LAMP primer combination for detecting two kinds of cryptococcus and application of LAMP primer combination

A primer combination and primer set technology, applied in the biological field, can solve the problems of high experimental operation requirements and long detection time (about 2.5h-3h).

Active Publication Date: 2019-07-09
CAPITALBIO CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently established related molecular diagnostic methods include common PCR method, pulsed field gel electrophoresis typing (PFGE), multilocus sequence typing (MLST), restriction fragment length polymorphism analysis (RFLP), real-time fluorescent quantitative PCR technology (RTFQ-PCR), etc., the common problem is that the requirements for experimental operation are high, and the detection time is long (about 2.5h-3h)

Method used

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  • LAMP primer combination for detecting two kinds of cryptococcus and application of LAMP primer combination
  • LAMP primer combination for detecting two kinds of cryptococcus and application of LAMP primer combination
  • LAMP primer combination for detecting two kinds of cryptococcus and application of LAMP primer combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] Embodiment 1, the preparation of the primer combination that detects two kinds of cryptococci

[0110] The primer set for the detection of two Cryptococci consisted of two LAMP primer sets, each for the detection of one Cryptococcus.

[0111] The primer set for detecting Cryptococcus neoformans is as follows (5'→3'):

[0112] Primer I-F3 (SEQ ID NO: 1): CGAGAAATGTAGGTCCCAATT;

[0113] Primer I-B3 (SEQ ID NO: 2): GCTGCTGATACAAAGATCTAGTG;

[0114] Primer I-FIP (SEQ ID NO: 3): AGCCAAGGCTGGAGCCATTCAAAGTGCTGTCCTCAAATCTACC;

[0115] Primer I-BIP (SEQ ID NO: 4): ATCAGGAAATCTACAACGTCCACGGGAGGCATCGAGCATAGC;

[0116] Primer I-LF (SEQ ID NO: 5): GGACATGAATTCAGTGTAAA;

[0117]Primer I-LB (SEQ ID NO: 6): CTTCTTCTCTGGTCCTTC.

[0118] The primer set for detecting Cryptococcus gattii is as follows (5'→3'):

[0119] Primer I-F3 (SEQ ID NO: 7): AATCTACAACAATAGGCTCTG;

[0120] Primer I-B3 (SEQ ID NO: 8): GGCTGCTGATACAAGGTTC;

[0121] Primer I-FIP (SEQ ID NO: 9): TGGGAAATGGGGATGTAA...

Embodiment 2

[0130] Embodiment 2, specificity

[0131] 1. Preparation of samples to be tested

[0132] Test sample 1: Genomic DNA of Cryptococcus neoformans (ATCC, strain number: 208821).

[0133] Sample 2 to be tested: Genomic DNA of Cryptococcus gattii (ATCC, strain number: 14248).

[0134] 2. Detection of samples to be tested

[0135] Using the samples to be tested in step 1 as templates, the primer set I and primer set II pairs prepared in Example 1 were used for loop-mediated isothermal amplification detection.

[0136] Reaction system (10 μL): 7.0 μL reaction solution (product of Boao Bio Group Co., Ltd., catalog number CP.440020), 1 μL primer mixture, 1 μL template DNA (5pg-50pg), make up to 10 μL with water. The primer mixture is the mixture composed of each primer in primer group I or primer group II. In the reaction system, the final concentrations of outer primer F3 and outer primer B3 were both 0.5 μM, the final concentrations of inner primer FIP and inner primer BIP were b...

Embodiment 3

[0143] Embodiment 3, sensitivity

[0144] Sample to be tested 1: Genomic DNA of Cryptococcus neoformans;

[0145] Sample to be tested 2: Genomic DNA of Cryptococcus gattii;

[0146] 1. The sample to be tested is diluted with sterile water to obtain each dilution.

[0147] 2. Using the dilution solution obtained in step 1 as a template, respectively use primer set I and primer set II prepared in Example 1 to perform loop-mediated isothermal amplification.

[0148] When the sample to be tested is the sample to be tested 1, the loop-mediated isothermal amplification is performed using primer set I. When the sample to be tested is the sample to be tested 2, loop-mediated isothermal amplification is performed using primer set II.

[0149] Reaction system (10 μL): 7.0 μL reaction solution (product of Boao Biological Group Co., Ltd., its catalog number is CP.440020), 1 μL primer mixture, 1 μL diluent (1 μL of the genome copy number contained in the diluent is 10 3 , 5×10 2 、10 ...

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Abstract

The invention discloses LAMP primer combination for detecting two kinds of cryptococcus and application of the LAMP primer combination. The primer combination provided by the invention consists of 12primers as shown from sequence 1 to sequence 12. The primer combination provided by the invention can be applied to detect whether a sample to be detected contains cryptococcus neoformans and / or cryptococcus gattii. The primer combination identification provided by the invention is used for detecting two kinds of cryptococcus of cryptococci, has high specificity and high sensitivity, and can realize simple, convenient, fast and accurate detection. The LAMP primer combination has a significant promotional value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a combination of LAMP primers for detecting two cryptococci and its application. Background technique [0002] Invasive fungal disease (IFD), also known as invasive fungal infection (IFI) or deep fungal infection (DFI), refers to the invasion of fungi into human tissues and blood, where they grow and reproduce. A pathophysiological process that causes an inflammatory response and leads to tissue damage and organ dysfunction. In the past 20 to 30 years, with the wide application of new technologies such as solid organ and hematopoietic stem cell transplantation, tumor chemotherapy, and immunosuppressants, the morbidity and mortality of invasive fungal diseases have increased year by year, and it has increasingly become the leading cause. One of the leading causes of death in hospitalized patients. Invasive mycoses usually occur in the ICU, advanced age, diabetes, malignant blood sys...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/6844C12Q2531/119Y02A50/30
Inventor 张岩王颢婷单万水邢婉丽高占成程京
Owner CAPITALBIO CORP
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