阅读说明：本技术 一种用于克柔念珠菌核酸检测的试剂盒及检测方法 (Kit and method for detecting candida krusei nucleic acid ) 是由 郑锐 金明兰 张洪涛 于 2019-09-27 设计创作，主要内容包括：本发明提供了一种用于克柔念珠菌核酸检测的试剂盒及检测方法,其包括微流控芯片,所述微流控芯片包括芯片反应池,所述芯片反应池内包被固定有引物组,所述引物组包含克柔念珠菌的至少两个不同基因位点的特异性引物组。本发明提供的检测试剂盒和检测方法可检出的克柔念珠菌具有灵敏度高、特异性强、方便快捷、适用范围广等优点,同时,还有试剂消耗量小,污染小等优点,该实验全程为闭盖式反应,减少了污染的可能性,成本低、且便携。(The invention provides a kit and a detection method for candida krusei nucleic acid detection, which comprises a microfluidic chip, wherein the microfluidic chip comprises a chip reaction tank, a primer group is coated and fixed in the chip reaction tank, and the primer group comprises specific primer groups of at least two different gene loci of candida krusei. The candida krusei detectable by the detection kit and the detection method provided by the invention has the advantages of high sensitivity, strong specificity, convenience, rapidness, wide application range and the like, and simultaneously has the advantages of small reagent consumption, small pollution and the like.)

1. A nucleic acid detection kit for Candida krusei is characterized in that: the primer set comprises specific primer sets of at least two different gene sites of candida krusei.

2. The kit for candida krusei nucleic acid detection according to claim 1, wherein: the microfluidic chip is internally provided with a control group and comprises a sample inlet hole, a reaction hole and an air outlet hole.

3. The kit for candida krusei nucleic acid detection according to claim 2, wherein: the control group includes an array of amplification controls, blank controls, and extraction controls.

4. The kit for candida krusei nucleic acid detection according to claim 3, wherein: the at least two different gene loci of C.krusei comprise at least two of the cytb gene, ITS gene and ERG11 gene.

5. The kit for candida krusei nucleic acid detection according to claim 4, wherein: the primer group comprises a cytb gene locus primer group, an ITS gene locus primer group and an ERG11 gene locus primer group;

the primer group of the cytb gene locus comprises a primer shown as SEQ ID NO.1 ~ 5, the primer group of the ITS gene locus comprises a primer shown as SEQ ID NO.6 ~ 10, and the primer group of the ERG11 gene locus comprises a primer shown as SEQ ID NO.11 ~ 16.

6. The kit for candida krusei nucleic acid detection according to claim 4, wherein: the kit comprises a nucleic acid amplification reaction solution, wherein the nucleic acid amplification reaction solution comprises 10 x Thermomal Buffer, MgSO 42-10 mmol/L, BSA0.2-2mg/ml, dATP 0.2-3mmol/L, dGTP0.2-3mmol/L, dCTP0.2-1.5mmol/L, dTTP 0.2-1.5mmol/L, Betaine 0.2-1mol/L, EvaGreen 0.2-2 x and Bst polymerase0.05-2U/μ L.

7. A detection method for detecting candida krusei nucleic acid is characterized by comprising the following steps: the method comprises the following steps:

step S1, sample processing and template extraction are carried out to obtain a template to be detected;

step S2, dissolving, diluting and mixing single primers in the primer group to obtain a mixed primer group, and fixing the mixed primer group in a chip reaction pool of the microfluidic chip;

step S3, mixing the amplified control DNA and the primer, and fixing the mixture in a chip reaction pool;

step S4, nucleic acid amplification, taking amplification reaction liquid, and adding the amplification reaction liquid into a template to be detected;

step S5, vibrating, mixing evenly, centrifuging, injecting the centrifuged liquid into the microfluidic chip, and putting the microfluidic chip into a nucleic acid amplification analyzer for amplification;

in step S6, the detection of the product of the nucleic acid amplification reaction is performed under a nucleic acid amplification analyzer.

8. The detection method for candida krusei nucleic acid detection according to claim 7, wherein: the sample comprises throat swabs, sputum, alveolar lavage fluid, thoraco-abdominal, blood, cerebrospinal fluid, urine, pus and human tissues.

9. The detection method for candida krusei nucleic acid detection according to claim 7, wherein: the template extraction is manual extraction outside the chip or automatic extraction by directly injecting a sample into the microfluidic chip.

10. The detection method for candida krusei nucleic acid detection according to claim 9, wherein: the manual extraction includes: adding the to-be-detected bacterium liquid and the inactivated extracted quality control bacterium into a centrifugal tube, centrifuging, discarding the supernatant, adding a cleaning solution for cleaning once, centrifuging again, discarding the supernatant, adding an extracting solution into the centrifugal tube, mixing uniformly, adding the mixture into the centrifugal tube filled with glass beads, vibrating, heating at a constant temperature of 100 ℃ for 5min, centrifuging, and taking the supernatant as a template to be detected.

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a kit and a detection method for candida krusei nucleic acid detection.

Background

The candida is widely distributed, and most of the candida in a human body parasitize skin, oral cavity, respiratory tract, lower urinary tract and the like, and can cause infection of skin, mucous membrane and deep organs when the resistance of the human body is reduced. There are at least 17 candida species that cause human diseases, and among them, infection caused by candida albicans is the most common, but in recent years, with the increasing application of immunosuppressive agents, broad-spectrum antibiotics and glucocorticoids in organ transplantation, the clinical common candida spectrum has changed, and non-candida albicans infection such as candida krusei is more common. Candida krusei is a common non-candida albicans, widely existing in nature, and is particularly common in dairy products and easily fermented foods. Common symptoms include pulmonary candida infection, urinary candida infection, peritoneal candida infection, candidemia, and the like. The clinical routine analysis of biochemical indexes such as blood and the like can only preliminarily judge the etiology and cannot determine the species of pathogenic bacteria.

At present, the method for clinically identifying the pathogenic bacteria is mainly a culture method. The method mainly comprises the steps of sampling, culturing and identifying. The culture method has many defects and cannot meet clinical requirements. Firstly, the culture method takes long time for detection, the culture time is related to the type of the infected microorganism, the fungus is longer when the culture method is used for at least 2-3 days, and the culture method cannot be applied when acute diseases appear clinically; secondly, the method has low accuracy, and particularly when batch identification is carried out, cross contamination among samples is easy to occur; the method is limited by the safety protection conditions of the domestic laboratory, and laboratory workers have the possibility of high risk of infection when carrying out separation culture on pathogenic bacteria; the final culture method has higher requirements on experimental conditions, more types of required culture media, and the detection method of bacterial culture cannot be used in rural hospitals, community medical units, outdoors and the like, so that the patient is easily delayed. The method for identifying pathogenic microorganisms by using a common PCR (Polymerase chain reaction) technology has certain defects in the aspect of detection accuracy because the detection gene site is single, and the gene of pathogenic bacteria has polymorphism and variable anisotropy.

Disclosure of Invention

Aiming at the technical problems, the invention discloses a kit and a detection method for detecting candida krusei nucleic acid, which can detect a plurality of gene loci of candida krusei simultaneously and can detect the candida krusei rapidly.

In contrast, the technical scheme adopted by the invention is as follows:

a kit for detecting candida krusei nucleic acid comprises a microfluidic chip, wherein the microfluidic chip comprises a chip reaction tank, a primer group is coated and fixed in the chip reaction tank, and the primer group comprises specific primer groups of at least two different gene loci of candida krusei. Wherein the primer of the primer set is complementary to a part of the nucleotide sequence of the target gene or a complementary strand thereof.

By adopting the technical scheme of the invention, the detection of the gene locus is realized by coating the primer group fixed in the chip reaction tank, the technical scheme of the invention combines the nucleic acid amplification technology and the micro-fluidic chip technology, simultaneously detects a plurality of gene loci of candida krusei, and has the advantages of good specificity, high sensitivity, simple operation and rapid detection.

As a further improvement of the invention, a control group is arranged in the microfluidic chip, and the microfluidic chip comprises a sample inlet hole, a reaction hole and an air outlet hole.

As a further improvement of the invention, the control group comprises an array of amplification controls, blank controls and extraction controls. The amplification control uses a DNA fragment irrelevant to the candida krusei as a template for amplification, a blank control has no primer group, and an extraction control uses extracted quality control bacterium nucleic acid as a template for amplification. And an internal contrast is set, so that the whole process of using the product can be effectively controlled.

As a further improvement of the invention, the at least two different genetic loci of Candida krusei comprise at least two of the cytb gene, ITS gene, ERG11 gene.

As a further improvement of the invention, the primer group comprises a cytb gene locus primer group, an ITS gene locus primer group and an ERG11 gene locus primer group;

the primer group of the cytb gene locus comprises primers shown as SEQ ID NO. 1-5, the primer group of the ITS gene locus comprises primers shown as SEQ ID NO. 6-10, and the primer group of the ERG11 gene locus comprises primers shown as SEQ ID NO. 11-16.

As a further improvement of the invention, the kit comprises a nucleic acid amplification reaction solution, wherein the nucleic acid amplification reaction solution comprises 10 x Thermomal Buffer, MgSO 42-10 mmol/L, BSA0.2-2mg/ml, dATP 0.2-3mmol/L, dGTP0.2-3mmol/L, dCTP0.2-1.5mmol/L, dTTP 0.2-1.5mmol/L, Betaine 0.2-1mol/L, EvaGreen 0.2-2 x and Bst polymerase0.05-2U/μ L.

The invention also discloses a detection method for detecting the candida krusei nucleic acid, which comprises the following steps:

step S1, sample processing and template extraction are carried out to obtain a template to be detected;

step S2, dissolving, diluting and mixing single primers in the primer group to obtain a mixed primer group, and fixing the mixed primer group in a chip reaction pool of the microfluidic chip;

step S3, mixing the amplified control DNA and the primer, and fixing the mixture in a chip reaction pool;

step S4, nucleic acid amplification, taking amplification reaction liquid, and adding the amplification reaction liquid into a template to be detected;

and step S5, vibrating, mixing uniformly, centrifuging, injecting the centrifuged liquid into the microfluidic chip, and putting the microfluidic chip into a nucleic acid amplification analyzer for amplification.

In step S6, the detection of the product of the nucleic acid amplification reaction is performed under a nucleic acid amplification analyzer. According to the color reaction, compared with a blank control hole, the detection hole has an S-shaped curve on a nucleic acid amplification analyzer to be judged as positive, and the S-shaped curve is not found to be negative.

Wherein, the amplification comprises constant temperature amplification and variable temperature amplification.

By adopting the secondary technical scheme, primers are designed aiming at a plurality of gene loci of the pathogen or a plurality of gene loci of the pathogen, a nucleic acid amplification technology is utilized, a micro-fluidic chip is taken as a carrier for amplification, and result interpretation is carried out according to a color reaction, so that the method is simple and the detection is rapid.

As a further improvement of the invention, the sample comprises a throat swab, sputum, alveolar lavage fluid, thoraco-abdominal, blood, cerebrospinal fluid, urine, pus, and human tissue.

As a further improvement of the invention, the template extraction is manual extraction outside the chip or automatic extraction by directly injecting the sample into the microfluidic chip.

As a further improvement of the present invention, the manual extraction comprises: adding the to-be-detected bacterium liquid and the inactivated extracted quality control bacterium into a centrifugal tube, centrifuging, discarding the supernatant, adding a cleaning solution for cleaning once, centrifuging again, discarding the supernatant, adding an extracting solution into the centrifugal tube, mixing uniformly, adding the mixture into the centrifugal tube filled with glass beads, vibrating, heating at a constant temperature of 100 ℃ for 5min, centrifuging, and taking the supernatant as a template to be detected.

Compared with the prior art, the invention has the beneficial effects that:

by adopting the technical scheme, the invention provides 3 primer groups designed aiming at different gene loci of the candida krusei, fixes the primer group microfluidic chip and a kit containing the amplification reaction liquid to detect the locus of the cytb gene, the ITS gene and the ERG11 gene of the candida krusei in a sample. The candida krusei detectable by the detection kit and the detection method provided by the invention has the advantages of high sensitivity, strong specificity, convenience, rapidness, wide application range and the like, and simultaneously has the advantages of small reagent consumption, small pollution and the like.

Drawings

Fig. 1 is a schematic layout of a microfluidic chip according to the present invention.

FIG. 2 is a graph showing the results of the detection of a sample of Kluyveromyces strain in example 1 of the present invention, in which 3 different gene loci were detected.

FIG. 3 is a graph showing the results of the test of sample 1 of Kluyveromyces strain with a partial site mutation according to example 1 of the present invention, where the ERG11 gene and the cytb gene were detected.

FIG. 4 is a graph showing the results of the examination of a sample 2 of a Kluyveromyces strain having a partial site mutation according to example 1 of the present invention, in which the cytb gene was detected.

In the above-mentioned FIGS. 2 to 4, the abscissa of the graph is in minutes (min), the ordinate has no unit, "+" indicates that the corresponding index is detected, and "-" indicates that the corresponding index is not detected.

Detailed Description

Preferred embodiments of the present invention are described in further detail below.