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LAMP primer composition used for detecting candida parapsilosis and LAMP detection kit and application method thereof

A technology of Candida and primer composition, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of complicated PCR operation process, long amplification time, and expensive PCR instrument , to achieve the effect of high sensitivity, simple operation and strong strain specificity

Inactive Publication Date: 2017-03-22
SHANGHAI IGENETEC DIAGNOSTICS CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the PCR operation process is relatively complicated and requires professionals to operate, and not all hospitals have such professional technicians
In addition, the PCR instrument is expensive and the amplification time is long

Method used

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  • LAMP primer composition used for detecting candida parapsilosis and LAMP detection kit and application method thereof
  • LAMP primer composition used for detecting candida parapsilosis and LAMP detection kit and application method thereof
  • LAMP primer composition used for detecting candida parapsilosis and LAMP detection kit and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] This embodiment provides a LAMP detection kit and detection method for Candida parapsilosis. The kit includes primer solution, master mix, DNA polymerase and controls. The primer solution includes 50 μM forward outer primer F3, 50 μM reverse outer primer B3, 50 μM forward inner primer FIP and 50 μM reverse inner primer BIP, and the primer sequences are as follows:

[0048] Forward outer primer F3: 5'-TGATGTGAATCGAATGAGTATAGCA-3' (SEQ ID NO: 1);

[0049] Reverse outer primer B3: 5'-TGCATAGAGAGGGATTTAATGTGAA-3' (SEQ ID NO: 2);

[0050] Forward internal primer FIP: 5'-TGGGAGTTTGAGAAAAGCTCAAGCCGTGACATGTGTAGATCCTTGGTTA-3' (SEQ ID NO: 3);

[0051] Reverse internal primer BIP: 5'-TTGGGCATATTGGCAATCCCTGTATAGCATGGCATAAACACAACG-3' (SEQ ID NO: 4);

[0052] The master mix includes 12mM dNTP: 10×Isothermal Amplification reaction buffer: 150mMMgSO4 aqueous solution, the volume ratio is 8:5:2; the DNA polymerase is Bst DNA polymerase, the concentration is 8U / μl; the control include...

Embodiment 2

[0057] This embodiment provides a LAMP detection kit and detection method for Candida parapsilosis containing a fluorescent dye. The kit includes primer solution, master mix, DNA polymerase, controls and fluorescent dyes. The primer solution includes 50 μM forward outer primer F3, reverse outer primer B3, forward inner primer FIP, and 50 μM reverse inner primer BIP. The primer sequences are as follows:

[0058] Forward outer primer F3: 5'-TGATGTGAATCGAATGAGTATAGCA-3' (SEQ ID NO: 1);

[0059] Reverse outer primer B3: 5'-TGCATAGAGAGGGATTTAATGTGAA-3' (SEQ ID NO: 2);

[0060] Forward internal primer FIP: 5'-TGGGAGTTTGAGAAAAGCTCAAGCCGTGACATGTGTAGATCCTTGGTTA-3' (SEQ ID NO: 3);

[0061] Reverse internal primer BIP: 5'-TTGGGCATATTGGCAATCCCTGTATAGCATGGCATAAACACAACG-3' (SEQ ID NO: 4);

[0062] The master mix includes 12mM dNTP: 10×Isothermal Amplification reaction buffer: 150mMMgSO4 aqueous solution, the volume ratio is 8:5:2; the DNA polymerase is Bst DNA polymerase, the concentrati...

Embodiment 3

[0067] This embodiment provides a LAMP detection kit and detection method for Candida parapsilosis containing a fluorescent indicator. The kit includes primer solution, master mix, DNA polymerase, control and fluorescent indicator. The primer solution includes 50 μM forward outer primer F3, reverse outer primer B3, forward inner primer FIP, and 50 μM reverse inner primer BIP. The primer sequences are as follows:

[0068] Forward outer primer F3: 5'-TGATGTGAATCGAATGAGTATAGCA-3' (SEQ ID NO: 1);

[0069] Reverse outer primer B3: 5'-TGCATAGAGAGGGATTTAATGTGAA-3' (SEQ ID NO: 2);

[0070] Forward internal primer FIP: 5'-TGGGAGTTTGAGAAAAGCTCAAGCCGTGACATGTGTAGATCCTTGGTTA-3' (SEQ ID NO: 3);

[0071] Reverse internal primer BIP: 5'-TTGGGCATATTGGCAATCCCTGTATAGCATGGCATAAACACAACG-3' (SEQ ID NO: 4);

[0072] The master mix includes 12mM dNTP: 10×Isothermal Amplification reaction buffer: 150mMMgSO4 aqueous solution, the volume ratio is 8:5:2; the DNA polymerase is Bst DNA polymerase, the c...

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Abstract

The invention discloses an LAMP primer composition used for detecting candida parapsilosis, an LAMP detection kit based on the primer composition and a detection method. The detection kit comprises the LAMP primer composition, Bst DNA polymerase, a color developing agent and the like; the color developing agent is fluorochrome SYBGreen or a fluorescent indicator Syto 9. According to the LAMP primer composition, four primers are designed on a peculiar endogenous gene place of the candida parapsilosis, thus the LAMP primer composition has very strong cultivar specificity, and successful proceeding of a reaction is guaranteed. In addition, the detection kit has the advantages of being convenient to operate, fast and efficient in reaction, high in specificity and sensitivity, various in identification method and the like, and is suitable for application and popularization in the clinical practice.

Description

technical field [0001] The invention relates to the technical field of molecular detection of fungal pathogens, in particular to a LAMP primer composition for detecting Candida parapsilosis, a LAMP detection kit and a use method thereof. Background technique [0002] In recent years, with the wide application of broad-spectrum antibiotics, antineoplastic drugs and immunosuppressants, etc., the widespread development of organ transplantation, bone marrow transplantation, catheterization, etc., especially the prevalence of AIDS, resulting in fungi, especially Candida Chances of deep infection are increasing. [0003] Candida is a budding yeast-like fungus. Common Candida species that are known to cause disease are: Candida albicans, tropics, nearly smooth, Crews, stellate, Ji Yemeng and Candida glabrata. These bacteria not only widely exist in nature, but also can parasitize on normal human skin, oropharynx, gastrointestinal tract, anus and vaginal mucosa without causing dis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 方雪恩王丽娟吴文娟郭建李杨孔继烈
Owner SHANGHAI IGENETEC DIAGNOSTICS CO LTD
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