221 results about "Dna template" patented technology

Provided is a circular multimeric tandem RNA sense strand, comprising at least one sense strand sequence and at least one spacer sequence. The circular RNA is derived from an engineered parental DNA template containing all essential sequences, and sequentially comprises a first cyclization element, optionally at least one first restriction enzyme recognition sequence, at least one target sequence, optionally at least one second restriction enzyme recognition sequence, and a second cyclization element. The circular multimeric tandem RNA sense strand can bind and deliver a plurality of antisense strand RNA, increasing the binding of the sense strand and the antisense strand while utilizing the stability advantage of circular RNA.

The invention provides annular multi-tandem PCSK9 siRNA, which comprises at least one positive-sense strand sequence and at least one spacer sequence. The sense strand of the cyclic siRNA is derived from an engineered parent DNA template containing all essential sequences, comprising in the following order a first cyclization element, optionally at least one first restriction enzyme recognition sequence, at least one target sequence, optionally at least one second restriction enzyme recognition sequence, and a second cyclization element. The annular multi-tandem RNA positive-sense strand can combine and deliver a plurality of antisense strand RNAs, and the combination of the positive-sense strand and the antisense strand is increased while the stability advantage of the annular RNA is utilized.

Provided is a circular concatemeric PCSK9 siRNA, comprising at least one sense strand sequence and at least one spacer sequence. The sense strand of the circular siRNA is derived from an engineered parental DNA template containing all essential sequences, which comprises, in the following order: a first cyclization element, optionally at least one first restriction enzyme recognition sequence, at least one target sequence, optionally at least one second restriction enzyme recognition sequence, and a second cyclization element. The sense strand of the circular concatemeric RNA can bind and deliver a plurality of antisense strand RNAs, enhancing the binding between the sense and antisense strands while leveraging the stability advantage of the circular RNA.

The invention relates to the technical field of phytopathogen detection, and particularly discloses a primer, a kit and a method for detecting crop root rot fusarium solani and application, the primer comprises a specific RPA primer RPA-F and a specific RPA primer RPA-R; the specific RPA primer RPA-F is as shown in SEQ ID NO. 1, and the specific RPA primer RPA-R is as shown in SEQ ID NO. 2; the kit further comprises a guide RNA primer crRNA, and the guide RNA primer crRNA is as shown in SEQ ID NO. 3. The kit comprises a specific RPA primer RPA-F, a specific RPA primer RPA-R, a Cas12 protein, a T7 transcriptase, a crRNA DNA template, a DNA polymerase and a Cas12 / 13 nucleic acid detection test strip. The kit has the characteristics of good sensitivity, relatively good specificity and enough reliability.

The invention belongs to the field of detection, and relates to an identification method for determining adulteration of twelve animal source components in edible animal viscera and products thereof based on a DNA (Deoxyribose Nucleic Acid) micro bar code technology of mitochondrial Cytb. The method comprises the following steps: deeply cleaning a sample by using a 70% ethanol solution and normal saline, mixing by combining a liquid nitrogen grinding technology and a vacuum freeze drying technology, extracting a DNA template by using an improved CTAB method, amplifying a target fragment by using a pair of designed universal primers Ctyb-A, and detecting the target fragment. Carrying out qualitative and quantitative analysis on an amplification product by utilizing a QIAxcel high-resolution capillary gel electrophoresis analysis system, and finally carrying out adulteration analysis on a sequencing result of the amplification product through an established adulteration visual model.

The present invention relates to the field of in vitro transcription, and particularly to a plasmid backbone capable of improving the stability of polyadenylate tails. The present invention provides an expression cassette, which sequentially includes: the rop gene, the replicon ori, and the resistance gene; the rop gene, the replicon ori, and the resistance gene are connected by a linking fragment. The present invention has established a low-copy pmRVacSL and a medium-copy pmRVacM plasmid vector system through artificial modification. Since pmRVacSL is a low-copy vector, the yield of plasmid DNA is relatively low. The pmRVacM plasmid backbone solves the problem of relatively low plasmid DNA yield of pmRVacSL. This vector system can not only maintain the stable replication of polyA tail sequences of more than 150 bp, but also quickly add genes of interest and polyadenylate (polyA) sequences, and efficiently and rapidly obtain the DNA template for in vitro transcription of the target mRNA.

The invention provides a method for producing a DNA template for mRNA containing a polyadenylate tail. This method is applicable to in vitro transcription reactions, generating mRNA with a specific polyadenylate tail length. Additionally, the invention further provides a method for extending the polyadenylate tail sequence using terminal deoxynucleotidyl transferase and combinations of restriction enzymes. This ensures that in in vitro transcription reactions, the mRNA can precisely terminate at the polyadenylate tail, thereby improving the accuracy and efficiency of mRNA synthesis.

Provided herein are compositions of conjugates of a guide RNA(s)-CRISPR Cas protein (RNP) complex. The conjugate comprises a guide RNA(s)-CRISPR Cas protein (RNP) complex and one or more molecules selected from PEG, non-PEG polymers, ligands for cellular receptors, lipids, oligonucleotides, polysaccharides and peptides and chemically linked to the Cas protein and / or guide RNA(s). Also provided a guide RNA-DNA template conjugate comprises a DNA template with 5-methylcytosines at specific positions for epigenome editing by DNA methylation at a single nucleotide resolution level. The conjugates are delivered to targeted cells as RNP complexes, or formed in targeted cells from guide RNA conjugates and an mRNA or a viral vector encoding a Cas protein, or formed in targeted cells from a crRNA conjugate(s) and a viral vector encoding both a Cas protein and a tracrRNA. Also provided are preparation methods and uses of these conjugates.

The present invention provides a process for the production of a closed linear DNA comprising the steps of (a) providing a DNA template comprising a DNA sequence of interest; (b) amplifying DNA from the DNA template of step (a) wherein the amplification is primed with a primase / polymerase enzyme; (c) generating a closed linear DNA with the amplified DNA produced in step (b); and (d) purifying the closed linear DNA produced in step (c). The invention also provides a closed linear DNA obtainable according to the process of the invention, a pharmaceutical composition comprising a therapeutically effective amount of the closed linear DNA of the invention, and a concatameric DNA comprising repeats of a DNA sequence of interest.

Methods, systems, and non-transitory computer readable media are disclosed for efficiently designing massively multiplex PCR primer sets via Degenerate Incomplete Multiplex Primer List Expansion (DIMPLE). DIMPLE has shorter runtime, allowing it to scale to massive multiplexing. DIMPLE is applicable to designing PCR primers for both standard DNA templates and to post-conversion DNA templates with degenerate nucleotides. DIMPLE allows simultaneous design of multiple primers to each DNA template of interest, to facilitate experimental optimization and to enable nested PCR. Finally, DIMPLE can avoid non-suitable templates that can result in primers with high experimental failure rates.

The invention discloses a method for preparing RNA (Ribonucleic Acid) through in-vitro transcription and application. The method comprises the following steps: (1) preparing a linearized DNA template; (2) constructing an in-vitro transcription reaction system, and performing in-vitro transcription under a reaction condition suitable for transcription; and (3) purifying the prepared mRNA. According to the method, RNA polymerase can reduce 3 '-extension and non-specific transcription initiation of an IVT product under the condition that the temperature is increased, and an appropriate amount of L-arginine or salt thereof is added into a reaction system, so that the RNA structure can be effectively prevented from being damaged while the mRNA yield is not reduced, and the generation of dsRNA is further reduced. In addition, the reduction of dsRNA can significantly reduce the immunogenicity of mRNA.

The invention belongs to the field of plant molecular genetics, and relates to a spirodela polymorphic microsatellite molecular marker primer and application thereof. The kit comprises 10 pairs of microsatellite primers which form 5 groups of duplex PCR systems, and the kit can be applied to population genetic diversity analysis of spirodela polyrhiza. The population genetic diversity analysis comprises the following steps: collecting a sample of spirodela polyrhiza to be identified; extracting spirodela polyrhiza genome DNA from the spirodela polyrhiza sample; taking the extracted DNA as a template, and carrying out PCR (Polymerase Chain Reaction) amplification on the DNA template by using the screened 10 pairs of microsatellite primers; carrying out electrophoresis detection on a PCR product, and then carrying out silver staining; the silver staining result is digitally analyzed, the UPGMA clustering analysis chart is drawn by genetic analysis software, the clustering analysis chart of the spirodela polyrhiza is identified according to the clustering analysis result, and the method has huge popularization and utilization value.

The invention provides a microsatellite genotyping method, and belongs to the technical field of genotyping. According to the microsatellite genotyping method, a bar code primer is introduced into a microsatellite primer, and the bar code primer comprises a bar code and a universal primer. The universal primer can be used for increasing PCR products obtained by the microsatellite primer so as to supplement a target product obtained by the microsatellite primer, the problem of amplification of a low-quality DNA template is solved, the library building success rate is remarkably increased to 90-100% from 60-70%, the library building and sequencing times are remarkably reduced, the sample number of each time of library building and sequencing is increased, and the library building and sequencing efficiency is improved. The method has the remarkable advantages of low cost and high accuracy.

The invention belongs to the technical field of microbiological detection, and particularly relates to a real-time fluorescent quantitative PCR (polymerase chain reaction) primer, a probe and a kit for detecting a special drug-resistant pedigree of candida albicans. The nucleotide sequence of the specific primer F is as shown in SEQ ID NO. 1; the nucleotide sequence of the specific primer R is as shown in SEQ ID NO. 2; the nucleotide sequence of a probe matched with the primer for use is as shown in SEQ ID No.3. The kit prepared by the invention ensures high specificity and high sensitivity in a PCR amplification process, effectively distinguishes drug-resistant mutant strains from sensitive strains, keeps efficient detection capability in a low-copy-number DNA template, reduces non-specific amplification and background signal interference, and improves the reliability and accuracy of a detection result.

The present application relates to the field of genetic engineering technology, and specifically, to a method for preparing a transcription reaction solution, a locked nucleic acid-modified RNA, and an application of a mutated T7 RNA polymerase. A transcription reaction solution for preparing locked nucleic acid-modified RNA comprises a DNA template, a mutated T7 RNA polymerase, LNA-NTPs, and a buffer; wherein the amino acid sequence of the mutated T7 RNA polymerase is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, or SEQ ID NO.7. The present application can significantly improve the transcription yield of locked nucleic acid-modified RNA by adding the above-mentioned mutated T7 RNA polymerase to the transcription reaction solution for synthesizing locked nucleic acid-modified RNA, and the operation is simple, which is conducive to industrial production.

The invention provides an ISSR-PCR reaction system for identifying kiwi fruit varieties with different colors and a method and application of the ISSR-PCR reaction system, and belongs to the technical field of molecular markers. The ISSR-PCR reaction system for identifying the kiwi fruit varieties with different colors is as follows: the DNA template is 70ng / mu L, the primer is 1.0 mu mol / L, the Mg < 2 + > is 2mM, the mix is 10mu L, and the remaining ddH2O is prepared into 20 mu L of the system. The amplification procedure is as follows: the pre-denaturation temperature is 94 DEG C, and the time is 5 minutes; the denaturation temperature is 94 DEG C for 30 seconds; the annealing temperature is set according to different primer conditions, and the time is 50s; the extension temperature is 72 DEG C, and the extension time is 1 min; circulation is performed for 35 times; and then extending at 72 DEG C for 7 minutes, and storing at 10 DEG C. The system is used for amplifying 42 kiwi fruit germplasm resources with different colors, the amplification result shows that the established ISSR-PCR reaction system is good in repeatability and high in stability, and the established ISSR molecular fingerprint spectrum can be used for variety identification.

The present application relates to the field of genetic engineering technology, and more specifically, to a method for synthesizing capped RNA and a capped RNA transcription reaction solution. The method for synthesizing capped RNA comprises mixing and incubating raw materials; the raw materials include a DNA template, a capping polynucleotide, NTPs, an RNA polymerase, and a solution containing magnesium ions; the DNA template includes an RNA promoter, and the base at position ‑1 of the template chain of the RNA promoter is cytosine; the bases at positions +1 to +n of the template chain of the RNA promoter are respectively connected to the bases B1 to B2 in the capping polynucleotide; n The present invention regulates the base at position ‑1 of the template strand of the RNA promoter to be cytosine, and at the same time regulates the bases at positions +1 to +n of the template strand of the RNA promoter to be respectively aligned with the bases at positions B1 to B2 in the capping polynucleotide. n Complementary pairing significantly improves the efficiency of RNA capping and is beneficial to improving the stability of RNA after transcription and synthesis.

The invention discloses a novel method for preparing circular RNA in vitro based on magnetic beads. The novel method comprises the following steps: 1) designing and synthesizing a DNA template for generating target RNA; designing and synthesizing RNA (Ribonucleic Acid) which is complementary with a target RNA sequence and is provided with a biotin label; 2) carrying out in vitro transcription by using the DNA template to obtain target RNA (Ribonucleic Acid); (3) purifying the RNA obtained by in-vitro transcription; 4) annealing: annealing the target RNA and the DNA with the biotin label to form a complementary double-stranded RNA structure with a cohesive end; 5) connection: connecting the linear double-stranded RNA structure into an annular double-stranded RNA structure by using T4RNA ligase 2; 6) magnetic separation: fully mixing the streptavidin magnetic beads with the double-stranded RNA; performing high-temperature dissociation on the annular hybrid double strands, and separating the RNA with the biotin label from the target annular RNA by streptavidin magnetic beads; (7) removing the annular double-stranded RNA by using RNase III (Ribonucleic Acid); (8) degrading the linear DNA; (9) degrading the linear RNA by using RNase R; (10) purifying the circular RNA; 11) qRT-PCR (quantitative reverse transcription-polymerase chain reaction) identification: designing and synthesizing primers for amplifying annular and linear RNA (ribonucleic acid), amplifying by using a fluorescent quantitative PCR instrument, and analyzing an amplification curve and a dissolution curve; and 12) analyzing the size and purity of the circular RNA by agarose gel electrophoresis.

The present application relates to the application of Klenow mutant fragments in the synthesis of nucleic acids, and belongs to the technical field of biological modification and synthesis.The present application provides the application of Klenow mutant fragments with an amino acid sequence as shown in SEQ ID NO.8 or SEQ ID NO.14 in the synthesis of nucleic acids.The mutant protein F762A expressed by the Klenow mutant fragments of the present application can efficiently synthesize deoxyribonucleic acid, ribonucleic acid, deoxyribonucleic acid analogs and ribonucleic acid analogs with different lengths of DNA templates and DNA / RNA primers at 37 DEG C, and has a wide synthesis range and universality.

The invention relates to the technical field of gene engineering, in particular to a method for rapidly and economically carrying out gene function analysis on lepidoptera insects and application. The method comprises the following steps: designing four different sgRNAs by aiming at an exon region of a target gene, and constructing a sgRNAs synthetic primer mixing pool; 4 different sgRNA-DNA templates are synthesized at the same time, 4 different sgRNAs are further synthesized at the same time, and a sgRNAs mixed pool is formed; the sgRNAs-Cas9 protein and Cas9 protein are prepared into an sgRNAs-Cas9 compound, and the sgRNAs-Cas9 compound is microinjected into newly laid eggs; after the injected eggs develop into larvae, genome DNA is extracted, sgRNAs targeted areas are amplified, the mutation rate of target genes is determined through T7E1 enzyme digestion, and phenotype analysis is conducted on the larvae. According to the method, the mutation rate of the target gene is increased through redundant targeting of the four sgRNAs, so that functional analysis of the target gene is quickly realized in the G0 generation, and the research period of the gene function of the lepidoptera insects is greatly shortened. Besides, four different sgRNAs are synthesized at the same time by constructing a primer mixing pool of a plurality of sgRNAs, so that the economic cost for synthesizing the sgRNAs is greatly reduced.

The invention relates to a kit for detecting Tau-181 protein and application, and relates to the technical field of biological detection.The kit comprises a first antibody coupled with a first component of DNAzyme with catalytic activity, a second antibody coupled with a second component of DNAzyme with catalytic activity, phi29DNA polymerase, dNTPs, an annular DNA template, an initiation primer, a fluorescent beacon probe and a reaction buffer solution; the Tau-181 protein detection technology based on double-antibody sandwich coupling DNAzyme is used for quantitatively detecting the content of the Tau-181 protein in a biological sample in vitro, and can be applied to the fields of neuroscience research and clinical diagnosis, especially in the processes of early diagnosis, disease monitoring and drug research and development of neurodegenerative diseases such as Alzheimer's disease. And the method has important significance in the aspects of detection and analysis of a disease-related biomarker Tau-181 protein and the like.

The present invention relates to compounds, methods and compositions for improving nucleic acid polymerization, including DNA replication by in vitro primer extension to produce polymers for use, for example, in nanopore-based single-molecule sequencing of DNA templates. The nucleic acid polymerase reaction composition has a polymerization enhancing moiety that allows for enhanced activity of DNA polymerase with nucleotide analogs, resulting in increased length of primer extension products for sequencing applications.

The invention provides an in-vitro transcription method based on RNA polymerase and a kit, which are suitable for the fields of mRNA synthesis, gene research and biological pharmacy. According to the method, the problems of low efficiency, poor stability and the like of traditional in-vitro transcription are solved through an optimization technology. The method comprises the following specific steps: providing a linear DNA template containing a T7 promoter; according to the method, site-directed mutagenesis or fusion protein modified high-activity T7RNA polymerase is combined with chemically modified NTPs and a spermine-BSA compound stabilizer to react under the conditions that the temperature is 37-42 DEG C and the pH is 7.5-8.5, and formation of double-stranded RNA is promoted through dynamic temperature control. A reaction system contains Mg < 2 + > / Mn < 2 + > and DTT, and enzyme activity and product integrity are optimized. The kit comprises an improved reagent and a standardized process, the transcription efficiency is improved by 2-5 times, the product yield is greater than or equal to 80%, and the activity is not lost after the kit is stored at 4 DEG C for 1 month. The method has the technical advantages that NTPs degradation resistance is achieved, template preparation is simplified, universality is high, the method is suitable for large-scale production of mRNA vaccines, gene therapy and the like, the production cost is remarkably reduced, and wide application of the in-vitro transcription technology is promoted.

The invention relates to the field of DNA (Deoxyribose Nucleic Acid) sequencing, in particular to a pathogenic microorganism high-throughput sequencing detection method and a pathogenic microorganism high-throughput sequencing detection system based on multiple PCR (Polymerase Chain Reaction) detection. The method comprises the following steps: quantitatively adding an internal reference nucleic acid marker into a first microorganism sample to be detected to generate a standardized DNA template library; performing multiple PCR amplification and quality control judgment and evaluation on the standardized DNA template library to generate a PCR quality control result table; constructing a sequencing library based on the PCR quality control result table to generate sequencing library original data; performing host reference genome comparison and removal on the original data of the sequencing library, and extracting a data set to be compared; and performing pathogenic species comparative analysis according to the to-be-compared data set, and outputting a final pathogenic microorganism detection list. The invention realizes rapid and accurate detection of pathogenic microorganisms.

The invention discloses a primer, a kit and an identification method for specifically identifying sheepskin, and belongs to the technical field of leather identification. The specific primer comprises a KLHL17 forward primer as shown in SEQ ID NO. 1 and a KLHL17 reverse primer as shown in SEQ ID NO. 2. The invention also discloses a kit for detecting the specific primer. The identification method specifically comprises the following steps: 1) extracting DNA of a cortex sample to be detected to obtain a total DNA template; (2) carrying out PCR (Polymerase Chain Reaction) amplification reaction by adopting a KLHL17 forward primer, a KLHL17 reverse primer and the total DNA template to obtain an amplified DNA fragment; and (3) detecting and amplifying the DNA fragment by adopting agarose gel electrophoresis, and judging that the sample is sheep skin if the size of a PCR (Polymerase Chain Reaction) amplified band is 106bp. The method disclosed by the invention has the characteristics of rapidness, simplicity, convenience, accuracy, wide application range and low requirements on equipment, and can be used for remarkably improving the identification efficiency and reducing the detection cost. In addition, the wide application of the method is of great significance to standardize the leather market order and improve the accuracy of leather product traceability.

The present invention provides a reagent for detecting the DNA barcode of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. and a fluorescence detection method for Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. The reagent of the present invention includes a specifically upstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. modified with AuNPs and a specifically downstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. modified with a fluorescent label. The sequences of the specifically upstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. and the specifically downstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. modified with a fluorescent label are respectively: 5’-SH-(CH2)6-ATTGACCACGCGAACTTGTGA-3’; 5’-fluorescent label-ACCGTCAACAAACCTGATTGCT-3’. Detection method: Mix the specifically upstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. modified with AuNPs and the specifically downstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. modified with a fluorescent label with a DNA template solution of the to-be-detected Asarum species for PCR reaction amplification, and detect the fluorescence spectral value of the obtained product to achieve qualitative or quantitative detection of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag.

The invention provides a plasmid and application thereof. The plasmid comprises a recognition site of a first endonuclease, a target gene and a recognition site of a second endonuclease from a 5 '-3' direction. According to the invention, the plasmid is amplified by using a rolling circle amplification method, the rolling circle amplification product is cleaved by using the first endonuclease and the second endonuclease, the enzyme cleaved product is digested by using exonuclease, and the product is further purified by using oligonucleotide. The obtained product can be used for in vitro transcription or directly applied to eukaryotic expression. According to the scheme provided by the invention, a simple, high-yield, economic, rapid and amplifiable preparation process of the full chemical synthesis DNA template for escherichia coli fermentation is established.

Provided are a circular functional nucleic acid, a preparation method therefor and a use thereof. The method involves combining a functional nucleic acid with a circular nucleic acid, using a method combining DNA template-directed assembly with enzymatic conjugation to first prepare a single-stranded circular DNA, synthesize a corresponding outer short strand, and conjugate a functional nucleic acid drug onto the outer short strand, and optimizing and screening reaction conditions, thereby preparing a circular functional nucleic acid. The circular functional nucleic acid has the characteristics of adjustable size, adjustable valence state (multivalent or polyvalent), high stability, and the capability for chemical conjugation with drugs or drug incorporation, etc.; a novel circular multivalent functional nucleic acid technology is created, which significantly enhances the targeting ability and stability of nucleic acid drugs; thus, a nucleic acid drug development platform o-FLARE having efficient targeting, immune agonism, and protein degradation functions can be established.

A method and composition for preparing a DNA library for replicating a target nucleic acid sequence are provided. A target DNA template containing the target sequence is circularized via a terminal adapter to form a circular construct, which is extended bidirectionally by polymerase-mediated elongation initiated at a nick site on the terminal adapter. Following polymerase-mediated elongation, a diploid DNA template is formed containing two copies of the target DNA template (and thus two copies of the target sequence). Each strand of the diploid DNA template contains a parent polynucleotide chain ligated to a newly synthesized daughter strand copy of the parent polynucleotide chain. Predetermined sequences, such as primer sequences, unique molecular identifiers, and sequence indices, can be included in the diploid DNA template. Sequencing of the diploid DNA template can reveal genetic / epigenetic information related to the target sequence. A method for constructing asymmetric and multiploid DNA template constructs is also provided.

The present invention provides a circular DNA template precursor for detecting Clostridium difficile, a preparation method thereof and an application thereof. The circular DNA template precursor includes a three-dimensional self-assembly reaction using a reaction system composed of H1 and H2 hairpin probes and a trigger strand Trigger. The three-dimensional self-assembly reaction includes Trigger triggering the H1 hairpin structure to form a T / H1 intermediate, the H1 hairpin structure being unfolded and binding to H2 to produce an H1 / H2 structure, and the DNA template of H2 being cyclized to obtain the circular DNA template precursor. When Clostridium difficile toxin is present, the trigger strand Trigger can be released again, and can continuously catalyze the hairpin structure of the H1 DNA hairpin probe without being consumed. By adopting the technical scheme of the present invention, signal cascade three-stage amplification is realized through three-dimensional self-assembly-amplification reaction and silver-enhanced gold nanoparticles, so as to realize highly sensitive and highly specific detection of Clostridium difficile infection.