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LAMP (loop-mediated isothermal amplification) primer composition for detecting Candida krusei, LAMP detection kit based on LAMP primer composition, and application method of LAMP detection kit

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 long amplification time, complicated PCR operation process, expensive PCR instrument, etc. , to achieve the effect of simple operation, high sensitivity and strong strain specificity

Inactive Publication Date: 2017-03-22
SHANGHAI EAST HOSPITAL +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 (loop-mediated isothermal amplification) primer composition for detecting Candida krusei, LAMP detection kit based on LAMP primer composition, and application method of LAMP detection kit
  • LAMP (loop-mediated isothermal amplification) primer composition for detecting Candida krusei, LAMP detection kit based on LAMP primer composition, and application method of LAMP detection kit
  • LAMP (loop-mediated isothermal amplification) primer composition for detecting Candida krusei, LAMP detection kit based on LAMP primer composition, and application method of LAMP detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] This embodiment provides a LAMP detection kit and detection method for Candida krusei. 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'-TAGTACTACACTGCGTGAG-3' (SEQ ID NO: 1);

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

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

[0051] Reverse inner primer BIP: 5'-GCAGCGAAATGCGATACCTATGTGCGTTCAAGAACTC-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 includes positive control and negative control...

Embodiment 2

[0057] This embodiment provides a LAMP detection kit and detection method for Candida krusei containing fluorescent dyes. 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'-TAGTACTACACTGCGTGAG-3' (SEQ ID NO: 1);

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

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

[0061] Reverse inner primer BIP: 5'-GCAGCGAAATGCGATACCTATGTGCGTTCAAGAACTC-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 concentration is 8U / μl; the control includes positi...

Embodiment 3

[0067] This embodiment provides a LAMP detection kit and detection method for Candida krusei 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'-TAGTACTACACTGCGTGAG-3' (SEQ ID NO: 1);

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

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

[0071] Reverse inner primer BIP: 5'-GCAGCGAAATGCGATACCTATGTGCGTTCAAGAACTC-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 concentration is 8U / μl; the control incl...

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Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) primer composition for detecting Candida krusei, a detection kit based on the primer composition, and a method for detecting Candida krusei; the kit comprises the LAMP primer composition, BstDNA polymerase, dNTP, 10*Isothermal Amplification reaction buffer solution, MgSO4, H2O and a color developing agent; the color developing agent includes fluorescent dye SYBGreen or fluorescent indicator Syto 9. The LAMP primer composition of the invention designs four primers at specific endogenous gene of Candida krusei, and has high cultivar specificity so that smooth reaction is guaranteed. In addition, the kit of the invention also has the advantages of good operation simplicity, high reaction speed, high reaction efficiency, high specificity and sensitivity, good identification process diversity and the like, and is suitable for clinical popularization and application.

Description

technical field [0001] The invention relates to the technical field of molecular detection of fungal pathogens, in particular to a LAMP primer composition of Candida krusei, 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 disease. They are ty...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6895C12Q1/6844C12Q2531/119C12Q2563/107
Inventor 吴文娟方雪恩王丽娟郭建李杨孔继烈
Owner SHANGHAI EAST HOSPITAL
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