Application of gene TSA in detection of collybia albuminosa derived components

A technology of derived components and genes, applied in the field of biological species identification and PCR detection, to achieve the effect of less time-consuming, good specificity and simple operation

Pending Publication Date: 2021-06-29

KUNMING UNIV OF SCI & TECH

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AI-Extracted Technical Summary

Problems solved by technology

And the internal standard gene of chicken fir has not been seen relevant report, in order to establish a kind of method t...

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Method used

1, for the internal gene TSA of embodiment 1 screening, utilize Primer Premier 5.0 software design primer, and carry out BLAST comparative analysis to designed pri...

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Abstract

The invention discloses an application of gene TSA in detection of collybia albuminosa derived component. A set of qualitative and quantitative PCR detection method for collybia albuminosa is established, a deep processing product of collybia albuminosaisdetected and verified, analysis finds that a BLAST comparison result graph of the TSA gene shows that all sequences of other genes are not the same as or similar to the sequence of the TSA gene, which indicates that the TSA gene does not have homology with other genes and has extremely high species specificity, so that the TSA gene is selected as a candidate endogenous reference gene of thecollybia albuminosa, and the collybia albuminosa-derived components in food can be rapidly and sensitively detected. The method is high in sensitivity, high in specificity, simple to operate, low in cost, easy in reaction result observation and very suitable for on-site real-time detection, and has good development prospects in rapid biological source detection of collybia albuminosa and deeply processed products thereof.

Application Domain

Microbiological testing/measurementDNA/RNA fragmentation

Technology Topic

CollybiaMolecular biology +4

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Examples

Experimental program(2)
Effect test(1)

Example Embodiment

[0016] Example 1: Chicken Source Universal Internal Standard Gene TSA Screening

[0017] In the NCBI's Nucleotide database, the gene information of the chicken is searching, and the glycosidase is finally screened by the BLAST alignment analysis. TSA Gene (login number: jo124034.1) as an internal standard gene, TSA Gene's BLAST results and details figure 1 , TSA The BLAST alignment of the gene is shown in the drawing of all sequences of any other genes. TSA The sequence of the gene is the same or similar, this shows TSA Genes have no homology with other genes, which has strong species specificity.

Example Embodiment

[0018] Example 2: Verification of geosetic components generalized internal standard gene

[0019] 1. Internal gene screening for Example 1 TSA The primer is designed with Primer Premier 5.0 software, and BLAST comparison analysis is performed on the design of the primer to ensure the specificity of the primer, the primer sequence is shown in Table 1; the amplified sequence is the 213th base of SEQ ID NO: 1 ~ 546 bases;

[0020] Table 1 TSA Gene's PCR primer sequence

[0021] ;

[0022] 2, qualitative verification of standard gene primer species in chicken

[0023] Take the yellow boys, northern wind bacteria, black bovine liver, milk pump, green bacteria, drum, chicken oil, pine, red, white bolet, big red mushroom, yellow raft, chicken, old man , Coral bacteria, mushrooms, tea trees, apricot emerrenus, genomic DNA of the mushrooms as a template, use primers TSA -F / b conducts normal PCR amplification detection, qualitatively verifies the species specificity of standard genes in the pills. The amplification product was analyzed by 2% agarose gel electrophoresis (containing 0.1 μg / ml EB), the voltage was 120V, and the electrophoretic buffer was 1 × Tae. The amplification product was 5 μL, scanned to generate an image with a gel imager and image analysis, a normal PCR amplification program: predetermitability (95 ° C, 5 min); 30 cycles: denaturation (95 ° C, 30S), annealing (58 ° C, 30S), extended (72 ° C, 30S); termination extension (72 ° C, 5 min), PCR reaction system is shown in Table 2;

[0024] The results show that the chicken genome DNA amplification product is located at 334bp, successfully expanded TSA Gene, strip is obvious, although there is a strip, but is located at 650bp; and the remaining mushrooms have no amplification products, such as figure 2 Indicated;

[0025] Table 2 Qualitative PCR reaction system

[0026] ;

[0027] 3, standard gene qualitative detection system sensitivity verification in chicken

[0028] In order to detect chicken TSA The detection sensitivity of the gene primer is ligated to gradient dilution (128 ng / μL, 12.8 ng / μL, 1.28 ng / μL, 0.128 ng / μL, 0.0128 ng / μL) for qualitative detection, using primers. TSA -F / b conducts normal PCR amplification detection, using 2% agarose gel electrophoresis (containing 0.1 μg / ml EB) to analyze the amplification product, the electrophoretic buffer is 1 × Tae, the upper sample is 5 μL, which is condensed Glue imaging device scans generates images and image analysis, the PCR reaction system with Table 2;

[0029] The results show that when the concentration of the chicken DNA template is 128 ng / μL, 12.8 ng / μL and 1.28 ng / μL, the clear-specific strip appears on the electrophoretic map, and the brightness is gradually weakened, and when the template concentration drops to 0.128 ng / μL Unexpled the product, the detection limit of the qualitative PCR was 1.28 ng / μL, such as image 3 Indicated.

[0030] 4, standard gene quantitative detection system sensitivity verification in chicken

[0031] In order to detect chicken TSAThe detection sensitivity of the gene primer is diluted to gradient (20 ng / μL, 4 ng / μL, 0.2 ng / μL, 0.4 ng / μL, 0.2 ng / μL, 0.4 ng / μL, 0.2 ng / μl, 0.1 ng / μL), SYBR GreenI, real-time quantitative PCR Amplification detection, quantitatively determines the detection sensitivity of standard gene specific primers in the eucalyptus. Sybr GreenI real-time fluorescent quantitative PCR amplification procedure: predetermitability (95 ° C, 10min); 40 cycles: denaturation (95 ° C, 30S), annealed (58 ° C, 30S), extended (72 ° C, 30S), PCR reaction system See Table 3;

[0032] Table 3 Specific primer verified Sybr Green I real-time fluorescent quantitative PCR reaction system

[0033]

[0034] Quantitative PCR amplification curve and standard curve see Figure 4 , 5 ,by Figure 4 It can be seen that when the template concentration is 200 pg / μL, specific amplification can still occur, the CT value is significantly higher than the negative control, which means that the minimum detection limit of SYBR Green I quantitative PCR is 200 pg / μL. according to Figure 5 , Standard curve formula: y = - 2.949X + 28.762, correlation coefficient R 2 0.998.

[0035] 5. Detection of PCR detection of chickens deep processing samples

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Application of gene TSA in detection of collybia albuminosa derived components

A technology of derived components and genes, applied in the field of biological species identification and PCR detection, to achieve the effect of less time-consuming, good specificity and simple operation

AI-Extracted Technical Summary

Problems solved by technology

Method used

Abstract

Image

Examples

Example Embodiment

Example Embodiment

PUM

Description & Claims & Application Information

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