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Dimer mutating fluorescent primer quantative PCR method for synchronous quantifying and genetic typing of four aspergilli

A genotyping and fluorescent primer technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., to achieve good social and economic benefits, high sensitivity, and suitable for popularization and application.

Active Publication Date: 2017-05-31
HAINAN MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in order to prevent the probe from being destroyed by its own extension caused by hybridization with the target sequence, phosphorylation at the end of the probe is still required to block its extension. Therefore, it is not a true single-labeled probe technology

Method used

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  • Dimer mutating fluorescent primer quantative PCR method for synchronous quantifying and genetic typing of four aspergilli
  • Dimer mutating fluorescent primer quantative PCR method for synchronous quantifying and genetic typing of four aspergilli
  • Dimer mutating fluorescent primer quantative PCR method for synchronous quantifying and genetic typing of four aspergilli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] [Example 1] Quantitative and Genotyping Detection of 4 Kinds of Aspergillus

[0056] Preparation of the amplification system:

[0057] (1) Required reagents: PCR-mix buffer (Takara), template DNA

[0058] (2) Primer mixture: a mixture of three primers of 2.0 μM each

[0059] 1. Fungal Culture and DNA Extraction

[0060] The standard strains of Aspergillus fumigatus (ATCC 36607), Aspergillus niger (ATCC 16888), Aspergillus flavus (ATCC 16883) and Aspergillus terreus (ATCC 16792) were cultivated in sandcastle weak medium, and the DNA was extracted with a fungal DNA extraction kit.

[0061] 2. Construct quantitative standard plasmid

[0062] Amplify Aspergillus niger DNA sequence with common PCR method:

[0063] The upstream primer sequence is: 5'-CCCTACCTGATCCGAGGTCAACC-3' (SEQ ID No.1);

[0064] The downstream primer sequence is: 5'-AAAGTAAGACAGGAAATGTG-3' (SEQ ID No.2).

[0065] Amplification conditions and systems are the same as Real-time PCR.

[0066] The ampl...

Embodiment 2

[0078] [Example 2] The method is analyzed for specificity and repeatability

[0079] Specificity: Use this method to standard strains of Aspergillus and Candida albicans, Escherichia coli, Staphylococcus aureus, standard strains of Pseudomonas aeruginosa, Shigella, Staphylococcus epidermidis, Klebsiella pneumoniae And the Salmonella typhi DNA was extracted and tested with the established method to analyze the specificity of Real-time PCR detection. The amplification curve is shown in Figure 5 left.

[0080] Repeatability: The repeatability within the batch passed for 3 series of standard products (1×10 4 ~1×10 2 copies / ml) and 1.5×10 3 Concentration clinical samples were repeated to do 5 parallel tubes, and the quantitative results were calculated for SD and CV.

[0081] Standard plasmid 1×10 2 ~1×10 4Copies / ml, SD respectively: 0.707, 0.1845, 0.3172, CV respectively: 4.79%, 5.83%, 4.94%, see the amplification curve Figure 5 right.

[0082] The concentration of clini...

Embodiment 3

[0084] [Example 3] Determination of 4 clinical strains

[0085] According to the method described above, we collected four different types of Aspergillus strains that were biochemically verified from the clinic for detection. The results of the amplification curve and melting curve are as follows Figure 6 As shown, the present invention can simultaneously quantitatively detect 4 different types of Aspergillus.

[0086] Figure 6 Left: Amplification curves from left to right are Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus;

[0087] Figure 6 Right: Melting curves from left to right represent A. niger, A. flavus, A. fumigatus, A. terreus.

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Abstract

The invention belongs to the field of biological detection and provides a dimer mutating fluorescent primer quantative PCR method for synchronous quantifying and genetic typing of four aspergilla. The detection method comprises the following steps: 1) extracting DNA from a to-be-detected sample and four standard strains; 2) preparing a positive plasmid standard substance; 3) operating fluorescent quantitative PCR; and 4) performing data analysis. The invention also provides a detection kit for synchronous quantifying and genetic typing of aspergillus fumigates, aspergillus niger, aspergillus flavus and aspergillus terreus. The kit comprises an upstream primer marking a fluorescence reporter gene, an upstream complementary chain marked by a quenching gene and a downstream primer, the nucleotide sequences of which are as shown in SEQ ID No.1-3. The method is a novel real-time PCR technology which is high in universality, specificity and sensitivity, simple to operate and low in price, has clinical application value, and is expected to generate good social and economical benefits.

Description

technical field [0001] The invention belongs to the field of biological detection, and specifically relates to a method for quantitatively detecting aspergillus and synchronously genotyping a fluorescent quantitative PCR method established based on dimer mutation primers. Background technique [0002] Aspergillus includes 132 species and 18 variants, accounting for about 12% of fungi in the air. It is a common mold that spreads through the air. It was regarded as the source of infection as early as 1848. It not only affects the health of people and animals, but also It will cause huge losses to agricultural production. [0003] In recent years, with the increase of immunocompromised patients caused by organ, bone marrow and hematopoietic stem cell transplantation, HIV, tumor radiotherapy and chemotherapy, etc., the invasive fungal infection caused by opportunistic pathogenic bacteria-Aspergillus has increased year by year. The common ones are Aspergillus fumigatus (A.fumiga...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12R1/66C12R1/685C12R1/67
CPCC12Q1/6851C12Q1/6895C12Q2600/156C12Q2531/113C12Q2563/107Y02A50/30
Inventor 夏乾峰董素芳
Owner HAINAN MEDICAL COLLEGE
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