reach 100%. Therefore, the reverse transcription product contains both cDNA with successful template conversion and cDNA with failed template conversion (without TSO). However, direct concentration measurement can only measure the concentration of total cDNA but cannot accurately measure the concentration of cDNA that has been successfully converted into templates. Therefore, many methods for measuring reverse enzyme Tdt activity are characterized by cDNA indicators that can reflect the success of template conversion. Existing methods for measuring reverse enzyme Tdt activity include mass spectrometry coupled capillary electrophoresis, fluorescent dye-based qPCR and sequencing methods.

Among them, the mass spectrometry-coupled capillary electrophoresis method uses the reverse transcriptase to be tested and primers with fluorescent markers (FAM) to perform RACE of RNA of a specific length, and then uses mass spectrometry and capillary electrophoresis to analyze the obtained cDNA products. Finally, the template conversion efficiency was characterized based on the peak area of the cDNA that was successfully converted on the plate. The qPCR method based on fluorescent dyes first uses an ordinary PCR instrument to perform reverse transcription and template conversion, and then uses the cDNA product as a template to perform a qPCR reaction based on the fluorescent dye method. Finally, the Ct value is used to characterize the successful template conversion of cDNA. The sequencing method uses first-generation sequencing or second-generation sequencing to obtain the template conversion efficiency of reverse transcription by performing sequence analysis and statistics on cDNA samples. However, these technologies have more or less defects and shortcomings, which are likely to hinder the application of this detection method in industrial production.

The above-mentioned current methods for measuring the template conversion efficiency of reverse transcription reactions have a major problem: the existing technology only characterizes the template conversion efficiency by using cDNA indicators that reflect successful template conversion. However, the template conversion efficiency of the reverse transcription reaction cannot completely reach 100%, which means that there will be cDNA with failed template conversion in the reverse transcription product. Therefore, if the template conversion efficiency is only characterized by measuring the relevant indicators of cDNA with successful template conversion Not entirely true and accurate.

In addition, there are many factors that affect template conversion efficiency, such as the type of reverse transcriptase, buffer components, input amounts of templates and primers, etc. Therefore, if only cDNA with successful template conversion is used as a detection indicator, it cannot meet the requirements of system debugging.

Contents of the invention

The purpose of this disclosure is to provide a method and kit for detecting terminal transferase activity to truly and accurately detect the terminal transferase activity of reverse transcriptase, more truly and accurately reflect the template conversion efficiency of the reverse transcription reaction, and further provide a method for cellular mRNA to be Capture efficiency provides scientifically accurate evaluation. The proposal of this disclosure will help the success of ScRNA-seq and the application of this sequencing technology to industrial production. The method provided by the present disclosure is suitable for debugging the reverse transcription system, has simple operation steps, low cost, and short detection cycle, and can meet the needs of large-scale detection.

This disclosure is implemented as follows:

The present disclosure provides a method for detecting terminal transferase activity, which includes the following steps:

(a) Anneal the reverse transcription primer to the RNA template to generate a reverse transcription primer-RNA hybrid strand.

(b) Use reverse transcriptase to perform RNA template-dependent DNA extension to obtain an RNA-cDNA intermediate with sticky ends.

(c) The 3′ region on TSO anneals to the sticky end of the RNA-cDNA intermediate; and using TSO as a template to extend the 3′ end of the cDNA chain of the RNA-cDNA intermediate to obtain cDNA after the template switching reaction;

(d) Set a reverse primer in the reverse transcription primer region, set a forward inner primer in the RNA template, set a forward outer primer in the TSO, use the product of the above step (c) as a template, and obtain the forward inner primer through qPCR reaction With the Ct1 value of the amplification product of the reverse primer, obtain the Ct2 value of the amplification product of the forward outer primer and the reverse primer through qPCR reaction, and calculate the difference ΔCt between the Ct1 value and the Ct2 value to obtain the activity of terminal transferase; qPCR reaction Use the same probe. The inventor discovered that reverse transcription primers (RT primer) and reverse transcriptase are used to perform reverse transcription based on the template switching effect. The reverse transcription product can be directly used in qPCR reactions after dilution. The qPCR reaction includes two reaction systems, the inner reaction system (Inner primer system) composed of forward inner primer and reverse primer, the inner reaction system (Outer primer system) composed of forward outer primer and reverse primer, and the forward inner primer It anneals and amplifies only the RNA template region of cDNA, and the forward outer primer anneals and amplifies only the TSO region of cDNA.

The obtained Inner system Ct value can characterize the amount of total cDNA in the reverse transcription product, while the Outer system Ct value is used to characterize the amount of cDNA that has been successfully converted from the template. Therefore, the difference (ΔCt) between the Ct value of the Outer system and the Ct value of the Inner system can accurately reflect the template conversion efficiency. The smaller the difference (ΔCt), the higher the amount of cDNA that has been successfully converted to the template, that is, the higher the concentration of one-strand cDNA that has been successfully converted to the template (successfully carrying TSO), indicating that high Tdt activity indicates the template transcription efficiency of reverse transcription. high. When applied to ScRNA-seq, this means that the more efficiently cellular mRNA is captured, the greater the abundance and accuracy obtained after sequencing.

On the contrary, the larger the difference (ΔCt), the lower the amount of cDNA that has been successfully converted to the template, that is, the lower the concentration of one strand of cDNA that has been successfully converted to the template (successfully carrying TSO), indicating low Tdt activity, and the reverse transcription template Transcription efficiency is low. When applied to ScRNA-seq, this means that the amount of cellular mRNA captured is low, and the final sequencing result obtained after sequencing has a low degree of recovery of the sequence information of the cell sample.

Therefore, the difference (ΔCt) between the Ct value of the Outer system and the Ct value of the Inner system can accurately reflect the template conversion efficiency. The position of the external primer has a certain impact on the Ct value. Theoretically, if ΔCt is used to characterize the conversion efficiency of the template, the amplification efficiency of the outer primer and the inner primer should be as close as possible. Therefore, the closer the distance between the outer primer and the inner primer to each other, the better.

The probes involved in this disclosure should be close to the reverse primer, but the distance of the inner and outer primers from the probe does not have much impact.

In addition, the background technology mentioned above needs to degrade the RNA template and purify the cDNA product before measuring the template conversion efficiency. Otherwise, it will affect the accuracy of measuring the template conversion efficiency. However, the RNA template used in the existing technology is only 20-30nt. , so the cDNA obtained by reverse transcription is shorter, which will increase the difficulty of cDNA purification. Moreover, the steps of degrading RNA templates and purifying cDNA are relatively cumbersome, which limits the application of the background technology to large-scale industrial detection.

The detection method provided by the present disclosure does not require the degradation of the RNA template and the purification of the cDNA product after the reverse transcription reaction, which greatly simplifies the entire operation process. It is more suitable for debugging the reverse transcription system of template switching effect and large-scale industrial detection.

Existing mass spectrometry-coupled capillary electrophoresis and sequencing methods have cumbersome detection steps and high costs. Especially for sequencing methods, a series of tedious operations such as library construction or plasmid construction are required before sequencing, and the sequencing cycle time is also very high. period is relatively long. Therefore, these detection technologies cannot obtain detection results in a short time. It is difficult to apply to large-scale detection and debugging of reverse transcription systems.

The detection method provided by the present disclosure has the advantages of low cost and short detection cycle. Therefore, the present disclosure is suitable for debugging the reverse transcription system of template switching effect and large-scale industrial detection.

Both amplifications use the same reverse primer and use a Taqman probe to avoid differences in amplification efficiency due to different probes, reverse primer positions or sequences, which is conducive to more accurate Characterize template conversion efficiency.

In an optional embodiment, the forward inner (or outer) primer and the reverse primer can also be appropriately diluted as needed to perform subsequent qPCR reactions.

The reverse transcription primer in step (a) is a stem-loop reverse transcription primer or a Poly T reverse transcription primer. Poly T reverse transcription primers can be selected from universal Poly T reverse transcription primers. As shown in SEQ ID NO:8:

GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCATTTTTTTTTTTCAAGCT.

The RNA-cDNA intermediate with sticky ends in step (b) adds multiple identical deoxynuclei to the 3′ end of the cDNA chain of the RNA-cDNA intermediate through the terminal transferase activity of the reverse transcriptase itself. Obtained by glucoside method.

In steps (a)-(c), reverse transcriptase, dNTPs, RNase inhibitors, template-switching oligonucleotides (TSO), and buffers are used to perform RNA template-dependent DNA extension. The reverse transcription product can be directly used in qPCR reactions after dilution.

In the above step (b), the reverse transcriptase buffer contains MnCl ₂ , and the final concentration of MnCl ₂ is 4-16mM; the inventor found that in the reverse transcription system, after the concentration of MnCl ₂ is diluted to the above concentration, a better template is obtained conversion efficiency. For example, 8-16mM, 4-8mM, 4-6mM.

In an alternative embodiment, the final concentration of _MnCl2 is 4-8mM.

In a preferred embodiment of the present disclosure, the plurality of deoxynucleotides added at the end in step (b) are a plurality of cytosine deoxynucleotides (C).

In an optional embodiment, three cytosine deoxynucleotides (C) are added to the end in step (b).

In an alternative embodiment, the 3' annealing region on the TSO contains three ribonucleotide residues.

In an alternative embodiment, the 3' annealing region contains three nuclear guanine (rG) ribonucleotides.

In certain embodiments, the 5' end region of the TSO further includes one or more of the following: barcode sequence, unique molecular identifier (UMI), amplification primer sequence, sequencing primer sequence, capture primer sequence, sequence-specific nucleic acid Enzyme cleavage sites, modified nucleotides, biotinylated nucleotides, and 5' modifications, etc.

The cDNA product obtained in the above step (c) needs to be diluted 10-100 times for use in the qPCR reaction of step (d). Under the above dilution factors, Taqman-qPCR technology can be used to obtain accurate results of template conversion efficiency. This avoids the tedious steps of degrading the RNA template and purifying the cDNA product.

In an optional embodiment, the dilution factor of the cDNA product obtained in step (c) is 50 times.

Since there are many factors that affect template conversion efficiency, such as the type of reverse transcriptase, buffer components, input amounts of templates and primers, etc., the inventor also screened the concentrations of reverse transcription primers and TSO in the reverse transcription system, as follows :

In a preferred embodiment of the present disclosure, in the reverse transcription reaction system, when 10 nM miRNA is used as the template, the concentration of TSO is 2-100 μM, and the concentration of the reverse transcription primer is 2-100 μM.

The inventor found that after debugging the reverse transcription reaction system and optimizing the amounts of TSO and reverse transcription primers used in the reverse transcription reaction, more accurate template conversion efficiency results can be obtained. Further in an optional embodiment, Taqman-qPCR technology can also be used to obtain accurate results of template conversion efficiency. This avoids the tedious steps of degrading the RNA template and purifying the cDNA product.

In an alternative embodiment, the concentration of TSO is 20-100 μM, and the concentration of the stem-loop primer is 20-100 μM. With the above primer concentration, more accurate template conversion efficiency results can be obtained.

In an alternative embodiment, the concentration of TSO is 20-100 μM, and the concentration of the stem-loop primer is 20-100 μM. For example: TSO concentration is 20-50μM, stem loop primer concentration is 20-50μM.

In an optional embodiment, during the qPCR reaction, the final concentration of the forward outer primer is 0.3-0.5 μM, the final concentration of the forward inner primer is 0.3-0.5 μM, and the final concentration of the reverse primer is 0.3 -0.5μM, the final concentration of the probe is 0.1-0.3μM.

In an optional embodiment, the final concentration of the forward outer primer is 0.3 μM, the final concentration of the forward inner primer is 0.3 μM, the final concentration of the reverse primer is 0.3 μM, and the final concentration of the probe is 0.1 μM. .

In a preferred embodiment of the present disclosure, the RNA template is selected from: mRNA, non-coding RNA, miRNA, siRNA, piRNA, lncRNA or ribosomal RNA.

In an alternative embodiment, the RNA template is selected from: miRNA.

Preferably, the reverse transcriptase is M-MLV reverse transcriptase, HIV-1 reverse transcriptase, AMV reverse transcriptase or telomerase reverse transcriptase with reduced or eliminated RNase activity. Different reverse transcriptases have a certain impact on template conversion efficiency. When testing, the same reverse transcriptase should be selected as much as possible to reduce the impact of different reverse transcriptases on the detection of terminal transferase activity.

In a preferred embodiment of the present disclosure, the method for judging the activity of terminal transferase is as follows:

The smaller the ΔCt value, the higher the activity of terminal transferase; or

The larger the ΔCt value, the lower the activity of terminal transferase.

In a preferred embodiment of the present disclosure, the annealing step in step (a) adopts gradient annealing. Gradient annealing helps improve binding efficiency.

The initial annealing temperature of gradient annealing in step (a) is 65°C, and decreases by 0.1°C per second until 4°C, and the annealing time is 15-20 minutes;

In an optional implementation, the annealing time is 16 minutes.

In a preferred embodiment of the present disclosure, the reaction temperature conditions of steps (b) and (c) are determined based on the optimal reaction temperature of reverse transcriptase, and the reaction time is 90-180 minutes;

In an optional embodiment, the reaction time of step (c) is 120 min.

In a preferred embodiment of the present disclosure, the qPCR reaction in step (d) also includes a probe;

In an alternative embodiment, the probe is a Taqman probe. Since Taqman-qPCR technology has higher reaction sensitivity than other technologies, the inventors optimized the amount of primers and the dilution factor of the reverse transcription product (for example, 10-100 times) before using Taqman-qPCR technology. Accurate results of template conversion efficiency can be obtained. This avoids the tedious steps of degrading the RNA template and purifying the cDNA product.

The present disclosure also provides a kit, which includes: qPCR polymerase, reverse primer, forward inner primer, forward outer primer, probe, reverse transcription primer, RNA template, TSO, the reverse primer, forward inner primer, forward outer primer, The nucleotide sequences of the RNA template, TSO, and probe are shown in SEQ ID NO: 1-3 and SEQ ID NO: 5-7, and the nucleotide sequence of the reverse transcription primer is shown in SEQ ID NO: 4 or SEQ ID NO:8, see Table 1.

Table 1

In an optional embodiment, the above-mentioned kit also includes materials such as reverse transcription reaction buffer, dNTPs, qPCR buffer, and water.

Compared with the existing background technology, characterizing the template conversion efficiency only based on cDNA-related indicators of successful template conversion has the problem of not being objective and accurate enough. The detection method provided by this disclosure: divide the reverse transcription product into two parts, one part is used to obtain the Ct1 value of the forward inner primer and reverse primer amplification product through qPCR, and the other part is used to obtain the forward outer primer and reverse primer amplification product through qPCR The Ct2 value, where the Ct1 value can characterize the amount of total cDNA in the reverse transcription product, and the Ct2 value is used to characterize the amount of cDNA that has been successfully converted into a template. Therefore, the difference ΔCt between the Ct1 value and the Ct2 value can accurately reflect the template conversion efficiency.

Both amplifications use the same reverse primer and a Taqman probe to avoid differences in reverse transcription efficiency due to different probes, reverse primer positions or sequences, which is conducive to more accurate Characterize template conversion efficiency.

In addition, the detection method provided by the present disclosure does not require degradation of the RNA template and purification of the cDNA product after the reverse transcription reaction, which greatly simplifies the entire operation process. It is more suitable for debugging the reverse transcription system of template switching effect and large-scale industrial detection.

The detection method provided by the present disclosure has the advantages of low cost and short detection cycle. Therefore, the present disclosure is suitable for debugging the reverse transcription system of template switching effect and large-scale industrial detection.

Description of drawings

In order to explain the technical solutions of the embodiments of the present disclosure more clearly, the drawings needed to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present disclosure and therefore do not should be regarded as a counter-paradigm For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without exerting creative efforts.

Figure 1 is a detection principle diagram of steps (a)-(c) of the detection method provided in Embodiment 1 of the present disclosure;

Figure 2 is a detection principle diagram of step (d) of the detection method provided in Embodiment 1 of the present disclosure;

Figure 3 shows the ΔCt values of different reverse transcriptases at different primer concentrations in Experimental Example 1;

Figure 4 shows the ΔCt values of different reverse transcriptases at higher primer concentrations in Experimental Example 1;

Figure 5 shows the ΔCt values of different reverse transcriptases at different MnCl ₂ concentrations in Experimental Example 2;

Figure 6 is a schematic diagram of the detection of linear primers.

Implementation

Reference will now be provided in detail to embodiments of the present disclosure, one or more examples of which are described below. Each example is provided by way of explanation, not limitation of the disclosure. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the disclosure. For example, features illustrated or described as part of one embodiment can be used in another embodiment, to yield still further embodiments.

Unless otherwise indicated, practice of the present disclosure will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis ( M.J.Gait, ed., 1984); "Animal Cell Culture (Ed., R.I.Freshney, 1987)"; "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (edited by D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (edited by J.M. Miller and M.P. Calos, 1987); "Contemporary "Current Protocols in Molecular Biology" (edited by F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (edited by Mullis et al., 1994); and "Contemporary Current Protocols in Immunology (eds. J.E. Coligan et al., 1991), each of which is expressly incorporated by reference into the present disclosure.

In order to make the purpose, technical solutions, and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

The features and performance of the present disclosure will be described in further detail below in conjunction with examples.

Example 1 Detection of terminal transferase activity of reverse transcriptase

This embodiment provides a method for detecting terminal transferase activity of reverse transcriptase, which specifically includes the following steps.

(a) Use a piece of miRNA as an RNA template and perform gradient annealing with a stem-loop reverse transcription primer (Stem-loop primer). The dosage of each component and reaction conditions are shown in Table 2 and Table 3 below.

In other embodiments, the Poly T reverse transcription primer shown in Figure 6 can also be used to perform gradient annealing. The principle is shown in Figure 6.

Table 2

table 3

(b) Prepare the reverse transcriptase, buffer, template switching oligonucleotide (TSO), RNase preparation, MnCl ₂ and other components to be tested according to Table 4 to perform RNA-dependent DNA extension. When extended to the 3' end of the cDNA, reverse transcriptase exerts its terminal transferase activity to add several cytosine deoxynucleotides (C). The reaction temperature is set according to the type of reverse transcriptase to be tested, see Table 5.

Table 4

table 5

(c) The added cytosine deoxynucleotide (C) will complementary pair with the guanine nucleotide (rG) on the TSO, thereby realizing the switching of the template from RNA to DNA (template conversion), and then using reverse transcriptase The DNA-dependent DNA polymerase activity continues to extend using TSO as the DNA template, and finally amplifies a cDNA with Stem-loop primer, miRNA and TSO region, which is a cDNA with successful template conversion.

However, if the conversion fails, a cDNA with only the Stem-loop primer and miRNA regions will be generated, see Figure 1. Therefore, the cDNA generated by the reverse transcription reaction based on template switching includes both cDNA with successful template switching and cDNA with failed template switching. The higher the proportion of successfully converted cDNA to the total cDNA, the higher the template conversion efficiency, as shown in Figure 1.

(d) Dilute the reverse transcriptase product 50 times and use it as a template to perform TaqMan probes qPCR technology to measure the terminal transferase activity (template conversion efficiency) of the reverse transcriptase. The qPCR reaction includes two reaction systems, the inner reaction system composed of forward inner primer (Inner PCR-primer) and reverse primer (Inner primer system), and the inner reaction system composed of forward outer primer (Outer PCR-primer) and reverse primer. In the reaction system (Outer primer system), the forward inner primer only anneals and amplifies the RNA template region of cDNA, and the forward outer primer only anneals and amplifies the TSO region of cDNA. The component preparation and reaction conditions of qPCR are shown in Table 6-8 below:

Table 6

Table 7

Table 8

The obtained Inner system Ct value can characterize the amount of total cDNA in the reverse transcription product, while the Outer system Ct value is used to characterize the amount of cDNA that has been successfully converted from the template. Therefore, the difference (ΔCt) between the Ct value of the Outer system and the Ct value of the Inner system can accurately reflect the template conversion efficiency, that is, the terminal transferase activity of reverse transcriptase.

It should be noted that cDNA products with successful template conversion can be annealed and amplified with the forward outer primer and forward inner primer, while cDNA products with failed template conversion can only be annealed and amplified with the forward inner primer. Both amplifications used the same reverse primer and the same Taqman probe. During extension with the reverse primer, the 5′→3′ exonuclease activity of Taq enzyme can be used to cleave the probe, generating a fluorescent signal that is captured by the instrument, thereby generating the corresponding Ct value. Refer to Figure 2.

The detection method provided by the present disclosure does not require degradation of the RNA template and purification of the cDNA product after the reverse transcription reaction, greatly simplifying the entire operation process, and also has the advantages of low cost and short detection cycle. Therefore, the present disclosure is suitable for debugging the reverse transcription system of template switching effect and large-scale industrial detection.

Sequence information such as templates, primers, and probes involved in this disclosure is shown in Table 9 below:

Table 9

Experimental Example 2 Effects of Different Concentrations of Stem-Loop Reverse Transcription Primers and TSO

This experimental example explores the effect of different concentrations of stem-loop reverse transcription primers and TSO on template conversion efficiency in a reverse transcription reaction system.

Set the concentrations of the stem-loop reverse transcription primer and TSO to: 26nM, 0.1μM, 0.2μM, 2μM, 10μM and 20μM respectively. Carry out the reverse transcription reaction according to the detection method in Example 1, and use two different reverse transcriptases to participate in the reaction. To determine their terminal transferase activity (Tdt activity). The reverse transcriptases used are all MMLV enzymes, and their names are MM02 reverse transcriptase (Feipeng Biotech, product number MD311) and SDmmlv reverse transcriptase (Feipeng Biotechnology, product number MDAR013). Unless otherwise specified, the concentrations of the miRNA template and other primers are based on the standards in Example 1.

Referring to Figure 3, the results show that when the concentration of the stem-loop reverse transcription primer is 20 μM and the concentration of TSO is 20 μM, the ΔCt value is the smallest, which means that the amount of cDNA with successful template conversion is higher, that is, the amount of cDNA with successful template conversion is higher. The higher the concentration of strand cDNA (successfully loaded with TSO), the template conversion efficiency of the reverse transcription reaction is the highest at this primer concentration. And the Tdt activity of MM02 reverse transcriptase is higher than SDmmlv.

Further, 10 μM, 20 μM, 50 μM and 100 μM stem-loop reverse transcription primers and TSO were used to perform the reverse transcription reaction according to the detection method in Example 1, and different reverse transcriptases were used to participate in the reaction. The reverse transcriptases are MM02 reverse transcriptase and SDmmlv reverse transcriptase.

Referring to Figure 4, the results show that when the concentration of the stem-loop reverse transcription primer is 100 μM and the TSO primer concentration is 100 μM, the ΔCt value is the smallest, and the template conversion efficiency of the reverse transcription reaction is the highest at this concentration. However, when the concentration of the stem-loop reverse transcription primer and TSO was increased from 20 μM to 100 μM, the ΔCt did not decrease significantly, that is, the template conversion efficiency did not increase significantly. Therefore, from a cost perspective, the optimal concentration of stem-loop reverse transcription primer and TSO is 20 μM. At the same time, the results showed that the Tdt activity of MM02 reverse transcriptase was higher than that of SDmmlv.

Experimental Example 3 Effect of Different MnCl ₂ Concentrations

This experimental example explores the effect of different MnCl ₂ concentrations on template conversion efficiency in the reverse transcription reaction system.

MnCl ₂ concentrations of 0mM, 4mM, 8mM, 16mM and 23.25mM were respectively used to perform reverse transcription reactions according to the detection method of Example 1 to explore the impact of MnCl ₂ on template conversion efficiency, and different reverse transcriptases were used to participate in the reaction. The reverse transcriptases are MM02 reverse transcriptase and SDmmlv reverse transcriptase. Unless otherwise specified, the concentrations of the miRNA template and related primers are based on the standards in Example 1.

Referring to Figure 5, the results show that when the _MnCl concentration is 8mM and the reverse transcriptase is MM02, the ΔCt value is the smallest, which means that the amount of cDNA with successful template conversion is higher, that is, one strand of cDNA with successful template conversion (successful The higher the concentration of TSO), which indicates the high Tdt activity of reverse transcriptase, the higher the transcription efficiency of the reverse transcription template. And when the MnCl ₂ concentration is 8mM, the Tdt activity of MM02 reverse transcriptase is higher than that of SDmmlv reverse transcriptase.

The above descriptions are only preferred embodiments of the present disclosure and are not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of this disclosure shall be included in the protection scope of this disclosure.

Although the present disclosure has been illustrated and described in terms of specific embodiments, it will be appreciated that many other changes and modifications can be made without departing from the spirit and scope of the disclosure. It is therefore intended to cover in the appended claims all such changes and modifications that fall within the scope of this disclosure.

Industrial applicability

The present disclosure provides a method and kit for detecting terminal transferase activity. This detection method is suitable for debugging the reverse transcription system. It has simple operation steps, low cost and short detection cycle. It can meet the needs of large-scale detection and has broad application prospects and high market value.

Claims (10)

1. A method for detecting terminal transferase activity, wherein the method includes the following steps:

   (a) Annealing the reverse transcription primer to the RNA template to generate a hybrid strand of the reverse transcription primer-RNA;

   (b) Use reverse transcriptase to perform RNA template-dependent DNA extension to obtain an RNA-cDNA intermediate with sticky ends;

   (c) The 3′ region on the template switching oligonucleotide (TSO) is annealed to the sticky end of the RNA-cDNA intermediate; and the TSO is used as a template to extend the 3′ region of the cDNA chain of the RNA-cDNA intermediate. end to obtain cDNA after template switching reaction;

   (d) Set a reverse primer in the reverse transcription primer region, set a forward inner primer in the RNA template, set a forward outer primer in the TSO, use the product of the above step (c) as a template, and obtain the forward inner primer through qPCR reaction With the Ct1 value of the amplification product of the reverse primer, obtain the Ct2 value of the amplification product of the forward outer primer and the reverse primer through qPCR reaction, and calculate the difference ΔCt between the Ct1 value and the Ct2 value to obtain the activity of terminal transferase; qPCR reaction Use the same probe.
2. The method according to claim 1, wherein the reverse transcription primer in step (a) is a stem-loop reverse transcription primer or a Poly T reverse transcription primer;

   The RNA-cDNA intermediate with a sticky end in step (b) adds multiple residues to the 3′ end of the cDNA chain of the RNA-cDNA intermediate through the terminal transferase activity of the reverse transcriptase itself. Obtained by the same deoxyribonucleotide method;

   In the steps (a)-(c), reverse transcriptase, dNTPs, RNase inhibitors, template switching oligonucleotides (TSO), and buffers are used to perform DNA extension that depends on the RNA template;

   Preferably, the plurality of deoxynucleotides added at the end in step (b) are a plurality of cytosine deoxynucleotides (C);

   Preferably, the 3′ annealing region on the TSO contains three ribonucleotide residues;

   Preferably, the 3' annealing region contains three nuclear guanine (rG) ribonucleotides;

   Preferably, the reverse transcriptase buffer in steps (a)-(c) contains MnCl ₂ , and the final concentration of MnCl ₂ is 4-16mM;

   More preferably, the final concentration of _MnCl2 is 4-8mM;

   Preferably, the RNA template is selected from: mRNA, non-coding RNA, miRNA, siRNA, piRNA, lncRNA or ribosomal RNA;

   More preferably, the RNA template is miRNA;

   Preferably, when 10 nM miRNA is used as a template, the concentration of TSO is 2-100 μM, and the concentration of the reverse transcription primer is 2-100 μM;

   More preferably, when 10 nM of miRNA is used as the template, the TSO concentration is 20-100 μM, and the concentration of the reverse transcription stem-loop primer is 20-100 μM.
3. The method according to claim 1, wherein the cDNA product obtained in step (c) needs to be diluted 10-100 times for use in the qPCR reaction of step (d);

   More preferably, the dilution factor of the cDNA product obtained in step (c) is 50 times.
4. The method according to claim 1, wherein during the qPCR reaction, the final concentration of the forward outer primer is 0.3-0.5 μM, and the final concentration of the forward inner primer is 0.3-0.5 μM. The final concentration of the reverse primer is 0.3-0.5 μM, and the final concentration of the probe is 0.1-0.3 μM;

   Preferably, the final concentration of the forward outer primer is 0.3 μM, the final concentration of the forward inner primer is 0.3 μM, the final concentration of the reverse primer is 0.3 μM, and the final concentration of the probe is 0.1 μM.
5. The method according to claim 1, wherein the reverse transcriptase is M-MLV reverse transcriptase, HIV-1 reverse transcriptase, AMV reverse transcriptase or telomerase reverse transcriptase with reduced or removed RNase activity.
6. The method according to any one of claims 1 to 5, wherein the method for judging the activity of the terminal transferase is as follows:

   The smaller the ΔCt value, the higher the activity of terminal transferase; or

   The larger the ΔCt value, the lower the activity of terminal transferase.
7. The method according to claim 1, wherein the annealing step in step (a) adopts gradient annealing;

   Preferably, the initial annealing temperature of gradient annealing in step (a) is 65°C, decreases by 0.1°C per second until 4°C, and the annealing time is 15-20 minutes;

   More preferably, the annealing time is 16 minutes.
8. The method according to claim 1, wherein the reaction temperature of step (b) and step (c) is determined according to the optimal reaction temperature of reverse transcriptase, and the reaction time is 90-180min;

   Preferably, the reaction time of step (c) is 120 minutes.
9. The method of claim 1, wherein the probe in step (d) is a Taqman probe.
10. A kit, wherein the kit includes: qPCR polymerase, reverse primer, forward inner primer, forward outer primer, probe, reverse transcription primer, RNA template and TSO,

    Wherein, the nucleotide sequences of the reverse primer, forward inner primer, forward outer primer, RNA template, TSO, and probe are shown in sequence as SEQ ID NO: 1-3 and SEQ ID NO: 5-7, The nucleotide sequence of the reverse transcription primer is shown in SEQ ID NO:4 or SEQ ID NO:8.