High-throughput qPCR (quantitative polymerase chain reaction) standard substance as well as preparation method and application thereof

Abstract

The invention discloses a high-throughput qPCR (quantitative polymerase chain reaction) standard substance and a preparation method thereof, the qPCR standard substance adopts a recombinant nucleic acid molecule, and the recombinant nucleic acid molecule comprises a gene segment and a vector skeleton for cloning and copying the gene segment; the gene segment is formed by alternately connecting at least two different target segments and oligonucleotide spacer sequences between the adjacent target segments in series; the qPCR standard substance is used for simultaneously drawing a qPCR quantitative standard curve for the at least two different target fragments through one-time dilution operation; the method overcomes the problem of high cost caused by the need of constructing a large number of standard substances in the traditional mode, is simple and convenient to operate, is highly matched with an ultrahigh-flux PCR instrument, ensures the accuracy of detection data, and brings a more efficient, economical and accurate solution to the field of qPCR detection.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid determination or testing technology, specifically to a high-throughput qPCR standard, its preparation method, and its application. Background Technology

[0002] qPCR standards refer to known and accurately quantified nucleic acid templates used to construct standard curves in real-time quantitative PCR experiments. They serve as a reference for absolute quantification in the entire qPCR experiment. Their core function is to establish a mathematical relationship between the fluorescence signal and the initial copy number of the template, thereby calculating the absolute quantity of the target gene in an unknown sample.

[0003] Currently, qPCR standards are generally constructed by amplifying positive samples using PCR, followed by agarose gel electrophoresis to detect the amplified products, followed by gel extraction and recovery. The products are then inserted into plasmid vectors using TA cloning, as seen in patents CN202210703703.5 (A Plasmid Standard for Quantitative Detection of Functional Bacteria in Sludge Communities, Construction Method and Application) and 201711006858.9 (A Plasmid Standard Containing a Novel Human Thymus Gene). This method ensures that the qPCR standards contain only a single target gene fragment. For high-throughput qPCR detection of dozens or even hundreds of targets, such as antibiotic resistance genes, environmental functional genes, and virulence factors, this method would require constructing dozens or even hundreds of standards. This not only results in high construction costs but also necessitates at least five dilutions per standard during experiments, leading to a large workload and a high risk of inaccurate dilutions, causing standard curve construction failure. Furthermore, the SmartChip™ Real-Time PCR System, a high-throughput qPCR instrument, cannot be used with single-target qPCR standards constructed using traditional methods. This is primarily because the system's qPCR reaction system uses a nano-scale dispensing system to automatically dispense samples onto the microarray. Dispensing follows a fixed sample and primer combination pattern, such as 72 samples * 72 primers, meaning the microarray can perform 72 samples and 72 primer pairs in a single dispensing cycle. In this dispensing mode, if the qPCR standard is a traditional single-target recombinant plasmid, each standard would need to be diluted at least five times, occupying a large number of microarray wells and making it impossible to place the sample to be tested. Therefore, the current method for high-throughput qPCR detection using this instrument is to plot a standard curve for only the internal reference gene using the 96-channel qPCR instrument, and then calculate the absolute copy number of other genes based on the internal reference gene's standard curve. However, this method has a large margin of error, so there is an urgent need for a qPCR standard compatible with this instrument. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a high-throughput qPCR standard, its preparation method, and its application.

[0005] A high-throughput qPCR standard, wherein the qPCR standard is a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising a gene fragment and a vector backbone for cloning and replicating the gene fragment; The gene fragment is composed of at least two different target fragments and oligonucleotide spacer sequences alternately tandemly between adjacent target fragments; wherein, the target fragment is a DNA sequence that is identical to a portion of the target gene to be quantified and can be amplified by PCR using specific primers; the qPCR standard is used to simultaneously plot qPCR quantitative standard curves for the at least two different target fragments through a single dilution operation.

[0006] Note: The above method overcomes the high cost of constructing a large number of standards using traditional methods, significantly reducing experimental costs. Previous techniques required multiple serial dilutions to plot standard curves for multiple target genes, a cumbersome process. This method, however, requires only a single serial dilution to easily plot standard curves for all target genes, offering a simple and convenient operation. The oligonucleotide spacer sequence effectively inhibits mutual interference during amplification of different target primers. Furthermore, this standard is highly compatible with the ultra-high-throughput SmartChip™ Real-Time PCR System, occupying only 5 wells of the serially diluted sample during detection, enabling simultaneous standard curve and sample testing. This maximizes the accuracy of the detection data, providing a more efficient, economical, and accurate solution for the qPCR detection field.

[0007] Furthermore, the recombinant nucleic acid molecule is a recombinant plasmid.

[0008] Note: Recombinant plasmids are commonly used gene vectors. The technology is mature and stable, and they are easy to operate and preserve in the laboratory. They can effectively ensure the integrity and stability of gene fragments and reduce the risk of experimental failure.

[0009] Furthermore, the oligonucleotide spacer sequence is an oligonucleotide sequence containing GC bases.

[0010] Furthermore, the oligonucleotide sequence containing GC bases is (GC). n Repeating sequences, where n It is an integer between 3 and 10.

[0011] Note: This sequence has a high GC content, which can effectively suppress the mutual interference between different target primers during amplification.

[0012] Furthermore, the target fragment includes at least two of the following: antibiotic resistance gene, virulence factor gene, pathogen-specific gene, host reference gene, environmental functional gene, and mobile genetic element sequence.

[0013] Note: The various standard fragments listed above can meet the qPCR detection needs of different fields and scenarios. Whether it is studying the pathogenic mechanisms and drug resistance of pathogenic microorganisms, analyzing host gene expression regulation, or monitoring the function of environmental microbial communities, this standard can achieve accurate determination, providing an efficient and comprehensive solution for high-throughput, multi-target gene quantitative research.

[0014] Furthermore, the sequence of the gene fragment is shown in SEQ ID NO: 1.

[0015] Furthermore, the oligonucleotide spacer sequence is shown in SEQ ID NO: 2.

[0016] Note: The explicitly defined sequence numbers mentioned above can be used as a standard reference to ensure the reproducibility and comparability of experimental results; Furthermore, the vector backbone is a linearized T-vector with a 3'-T end protrusion; the gene fragment has a 3'-A end protrusion, and the gene fragment is linked to the T-vector via TA cloning.

[0017] Note: The TA cloning mechanism eliminates the need for complex enzyme digestion and ligase reactions, simplifying the cloning process and shortening the experimental cycle. Its highly specific ligation method effectively reduces non-specific ligation, improving the success rate and accuracy of recombinant plasmid construction. Furthermore, TA cloning has relatively relaxed requirements for gene fragments and vectors, making it suitable for inserting gene fragments of different sources and lengths. This enhances the versatility and flexibility of this qPCR standard construction method, providing a more convenient, efficient, and reliable standard preparation route for high-throughput qPCR detection.

[0018] A method for preparing high-throughput qPCR standards includes the following steps: The at least two different target fragments are synthesized into a complete nucleotide sequence using a gene synthesizer, and then oligonucleotide spacer sequences are inserted between the target fragments to form a gene fragment; Gene fragments were cloned into the vector backbone via TA to construct qPCR standards containing multiple targets.

[0019] Note: The above preparation method utilizes a gene synthesizer to directly synthesize multiple target fragments into complete nucleotide sequences and inserts oligonucleotide spacer sequences to form gene fragments. This method is not only precise and controllable but also efficiently integrates multiple targets, avoiding the cumbersome process of constructing individual target fragments separately, thus significantly improving preparation efficiency. The gene fragments are inserted into the vector backbone using TA cloning technology. This technology is simple to operate, has high ligation efficiency, and does not require complex enzyme digestion and ligase reaction systems, reducing experimental difficulty and cost. Finally, qPCR standards containing multiple targets are constructed, which can meet the needs of high-throughput qPCR detection of multiple target genes in one go, providing an efficient, convenient, and economical standard preparation solution for gene expression analysis, pathogen detection, and other research.

[0020] The application of a high-throughput qPCR standard in the precise quantification of a target gene involves introducing a recombinant plasmid qPCR standard into host cells, culturing, amplifying, and extracting the qPCR standard to obtain a high-purity recombinant plasmid standard stock solution; and then performing concentration quantification and sequence verification on the recombinant plasmid standard stock solution.

[0021] Explanation: The above application introduces recombinant plasmid-based qPCR standards into host cells and performs culture, amplification, and extraction operations. Leveraging the efficient replication mechanism of the host cells, a large quantity of recombinant plasmids can be rapidly obtained, resulting in high-purity recombinant plasmid standard stock solutions, ensuring an ample supply and stable quality of the standards. Quantitative analysis of the recombinant plasmid standard stock solutions allows for precise determination of their content, providing a reliable basis for accurate dilution and use of standards in subsequent experiments. Sequence validation ensures the accuracy and integrity of the target gene fragments in the recombinant plasmids, avoiding experimental result deviations due to sequence errors. This provides high-quality, reliable standards for high-throughput qPCR detection, effectively guaranteeing the accuracy and reproducibility of the detection results.

[0022] The beneficial effects of this invention are: This invention overcomes the high cost of constructing large quantities of standards using traditional methods, significantly reducing experimental costs. Previous technologies required multiple serial dilutions to plot standard curves for multiple target genes, a cumbersome process. This method, however, requires only a single serial dilution to easily plot standard curves for all target genes, offering a simple and convenient operation. The oligonucleotide spacer sequence effectively inhibits mutual interference during amplification of different target primers. Furthermore, this standard is highly compatible with the ultra-high-throughput SmartChip™ Real-Time PCR System, requiring only five wells from the serial dilution to simultaneously perform standard curve and sample detection, maximizing the accuracy of the data and providing a more efficient, economical, and precise solution for qPCR detection. Attached Figure Description

[0023] Figure 1 This is the standard curve of 16S rRNA in the internal reference gene of this invention embodiment; Figure 2 This is the ermB standard curve for the target detection ARGs categories in this embodiment of the invention; Figure 3 This is the lnuB standard curve for the target detection ARGs categories in this embodiment of the invention; Figure 4 This is the sul1 standard curve among the target detection ARGs in the embodiments of the present invention; Figure 5 This is the sul2 standard curve among the target detection ARGs in this embodiment of the invention; Figure 6 This is the tetO standard curve among the target detection ARGs categories in this embodiment of the invention; Figure 7 This is the tetW standard curve for the target detection ARGs categories in the embodiments of the present invention; Figure 8 This is the qnrA standard curve for the target detection ARGs categories in this embodiment of the invention; Figure 9 This is the qnrS standard curve for the target detection ARGs categories in this embodiment of the invention; Figure 10 This is the standard curve of blaOXA-1 among the target detection ARGs in the embodiments of the present invention; Figure 11 This is the blaTEM-1 standard curve among the target detection ARGs in this embodiment of the invention; Figure 12 This is the ant(6)-Ia standard curve for the target detection ARGs in the embodiments of the present invention; Figure 13 This is the standard curve of ant(9)-Ia among the target detection ARGs in the embodiments of the present invention; Figure 14 This is the IntI1 standard curve among the ARGs types of target detection objects in this embodiment of the invention. Detailed Implementation

[0024] To further illustrate the methods and effects of this invention, the technical solution of this invention will be clearly and completely described below in conjunction with experiments.

[0025] As can be seen from the background technology, traditional methods for constructing and using qPCR standards based on single-target recombinant plasmids are completely incompatible with the technical characteristics of next-generation ultra-high-throughput qPCR platforms (such as SmartChip), leading to a significant contradiction between cost, throughput, and accuracy. On the one hand, in-depth scientific research demands high-precision absolute quantification of hundreds of genes in complex systems (such as environmental microbiomes). On the other hand, the methods supporting quantitative accuracy, from construction to application, are incompatible with the high-throughput platforms and experimental designs required to achieve this goal, resulting in a multi-faceted dilemma of "too high cost, insufficient throughput, insufficient well positions, and decreased accuracy." Therefore, the innovation urgently needed in the field is a standard solution that can solve all the above problems; specifically, the following is a specific solution from an embodiment of the present invention: A high-throughput qPCR standard is a recombinant nucleic acid molecule comprising a gene fragment and a vector backbone for cloning and replicating the gene fragment; the gene fragment is composed of at least two different target fragments alternately tandemly with oligonucleotide spacer sequences located between adjacent target fragments; wherein the target fragment is a DNA sequence that is partially identical to the target gene to be quantified and can be amplified by PCR using specific primers; the qPCR standard is used to simultaneously plot qPCR quantitative standard curves for the at least two different target fragments through a single dilution operation; The target gene to be quantified refers to the gene to be detected in the environment or biological sample; specific primers refer to a pair of artificially synthesized short-chain DNA molecules (called upstream primers and downstream primers) used to uniquely identify and bind to a specific site of the "target gene to be quantified"; PCR amplification refers to the use of DNA polymerase to perform exponential replication, starting with primers and using the original DNA strand as a template. Specifically, the recombinant nucleic acid molecule is a recombinant plasmid; the oligonucleotide spacer sequence is an oligonucleotide sequence containing GC bases; the oligonucleotide sequence containing GC bases is (GC) n Repeating sequences, where n The value is an integer from 3 to 10; the target fragment includes at least two of the following: antibiotic resistance gene, virulence factor gene, pathogen-specific gene, host reference gene, environmental functional gene, and mobile genetic element sequence; For example, the sequence of the gene fragment is shown in SEQ ID NO: 1; the oligonucleotide spacer sequence is shown in SEQ ID NO: 2; The vector backbone is a linearized T-vector with a 3'-T end protrusion; the gene fragment has a 3'-A end protrusion and is linked to the T-vector via TA cloning.

[0026] This invention also provides a method for preparing the above-mentioned high-throughput qPCR standard, comprising the following steps: S1. Using a gene synthesizer, synthesize the at least two different target fragments into a complete nucleotide sequence, and then insert oligonucleotide spacer sequences between the target fragments to form a gene fragment; S2. The gene fragment is cloned into the vector backbone via TA to construct a qPCR standard containing multiple targets.

[0027] This invention also provides the application of the above-mentioned high-throughput qPCR standards in the accurate quantification of target genes, including S3: S3. Introduce the recombinant plasmid qPCR standard into host cells, culture, amplify and extract to obtain a high-purity recombinant plasmid standard stock solution; perform concentration quantification and sequence verification on the recombinant plasmid standard stock solution.

[0028] Combining the above steps, the specific preparation and application scheme includes the following steps (1) to (5); Step (1), obtaining the fusion gene fragment: Using the Primer blast function in the NCBI database, the target sequences of 12 antibiotic resistance genes and 1 mobile genetic element were identified based on the upstream and downstream primer sequences.

[0029] The fusion gene fragment for constructing the standard plasmid in this standard is synthesized by merging 13 target sequences using gene synthesis techniques (completed by Qingke Biotechnology).

[0030] Step (2): Utilizing the complementarity of AT bases, the fusion gene fragment is ligated into the T vector (pUC57 SimpleVector) to form a recombinant plasmid containing 12 antibiotic resistance genes and 1 mobile genetic element gene.

[0031] Add 4 μL of the synthesized fusion gene fragment and 1 μL of the T-vector to a sterile centrifuge tube, and immediately place the centrifuge tube on ice; gently shake to mix, and react at room temperature (20℃-37℃) for 5 minutes to allow the target gene to ligate with the vector, and the reaction is complete.

[0032] Step (3), Transformation: Add the ligation product to 50 μL of freshly thawed E. coil DH5α competent cells, gently tap to mix, and place on ice for 20-30 min; immediately after the ice bath, heat shock in a 42 ℃ water bath for 30 s, and then immediately place on ice for 2 min to transform the constructed plasmid DNA into competent cells. Then, add 250 μL of sterilized fresh LB liquid medium to this bacterial culture, and incubate in a 37 ℃ shaker at 200 rpm for 1 hour to revive the competent cells; after revival, take 100 μL of bacterial culture and spread it evenly on a prepared LB solid plate containing 100 µg / mL ampicillin, and incubate in a 37 ℃ incubator for 12-16 h. After the culture was completed, under aseptic conditions, white bacterial plaques were picked and inoculated into LB liquid medium containing 100 µg / mL ampicillin, and then placed in a shaker incubator at 200 rpm and cultured at 37 ℃ for 12-16 h to expand the culture of Escherichia coli containing the fusion gene.

[0033] Step (4) Sequencing and identification of positive recombinants. After overnight culture, plasmids were extracted from positive clones using a plasmid extraction kit (OMEGA). Then, using the extracted plasmid DNA as a template, the fragment inserted into the vector was amplified and sequenced using primers M13F and M13R provided in the vector kit (completed by Qingke Biotechnology). The fragment inserted at the multiple cloning site was found, and the obtained inserted sequence was compared with the fusion gene fragment to check whether it was consistent with the fusion gene sequence. This determined whether the preparation of the standard plasmid containing the fusion gene fragment was successful.

[0034] Finally, a standard plasmid containing 13 target analytes was constructed for subsequent experiments.

[0035] Step (5): Standard curve plotting; for each target analyte, a series of standard plasmids at different concentrations need to be prepared to construct a standard curve. The specific steps are as follows: a. Accurately quantify the DNA concentration of standard plasmids.

[0036] b. Calculate the copy number.

[0037] c. Perform 10-fold serial dilutions, typically from 10⁷ to 10¹ copies / μL.

[0038] d. qPCR detection was performed using a 25 μL qPCR reaction system (12.5 μL 2×TransStart Green qPCR Supermix, 1.25 μL Forward Primer, 1.25 μL Reverse Primer, 5 μL ddH2O, and 5 μL Template DNA). Three replicates were prepared for each DNA sample, along with a blank control. The reaction program was as follows: Pre-denaturation: 94℃, hold for 40s.

[0039] 40 cycles of amplification: 94℃, hold for 5s, 60℃ annealing for 15s, 72℃, hold for 10s.

[0040] Melting curve verification: from 72℃ to 94℃, the temperature is increased by 0.5℃ for each cycle, and the temperature is recorded once every 0.5℃, with a 30s pause in between, and the process is completed after 80 cycles.

[0041] e. Construct a standard curve using the logarithm of the Ct value and the copy number of the standard. The standard curve equations for the target analytes are shown in Table 1; the corresponding standard curve plots for each target analyte in Table 1 are shown in [the table below]. Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 , Figure 11 , Figure 12 , Figure 13 as well as Figure 14 ;in Figure 1 The corresponding internal reference gene 16S rRNA refers to a gene that is consistently and stably expressed in all test samples, providing a reference for the other target detection substances; Table 1 Standard Curve Equation of Target Detection Object

[0042] SEQ ID NO: 1: SEQ ID NO: 2 is: GCGCGCGCGC; In summary, this invention synthesizes a single gene fragment seq1 (SEQ ID NO: 1) from multiple target fragments using a gene synthesizer, and inserts a specific gene sequence seq2 (SEQ ID NO: 2) between the target fragments. Then, seq1 is inserted into a plasmid using TA cloning to construct a recombinant plasmid containing multiple targets. A single dilution of this recombinant plasmid is sufficient for plotting qPCR standard curves for all primers of the integrated target genes. This invention requires only the construction of one recombinant plasmid, resulting in lower experimental costs compared to constructing dozens or even hundreds of standards previously. Furthermore, the operation is simple, requiring only one serial dilution to plot standard curves for all target genes. Moreover, the standards described in this invention are perfectly compatible with the ultra-high-throughput qPCR instrument: SmartChip™ Real-Time PCR System, occupying only 5 sample wells in the serial dilution, allowing standard curve plotting to be performed simultaneously with sample detection.

Claims

1. A high-throughput qPCR standard, characterized in that, The qPCR standard is a recombinant nucleic acid molecule, which contains a gene fragment and a vector backbone for cloning and replicating the gene fragment; The gene fragment is composed of at least two different target fragments and oligonucleotide spacer sequences alternately tandemly between adjacent target fragments; wherein, the target fragment is a DNA sequence that is identical to a portion of the target gene to be quantified and can be amplified by PCR using specific primers; the qPCR standard is used to simultaneously plot qPCR quantitative standard curves for the at least two different target fragments through a single dilution operation.

2. The high-throughput qPCR standard as described in claim 1, characterized in that, The recombinant nucleic acid molecule is a recombinant plasmid.

3. The high-throughput qPCR standard as described in claim 1, characterized in that, The oligonucleotide spacer sequence is an oligonucleotide sequence containing GC bases.

4. A high-throughput qPCR standard as described in claim 3, characterized in that, The oligonucleotide sequence containing GC bases is (GC). n Repeating sequences, where n It is an integer between 3 and 10.

5. A high-throughput qPCR standard as described in claim 1 or 3, characterized in that, The target fragment includes at least two of the following: antibiotic resistance gene, virulence factor gene, pathogen-specific gene, host reference gene, environmental functional gene, and mobile genetic element sequence.

6. A high-throughput qPCR standard as described in claim 1 or 3, characterized in that, The sequence of the gene fragment is shown in SEQ ID NO:

1.

7. A high-throughput qPCR standard as described in claim 1 or 3, characterized in that, The oligonucleotide spacer sequence is shown in SEQ ID NO:

2.

8. A high-throughput qPCR standard as described in claim 1, characterized in that, The vector backbone is a linearized T-vector with a 3'-T end protrusion; the gene fragment has a 3'-A end protrusion and is linked to the T-vector via TA cloning.

9. A method for preparing a high-throughput qPCR standard as described in any one of claims 1 to 4, characterized in that, Includes the following steps: The at least two different target fragments are synthesized into a complete nucleotide sequence using a gene synthesizer, and then oligonucleotide spacer sequences are inserted between the target fragments to form a gene fragment; Gene fragments were cloned into the vector backbone via TA to construct qPCR standards containing multiple targets.

10. The application of a high-throughput qPCR standard as described in any one of claims 1 to 4 in the accurate quantification of a target gene, characterized in that, The recombinant plasmid qPCR standard was introduced into host cells, cultured, amplified, and extracted to obtain a high-purity recombinant plasmid standard stock solution; the concentration of the recombinant plasmid standard stock solution was quantified and the sequence was verified.

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