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Kit for rapidly detecting candida albicans

A technology of Candida albicans and a kit, applied in the field of molecular biology, can solve the problems of difficult diagnosis of invasive candidiasis, lack of timely treatment for patients, high fatality rate, etc., to eliminate product contamination, real-time detection, high-efficiency The effect of specific amplification

Pending Publication Date: 2019-12-13
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The diagnosis of invasive candidiasis is difficult. The early symptoms are usually persistent low-grade fever, and the traditional gold standard for identification, that is, the culture method, takes at least two days to obtain the experimental results, which often prevents patients from receiving timely treatment and leads to a high mortality rate.
Molecular biology techniques such as PCR, real-time fluorescent PCR, and immunoassays have also been used for the detection of Candida albicans, but these methods are relatively complicated

Method used

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  • Kit for rapidly detecting candida albicans
  • Kit for rapidly detecting candida albicans
  • Kit for rapidly detecting candida albicans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] 1. Primer Design

[0067] According to the full-length sequence of Candida albicans ITS2 gene (No.KP675666.1), design specific primer pairs for rapid detection of Candida albicans:

[0068] Ft: 5'-gtcaaagcgatcccgccttacTGAGCGTCGTTTCTCCCCT-3'; SEQ ID NO.1;

[0069] Bt: 5'-cattccgccctagcgaaactgGATATACGTGGTAGACGTTGC-3'; SEQ ID NO.2;

[0070] IF: 5'-CTCAACACCAAACCCAGCGG-3'; SEQ ID NO.3;

[0071] IB: 5'-ACATTGCTTGCGGCG-3'; SEQ ID NO.4.

[0072] Primer sequences and their positions on the target gene such as figure 1 As shown, the primers include main primers Ft, Bt and accelerated primers IF, IB, Ft and Bt's 3' end (F and B parts, capital letters) and the target sequence ITS2 gene complementary (sequence 282-299; 437-417), The 5' ends of Ft and Bt (Tr and T parts, lowercase letters) are complementary to the target sequence (sequence 359–379), and accelerating primers IF and IB (sequence 323–304, 402–416) were also used as auxiliary primers for the ITS2 gene Amplify.

[...

Embodiment 2

[0089]Comparing the sensitivity of real-time turbidimetric method and chromogenic method in detecting Candida albicans with the traditional PCR method in Example 1, the pure nucleic acid extracted from C.albicans ATCC 24433 was diluted tenfold, and diluted from 69.0ng / μl To 0.069pg / μl, as a DNA template.

[0090] Real-time turbidimetry and colorimetric detection are the same as in Example 1.

[0091] PCR reaction uses 0.5 μM upstream primer SAP-F (SEQ ID NO.5) (5'-CTGCTGATATTACTGTTGGTTC-3') and 0.5 μM downstream primer SAP-B (SEQ ID NO.6) (5'-CCACCAATACCAACGGTATC-3') To carry out, 12.5 μl Tiangen PCR reaction Mix, and add the same amount of DNA template as the reaction system in Example 1 of the present invention. The PCR reaction was carried out according to the conventional method, the annealing temperature was set at 55°C, and the products were gelled after electrophoresis.

[0092] The result is as Figure 5-7 Shown, the detection limit of real-time turbidity method and...

Embodiment 3

[0094] For evaluating the applicability of the primers, kits and detection methods of the present invention, select 100 suspected patients who are infected by Candida albicans, and evaluate 100 samples with 4 methods:

[0095] (1) Microscopic examination commonly used in clinical practice, that is, to observe the secretion sample under a microscope after simple treatment, and to see budding yeast and pseudohyphae, which can be judged as Candida infection;

[0096] (2) CHROMagar color culture method;

[0097] (3) embodiment 1 real-time turbidity method;

[0098] (4) Embodiment 1 color development method.

[0099] In these 100 clinical samples, the real-time nephelometric method and the chromogenic method ( Figure 8 ) all detected 42 positives and 58 negatives, the results were in agreement with CHROMagar chromogenic medium culture method, and corrected some omissions and false detections of the microscopic examination method used in clinical practice.

[0100] Some comparis...

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Abstract

The invention discloses a kit for rapidly detecting candida albicans. The kit comprises a reagent I, a reagent II and a sealing agent; the reagent I comprises primers Ft and Bt, primers IF and IB, BstDNA polymerase, dNTP and an indicator; the reagent II comprises Tris-HCl, ammonium sulfate, magnesium sulfate, potassium chloride, Tween-20 and betaine, and the pH of the reagent II is 8.8. The kit provided by the invention adopts a unique buffer system, has the advantage of strong amplification compatibility, can carry out high-efficiency specific amplification without thoroughly removing impurities such as protein and the like, and is sensitive, quick and accurate in detection.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a kit for rapidly detecting Candida albicans. Background technique [0002] Candida albicans (Candida albicans) is the most important human opportunistic pathogenic fungus. It usually exists on the surface of normal human skin and mucous membranes. Bacteria multiply and cause diseases, which can be summarized into two categories: mucocutaneous candidiasis and invasive or deep organ candidiasis. [0003] Mucocutaneous candidiasis mainly includes thrush, interdigital erosion, papuloid, paronychia and gynecological candidiasis. Among them, oral and vulvovaginal candidiasis is the most common, which is characterized by a white film of bacteria that is easily scraped off on the affected area. Oral candidiasis (also known as oral thrush) occurs mostly in infants and young children under 2 years old, and is less common in adults. Vulvovaginal candidiasis (VVC) is the second most comm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/725
CPCC12Q1/6895C12Q1/6844C12Q2527/125
Inventor 王静刘威慈颖张乔张晓龙杨燕孙筱霞
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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