CN110551840A

CN110551840A - Nucleic acid reagent, kit, system and method for detecting invasive fungi

Info

Publication number: CN110551840A
Application number: CN201910770369.3A
Authority: CN
Inventors: 林笑冬; 王晓艳; 王雷; 吴瑞倩; 张志强
Original assignee: Beijing Zhuo Chenghui Biological Polytron Technologies Inc
Current assignee: Beijing Zhuo Chenghui Biological Polytron Technologies Inc
Priority date: 2019-08-20
Filing date: 2019-08-20
Publication date: 2019-12-10

Abstract

The present disclosure relates to a nucleic acid reagent, a kit, a system and a method for detecting invasive fungi, wherein the nucleic acid reagent comprises primers shown in SEQ ID NO.1-14 and probes shown in SEQ ID NO.17-26, which are stored independently of each other or randomly mixed with each other. According to the primer and the probe, the nucleic acid reagent, the kit, the system and the method for detecting at least 9 invasive fungi such as candida albicans, candida tropicalis, candida glabrata, candida parapsilosis, mucor, aspergillus, cryptococcus neoformans, pneumocystis yeri, candida auriculata and the like are established, so that the rapid, comprehensive, sensitive, specific and automatic detection result judgment can be realized, and the sensitivity, the specificity and the simplicity of simultaneously detecting the target genome are obviously improved.

Description

nucleic acid reagent, kit, system and method for detecting invasive fungi

Technical Field

The disclosure relates to the field of biotechnology, and in particular relates to a nucleic acid reagent, a kit and a method for detecting invasive fungi.

Background

Invasive Fungal Infection (IFI) refers to the pathological changes and pathophysiological processes in which fungi invade human tissues and blood, grow and reproduce to cause tissue damage, organ dysfunction and inflammatory response. In the last 30 years, with the development and utilization of solid organ and hematopoietic stem cell transplantation technology and catheter intervention and indwelling technology, and the massive application of immunosuppressants and radiotherapy and chemotherapy, the incidence and mortality of invasive fungal infections have increased dramatically worldwide. Epidemiological studies show that the nosocomial invasive fungal infection rate is 7.5 percent, the tumor patients have the invasive fungal infection rate of 8 percent, the severe burn and trauma patients have the invasive fungal infection rate of 16 percent, the invasive fungal infection rate of 55 to 70 percent, and the invasive fungal infection becomes a disease seriously threatening the life and health of human beings.

Generally, a large number of invasive fungi are planted in human bodies, when the human bodies are in a healthy state, the invasive fungi cannot damage the functions of the human bodies, and when the immune mechanisms of the human bodies are disordered or seriously damaged, the invasive fungi planted in the human bodies can damage the functions of blood vessels, digestive tracts and other organs and endanger the lives of patients, so that the diagnosis of the invasive fungi has great clinical value for clinical treatment and reasonable medication guidance of the patients with the impaired immune functions.

Currently, invasive tests are commonly used, including microscopic examination, histopathological examination, culture, and serological tests. Wherein, the positive rate in the detection results of microscopic examination and histopathological examination is low, and the interference of false negative can not be eliminated; the culture method has long detection period, low sensitivity and higher requirements on technical conditions and professional levels of operators, and only can realize single-target detection; serological tests can only determine whether a patient has a fungal infection and cannot differentiate the level of invasive fungal species.

Polymerase Chain Reaction (PCR) technology and multiplex Real-Time fluorescence PCR (RT-PCR) technology are commonly used in invasive fungus detection in recent years. PCR detection requires a complex sample processing procedure at the early stage, a cumbersome test procedure and a complex result judgment. In addition, the detection targets covered by RT-PCR are limited, and multi-target detection can be realized only by multi-system setting, so that the complexity of operation is increased.

Disclosure of Invention

The purpose of the disclosure is to provide a nucleic acid reagent, a kit, a system and a method for detecting invasive fungi, which are rapid, accurate, integrated and highly sensitive.

In order to achieve the above objects, the present disclosure provides a nucleic acid reagent for detecting invasive fungi, wherein the nucleic acid reagent comprises primers represented by SEQ ID nos. 1 to 14 and probes represented by SEQ ID nos. 17 to 26, each of which is stored independently of each other or in any mixture with each other.

Alternatively, the content of the primers represented by SEQ ID NO.2 to 14 and the content of the probes represented by SEQ ID NO.17 to 26 are each 1 to 3. mu. mol, respectively, with respect to 1. mu. mol of the primer represented by SEQ ID NO. 1.

Optionally, the nucleic acid reagent further comprises a positive internal quality control;

the positive endoplasmic control contains a primer shown by SEQ ID NO.15-16 and a probe shown by SEQ ID NO. 27.

Optionally, the nucleic acid reagent comprises tube a, tube B, and tube C; the tube A contains a primer shown in SEQ ID NO.1-2 and a probe shown in SEQ ID NO. 17-20; the tube B contains primers shown in SEQ ID NO.3-4 and 7-12 and probes shown in SEQ ID NO.21 and 23-25; the C tube contains primers shown in SEQ ID NO.5-6 and 13-16 and probes shown in SEQ ID NO.22 and 26-27.

Optionally, the probes shown in SEQ ID NO.17, 21, 22 have a first fluorescent label; the probes shown in SEQ ID NO.18, 23 and 26 have a second fluorescent label; the probes shown in SEQ ID NO.19 and 24 have a third fluorescent label; the probes shown in SEQ ID NO.20, 25 and 27 have a fourth fluorescent label; the first fluorescent label, the second fluorescent label, the third fluorescent label and the fourth fluorescent label are different from each other and are each independently selected from one of a FAM fluorescent label, a VIC fluorescent label, a CY5 fluorescent label and a ROX fluorescent label.

Optionally, the invasive fungus comprises at least one of candida albicans, candida tropicalis, candida glabrata, candida parapsilosis, mucor, aspergillus, cryptococcus neoformans, pneumocystis yeri, and candida auriculata.

The present disclosure also provides a kit for detecting invasive fungi, which comprises the nucleic acid reagent of any one of the above, and optionally, at least one of a reaction system buffer, a template DNA, and water.

The present disclosure also provides a use of the nucleic acid reagent of any one of the above in preparing a kit for detecting invasive fungi.

the present disclosure also provides a system for detecting invasive fungi, the system comprising a PCR instrument having an a-tube detector, a B-tube detector, and a C-tube detector, a computing device, and an output device; the tube A detector, the tube B detector and the tube C detector are respectively a nucleic acid reagent storage container loaded with any one of the nucleic acid reagents; the PCR instrument comprises a first fluorescence channel, a second fluorescence channel, a third fluorescence channel and a fourth fluorescence channel; the first, second, third and fourth fluorescent channels are different from each other and each independently selected from one of FAM, VIC, CY5 and ROX fluorescent channels; the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the discrimination as follows:

If the blank control, the positive control and the positive internal control are established, the detection result is valid;

if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is less than 35, judging that the detection result is positive; if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is more than or equal to 35 and less than 40, re-extracting the sample and rechecking, if the amplification curve obtained by rechecking is S-shaped and the CT value is less than 40, judging that the detection result is positive, otherwise, judging that the detection result is negative;

If the detection result of the first fluorescence channel of the tube A is positive, the sample is judged to contain candida albicans; if the detection result of the second fluorescence channel of the tube A is positive, the Candida tropicalis is judged to be contained in the sample; if the detection result of the third fluorescence channel of the tube A is positive, judging that the sample contains candida glabrata; if the detection result of the fourth fluorescence channel of the tube A is positive, judging that the sample contains candida parapsilosis;

If the detection result of the first fluorescence channel of the tube B is positive, the sample is judged to contain aspergillus; if the detection result of the second fluorescence channel of the tube B is positive, determining that the sample contains cryptococcus neoformans; if the detection result of the third fluorescent channel of the tube B is positive, determining that the sample contains the pneumocystis yedoensis; if the detection result of the fourth fluorescence channel of the tube B is positive, judging that the sample contains candida auricula;

If the detection result of the first fluorescence channel of the tube C is positive, determining that the sample contains mucor; if the detection result of the C tube second fluorescence channel is positive, judging that the sample contains fungi; and if the detection result of the fourth fluorescence channel of the C tube is positive, judging that the sample extraction and detection process is normal.

The present disclosure also provides a method for detecting an invasive fungus, wherein the method comprises: carrying out PCR amplification on a nucleic acid sequence of a sample to be detected by adopting the nucleic acid reagent; the PCR instrument for carrying out the PCR amplification comprises a first fluorescence channel, a second fluorescence channel, a third fluorescence channel and a fourth fluorescence channel; the first, second, third and fourth fluorescent channels are different from each other and each independently selected from one of FAM, VIC, CY5 or ROX fluorescent channels; and the following discrimination is made:

The beneficial effect of this disclosure lies in:

The method can quickly realize screening and identification of 9 invasive fungi in a sample to be detected, identify whether the sample contains other fungi, quickly and accurately detect the invasive fungi infected at common positions, avoid complicated operations of methods such as serology, pathogen culture and the like, and achieve the following detection effects:

Higher multiple detection capability

the detection method established by the disclosure can be used for simultaneously screening and identifying 9 invasive fungi such as candida albicans, candida tropicalis, candida glabrata, candida parapsilosis, mucor, aspergillus, cryptococcus neoformans, pneumocystis yeri, candida auriculata and the like in 3 reaction systems, identifying general fungal infection, quickly and simply confirming the pathogenic type of the invasive fungi, overcoming the defects of long time consumption, complex operation or single detection target of the traditional method, saving time, manpower and reagent cost and playing a key role in prompting drug resistance analysis of downstream fungi.

(II) high sensitivity

The detection method established by the disclosure can realize simultaneous detection of more than 9 invasive fungi, and the detection sensitivity of each species gene of target fungi to be detected in different samples to be detected, such as blood, sputum, alveolar lavage fluid and the like, can reach 5 multiplied by 10copies/mL, which is equivalent to the single real-time fluorescence PCR detection sensitivity.

(III) high specificity

In the detection method established by the disclosure, all primers are subjected to BLAST comparison analysis, have high conservatism and specificity, can distinguish detection targets from each other, but also can be distinguished from other microorganisms which have homology with the nucleic acid sequence, are easy to cause the same or similar clinical symptoms, are normally parasitic on the sampling part or are easy to be complicated, these microorganisms include at least human coronavirus, cytomegalovirus, enterovirus, measles virus, human metapneumovirus, mumps virus, rhinovirus, Bordetella pertussis, Chlamydia pneumoniae, Haemophilus influenzae, nonvirulent Mycobacterium tuberculosis, Neisseria meningitidis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Corynebacterium, Escherichia coli, Lactobacillus, Moraxella catarrhalis, gonococcus, Pseudomonas aeruginosa, Staphylococcus epidermidis, and the like.

(IV) prevention of false negative results

In the detection method established by the disclosure, a false negative detection result can be effectively prompted by using a positive internal control (IAC).

Additional features and advantages of the disclosure will be set forth in the detailed description which follows.

Detailed Description

The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

The first aspect of the present disclosure provides a nucleic acid reagent for detecting invasive fungi, wherein the nucleic acid reagent comprises primers represented by SEQ ID Nos. 1 to 14 and probes represented by SEQ ID Nos. 17 to 26, which are stored independently of each other or in any mixture with each other.

The nucleic acid reagent provided by the disclosure adopts a multiple fluorescence PCR technology, can quickly, accurately and simply screen and identify 9 invasive fungi such as candida albicans, candida tropicalis, candida glabrata, candida parapsilosis, mucor, aspergillus, cryptococcus neoformans, pneumocystis yeri, candida auriculata and the like, and can also identify general fungal infection. The method can quickly and accurately screen the variety of various invasive fungi carried by a sample to be tested, has important significance for clinical treatment and medication guidance of patients with impaired immune function, and is favorable for improving the survival rate of infected patients and guiding the prevention and treatment work of susceptible patients.

In the multiplex fluorescence PCR technology, the combination effect of the probe and the primer has an important influence on the amplification effect, the primer and the probe not only consider the problem of co-amplification of the primer and the probe of different target genes in a reaction system in the design process, namely, the conditions of evaluating the consistency of Tm values and the homogenization of GC content, avoiding the occurrence of hairpin structures, primer dimers and the like, but also ensure that the alternative primer and the probe segment can respectively and comprehensively cover the at least 9 invasive fungi, and the primer and the probe have good specificity, high coverage and high sensitivity.

In accordance with the present disclosure, the relative amounts of each primer and/or probe in the above-described nucleic acid reagents can vary over a wide range. For example, the content of the primers shown by SEQ ID NO.2 to 14 and the content of the probes shown by SEQ ID NO.17 to 26 are 1 to 3. mu. mol, respectively, and 0.5 to 1. mu. mol, respectively, relative to 1. mu. mol of the primer shown by SEQ ID NO. 1.

According to the present disclosure, in order to Control the Internal quality of the reaction system and better determine whether the reaction is disturbed, the nucleic acid reagent may further include an Internal Amplification Control (IAC). Further, the positive internal quality control can contain primers shown in SEQ ID NO.15-16 and a probe shown in SEQ ID NO. 27. In this case, the content of each of the primers represented by SEQ ID NO.15 to 16 and the content of the probe represented by SEQ ID NO.27 may be 1 to 3. mu. mol and 0.5 to 1. mu. mol, respectively, relative to 1. mu. mol of the primer represented by SEQ ID NO. 1. The positive endoplasmic control can effectively prompt false negative detection results caused by misoperation, PCR inhibitors and the like. Wherein, the positive endoplasmic control primer and the probe can be designed by using a human ribonuclease P (RNaseP) gene as a template.

According to the present disclosure, in order to enhance the accuracy of the detection result, the nucleic acid reagent may be divided into three tubes, for example, the nucleic acid reagent may include a tube a, a tube B, and a tube C; the tube A contains a primer shown in SEQ ID NO.1-2 and a probe shown in SEQ ID NO. 17-20; the tube B contains primers shown in SEQ ID NO.3-4 and 7-12 and probes shown in SEQ ID NO.21 and 23-25; the C tube contains primers shown in SEQ ID NO.5-6 and 13-16 and probes shown in SEQ ID NO.22 and 26-27.

Further, in order to allow amplification of different probes in the same system to be recognized separately, the probes may be fluorescently labeled separately. For example, in one embodiment, the probes shown in SEQ ID NO.17, 21, 22 have a first fluorescent label; the probes shown in SEQ ID NO.18, 23 and 26 have a second fluorescent label; the probes shown in SEQ ID NO.19 and 24 have a third fluorescent label; the probes shown in SEQ ID NO.20, 25 and 27 have a fourth fluorescent label; the first fluorescent label, the second fluorescent label, the third fluorescent label and the fourth fluorescent label are different from each other and are each independently selected from one of a FAM fluorescent label, a VIC fluorescent label, a CY5 fluorescent label and a ROX fluorescent label. As a particularly preferred embodiment, the probes shown in SEQ ID NO.17, 21, 22 have FAM fluorescent labels; the probes shown in SEQ ID NO.18, 23 and 26 have VIC fluorescent labels; the probes shown in SEQ ID NO.19 and 24 have a CY5 fluorescent label; the probes shown in SEQ ID NO.20, 25 and 27 have ROX fluorescent labels. In the probe, FAM is 6-carboxyfluorescein, VIC is a dye purchased from ABI company, CY5 is 5H-indocyanine, and ROX is 6-carboxy-X-rhodamine.

according to the present disclosure, the invasive fungus includes at least one of candida albicans, candida tropicalis, candida glabrata, candida parapsilosis, mucor, aspergillus, cryptococcus neoformans, pneumocystis yeri, and candida auriculata. Preferably, the candida albicans target gene is 18SrRNA + ITS1+5.8s + ITS2 gene, the candida tropicalis target gene is 18SrRNA + ITS1+5.8s + ITS2 gene, the candida glabrata target gene is 18SrRNA + ITS1+5.8s + ITS2 gene, the candida parapsilosis target gene is 18SrRNA + ITS1+5.8s + ITS2 gene, the mucorales target gene is 18SrRNA + ITS1+5.8s + ITS2 gene, the aspergillus target gene is 18SrRNA + ITS1+5.8s gene, the cryptococcus neoformans target gene is ITS2 gene, the sporotrichum target gene is LSU gene, and the candida tara target gene is 18SrRNA + ITS1+5.8s + 2 gene. The target genes of the above-mentioned detection target bacteria are well known in the art and will not be described herein.

a second aspect of the present disclosure provides a kit for detecting invasive fungi, which comprises the nucleic acid reagent of any one of the above, and optionally, at least one of a reaction system buffer, a template DNA, and water.

Further, the configuration of the kit may be: the total amount is 25 mu L, wherein 11-13 mu L of 2 XPCR buffer solution (containing IAC template); 2-3 mu L of 10 multiplied primer probe mixture (the concentration of each primer is 200-400 nM, the concentration of each probe is 100-200 nM), 3-6 mu L of DNA template, and the balance ultrapure water.

As a particularly preferred embodiment, the kit is: 2 XPCR buffer 12.5. mu.L (containing IAC template); 2.5. mu.L of 10 Xprimer probe mixture (each primer concentration is 200-400 nM, each probe concentration is 100-200 nM), 5. mu.L of DNA template, and 25. mu.L of ultrapure water.

The kit disclosed by the invention can realize rapid, comprehensive, accurate, sensitive, specific and automatic detection result judgment, and remarkably improves the sensitivity, specificity and simplicity of simultaneously detecting at least 9 invasive fungi such as candida albicans, candida tropicalis, candida glabrata, candida parapsilosis, mucor, aspergillus, cryptococcus neoformans, pneumocystis yeri, candida auriculae and the like.

In a third aspect of the present disclosure, the present disclosure provides use of the nucleic acid reagent of any one of the above in the preparation of a kit for detecting an invasive fungus.

A fourth aspect of the present disclosure provides a system for detecting invasive fungi, the system comprising a PCR instrument having an a-tube detector, a B-tube detector, and a C-tube detector, a computing device, and an output device; the tube A detector, the tube B detector and the tube C detector are respectively a nucleic acid reagent storage container loaded with any one of the nucleic acid reagents; the PCR instrument comprises a first fluorescence channel, a second fluorescence channel, a third fluorescence channel and a fourth fluorescence channel; the first, second, third and fourth fluorescent channels are different from each other and each independently selected from one of FAM, VIC, CY5 and ROX fluorescent channels; the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the discrimination as follows:

A fifth aspect of the present disclosure provides a method for detecting an invasive fungus, wherein the method comprises: carrying out PCR amplification on a nucleic acid sequence of a sample to be detected by adopting the nucleic acid reagent; the PCR instrument for carrying out the PCR amplification comprises a first fluorescence channel, a second fluorescence channel, a third fluorescence channel and a fourth fluorescence channel; the first, second, third and fourth fluorescent channels are different from each other and each independently selected from one of FAM, VIC, CY5 or ROX fluorescent channels; and the following discrimination is made:

Wherein, the PCR amplification condition can be as follows:

a：95℃，5min；

b：95℃，15s，

c: 60 ℃ for 45 s; b-c cycles through 45 reactions and collects fluorescence.

The sample to be detected can be at least one of blood, sputum and alveolar lavage fluid of a clinical patient, and the sample to be detected can be processed by adopting a thermal cracking method to obtain DNA to be detected.

Preferably, the method for detecting invasive fungi of the present disclosure is not used for diagnosis, or the qualitative and quantitative results of invasive fungi do not have a one-to-one correspondence to the occurrence or non-occurrence of diseases, and do not belong to the diagnosis result, but the qualitative and quantitative detection results of invasive fungi can be used as intermediate information for reference of clinicians.

The method disclosed by the invention can quickly, sensitively and specifically realize the systematic screening of at least 9 invasive fungi such as candida albicans, candida tropicalis, candida glabrata, candida parapsilosis, mucor, aspergillus, cryptococcus neoformans, pneumocystis yeri, candida auriculata and the like, the detection process is simple, the result is automatically interpreted and reliable, and the time, the labor and the reagent cost are saved.

The present disclosure is further illustrated in detail below by way of examples, but the present disclosure is not limited thereto.

Example 1 detection method and determination of detection result

1. Primer and probe synthesis

Sequence synthesis was performed according to the primer sequences shown in Table 1 and the probe sequences shown in Table 2. In the probe, FAM is 6-carboxyfluorescein, CY5 is 5H-indocyanine, ROX is 6-carboxy-X-rhodamine, VIC is a dye purchased from ABI company, and BHQ1-3 is a quenching group.

TABLE 1

TABLE 2

Detecting an object	Probe code	Probe sequence	SEQ ID NO
				CA	CA-P	FAM-acgtggtggacgttaccgcc-BHQ1	17
CT	CT-P	VIC-tgtggtggccactagcaaaat-BHQ1	18
				CG	CG-P	CY5-tctgtctrcccagcacgc-BHQ3	19
CP	CP-P	ROX-caaacccgagggtttgagggagaaa-BHQ2	20
				Asp	Asp-P	FAM-tcctcgagcgtatggggct-BHQ1	21
Muc	Muc-P	FAM-caacttgcgctcattggta-BHQ1	22
				Cn	Cn-P	VIC-ctcgggttttaytacctgttggactt-BHQ1	23
Pnj	Pnj-P	CY5-cagacttcttgcgataagg-BHQ3	24
				C-au	C-au-P	ROX-tgtcgttattgttactactgactctgacggttc-BHQ2	25
Fungi	Fungi-P	VIC-agaacggccawgcacca-BHQ1	26
				IAC	IAC-P	ROX-gctcccgagcagccgcttgcgatatcaaaggg-BHQ2	27

note: CA-candida albicans; CT-Candida tropicalis; CG-Candida glabrata; CP-Candida parapsilosis; Asp-Aspergillus; muc-mucor; cn-cryptococcus neoformans; Pnj-Pneumocystis yeri; C-au-Candida auricular; fungi-fungal general; IAC-positive internal quality control; in the sequence, y represents a degenerate base T/C, and r represents a degenerate base A/G.

2. Sample processing

after blood, sputum and alveolar lavage fluid samples of patients are collected by a conventional method, the fungal genome is extracted by a thermal cracking method.

3. Construction of a detection System

The reaction system was prepared as follows: the total system is 25 mu L, and the specific configuration is as follows: 2 XPCR buffer (containing IAC template) 12.5. mu.L; 10 Xprimer probe mixture 2.5. mu.L (each primer concentration is 0.2-0.4. mu.M, each probe concentration is 0.1-0.2. mu.M), DNA template 5. mu.L, ultrapure water to 25. mu.L.

the kit is divided into 3 reaction tubes of tube A, tube B and tube C, wherein the tube A contains a primer shown by SEQ ID NO.1-2 in the table 1 and a probe shown by SEQ ID NO.17-20 in the table 2; the tube B contains primers shown by SEQ ID NO.3-4 and 7-12 in the above table 1 and probes shown by SEQ ID NO.21 and 23-25 in the above table 2; the C-tube contains the primers shown by SEQ ID Nos. 5-6 and 13-16 in Table 1 above and the probes shown by SEQ ID Nos. 22 and 26-27 in Table 2 above.

Placing the PCR tube into a fluorescent quantitative PCR instrument, selecting FAM, VIC, CY5 and ROX as reporter groups, and carrying out the following reaction procedures:

a：95℃，5min；

b：95℃，15s，

c: 60 ℃ for 45 s; b-c cycle 40 reactions and fluorescence was collected at this stage.

and (3) judging a detection result:

If the blank control, the positive control and the positive internal control are established, the detection result is valid, otherwise, the experiment is regarded as invalid.

If the amplification curve detected by the fluorescence channel is S-shaped and the CT value is less than 35, judging that the detection result is positive; if the amplification curve detected by the fluorescence channel is S-shaped and the CT value is more than or equal to 35 and less than 40, re-extracting the sample and rechecking, if the amplification curve obtained by rechecking is S-shaped and the CT value is less than 40, judging that the detection result is positive, otherwise, judging that the detection result is negative; if the amplification curve detected by the fluorescence channel is non-S-shaped but the CT value is reported, the detection is judged to have non-specific amplification, and the collected fluorescence may be a non-specific fluorescence signal released by probe degradation.

the specific determination method of the detection result is shown in Table 3.

TABLE 3

Example 2 minimum detection Limit validation

The test samples for evaluation were prepared by selecting Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Mucor, Aspergillus, Cryptococcus neoformans, Coccidioides Yersinia and Candida auriculata in blood, sputum or alveolar lavage fluid, extracting their respective genomes by thermal cracking method, and quantifying to 5 × 10 ⁴ copies/mL, and then performing gradient dilution to obtain templates for evaluation having concentrations of 5 × 10 ³ copies/mL, 5 × 10 ² copies/mL, 5 × 10copies/mL, respectively.

The evaluation template was examined for each concentration according to the examination method of example 1, and the examination was repeated 20 times for each concentration gradient, and the average was taken as the final examination result, as shown in Table 4.

TABLE 4

as can be seen from Table 4, the lowest detection limits of the kit for detecting the genome of the target invasive fungus in blood, sputum and alveolar lavage fluid all reach 5 × 10copies/mL, and the kit has high detection sensitivity.

Example 3 specificity verification

Selecting human coronavirus (purchased from the Wuhan virus institute of Chinese academy of sciences, numbered 2233), cytomegalovirus (purchased from the Wuhan virus institute of Chinese academy of sciences, numbered 303), enterovirus (purchased from the Wuhan virus institute of Chinese academy of sciences, numbered 977), measles virus (purchased from the Wuhan virus institute of Chinese academy of sciences, numbered 1513), human metapneumovirus (purchased from the Wuhan virus institute of Chinese academy of sciences, numbered 1768), mumps virus (purchased from the Wuhan virus institute of Chinese academy of sciences, numbered 3145), rhinovirus (purchased from the Wuhan virus institute of Chinese academy of sciences, numbered 2343), bordetella pertussis (purchased from the Wuhan virus institute of Chinese academy of sciences, numbered 23524), chlamydia pneumoniae (purchased from the Wuhan virus institute of Chinese academy of sciences, numbered 14127), mycoplasma pneumoniae (purchased from the Wuhan virus institute of Chinese academy sciences, numbered 14121), haemophilus influenzae (purchased from the medical strain preservation center, 58532), nontoxic Mycobacterium tuberculosis (purchased from China medical culture Collection, 93009), Neisseria meningitidis (purchased from China medical culture Collection, 21123), Staphylococcus aureus (purchased from China medical culture Collection, 31122), Streptococcus pneumoniae (purchased from China medical culture Collection, 31108), Streptococcus pyogenes (purchased from China medical culture Collection, 25341), Streptococcus salivarius (purchased from China medical culture Collection, 25346), Corynebacterium (purchased from China medical culture Collection, 32451), Escherichia coli (purchased from China medical culture Collection, 32516), Lactobacillus (purchased from China medical culture Collection, 34106), Moraxella catarrhalis (purchased from China medical culture Collection, 11256), Gonococcus (purchased from China medical culture collection center, with the number of 13251), pseudomonas aeruginosa (purchased from China medical culture collection center, with the number of 15204), staphylococcus epidermidis (purchased from China medical culture collection center, with the number of 26069) and other microorganisms which have homology with the nucleic acid sequence of the target invasive fungus, are easy to cause the same or similar clinical symptoms, and are normally parasitic or easily complicated at the sampling position are taken as specificity evaluation samples.

By applying the kit provided by the disclosure, the specificity evaluation sample is detected according to the detection method of the embodiment 1, and under the condition that negative control, positive control and positive internal control are all satisfied, no non-specific fluorescent signal appears in the target to be detected, which indicates that the kit provided by the disclosure can effectively distinguish non-detection target bacteria and has better specificity.

EXAMPLE 4 shelf-Life testing of kits

5X 10copies/mL of Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Mucor, Aspergillus, Cryptococcus neoformans, Coccidioides Yersinia, Candida auriculata, and fungus universal synthetic genes were used as test samples for evaluation, and the test samples were frozen in 9 portions at day 0 in a freezer at-70 ℃. And (3) storing the assembled kit at the temperature of-20 ℃, and performing storage period tests on the kit with the time periods of 0, 10, 15, 30, 60, 90, 120, 150, 180 and 360 days respectively.

The results show that the kit disclosed by the invention is stored in a refrigerator at the temperature of-20 ℃, and the detection is positive in different storage periods, which indicates that the storage period of the kit is at least one year.

Comparative example

1. Primer and probe synthesis

Sequence synthesis was performed according to the primer and probe sequences shown in tables 5 and 6.

TABLE 5

TABLE 6

2. Detection method

(1) Sample processing

(2) Construction of a detection System

The reaction system was prepared as follows: the total system is 25 mu L, and the specific configuration is as follows: PCR buffer 22.5. mu.L, Taq DNA Polymerase 0.3. mu.L, Uracil-DNA Glycosylase 0.2. mu.L, DNA template 2.0. mu.L.

putting the PCR tube into a fluorescent quantitative PCR instrument, wherein a detection fluorescent channel is an FAM channel, an internal reference fluorescent channel is a VIC channel, and the reaction program is as follows:

a：40℃，5min；

b：95℃，5min；

c：95℃，15s，

d: 60 ℃ for 50 s; b-c cycle 40 reactions and fluorescence was collected at this stage.

(3) Determination of results

Fungal (FAM) Ct value >32 or "undet." (ABI 7500) "blank" (LightCycler480), and internal reference (VIC) Ct value <40 negative; the Ct value of the Fungus (FAM) is less than or equal to 30, and a better logarithmic growth curve is positive; fungi (FAM)30 < Ct samples < 32 are gray-scale bands and repeat tests are recommended for confirmation.

3. Minimum detection limit verification

²the detection method is utilized to detect the template for evaluating the minimum detection limit of each concentration in the embodiment 2, each concentration gradient is repeatedly detected for 20 times, and the average value is taken as the final detection result.

4. Specificity verification

the detection method is used for detecting each specificity evaluation sample in the embodiment 3, and the result shows that the detection result of the contrast kit on haemophilus influenzae is positive. It is demonstrated that the disclosed kits have better detection specificity compared to the control kit.

As can be seen from comparison of the examples and comparative examples, the present disclosure can detect at least 9 invasive fungi such as Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Mucor, Aspergillus, Cryptococcus neoformans, Coccidioides Yersinia, and Candida auriculata at a time, and has a lower minimum detection limit and high specificity.

the preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, for example, the a-tube detection target and the B-tube detection target can be interchanged. In order to avoid unnecessary repetition, various possible combinations will not be separately described in this disclosure.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Sequence listing

<110> Beijing Zhuozcheng Biotech GmbH

<120> nucleic acid reagent, kit, system and method for detecting invasive fungi

<130> 13930-K-ABT

<160> 35

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gactaggata tagctggtt 19

<210> 2

<211> 14

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ttctgggctg tttc 14

<210> 3

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ctgtccgagc gtcatt 16

<210> 4

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

acctgatccg aggtcacc 18

<210> 5

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tcgagtcttt gaacgca 17

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tgtgggttgt tgttgatact g 21

<210> 7

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctgtttgaga gtcatgaaa 19

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcaccttccc actaacacat tt 22

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cggactagga tatagctggt t 21

<210> 10

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ttctgggctg tttccct 17

<210> 11

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tcgttactac tgactctgac ggttcatt 28

<210> 12

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gtactgatac caacggcaac ttgatca 27

<210> 13

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ggraaactca ccaggtcca 19

<210> 14

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gcyctatccc cagcacga 18

<210> 15

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cgaagggttg ccgcttttcg gtgg 24

<210> 16

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gaccgaaccc ttctggtcag gcacc 25

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

acgtggtgga cgttaccgcc 20

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tgtggtggcc actagcaaaa t 21

<210> 19

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tctgtctrcc cagcacgc 18

<210> 20

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

caaacccgag ggtttgaggg agaaa 25

<210> 21

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tcctcgagcg tatggggct 19

<210> 22

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

caacttgcgc tcattggta 19

<210> 23

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ctcgggtttt aytacctgtt ggactt 26

<210> 24

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

cagacttctt gcgataagg 19

<210> 25

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

tgtcgttatt gttactactg actctgacgg ttc 33

<210> 26

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

agaacggcca wgcacca 17

<210> 27

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

gctcccgagc agccgcttgc gatatcaaag gg 32

<210> 28

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

actttyaaca ayggatctct tggytc 26

<210> 29

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

tgacrctsrr acaggcatg 19

<210> 30

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

tgatactgaa rcaggcgtrc tc 22

<210> 31

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

caagtcgaac ggtaacagga agaa 24

<210> 32

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

tcttgcgacg ttatgcggta t 21

<210> 33

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

catcgatgaa gaacgcagcg aaatgc 26

<210> 34

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

catcgatgaa gaacgyagcr aartgc 26

<210> 35

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

cttgcttctt tgctgacgag 20

Claims

1. a nucleic acid reagent for detecting invasive fungi, wherein the nucleic acid reagent comprises primers represented by SEQ ID nos. 1 to 14 and probes represented by SEQ ID nos. 17 to 26, which are stored independently of each other or are optionally mixed with each other.

2. The nucleic acid reagent according to claim 1, wherein the content of the primers represented by SEQ ID nos. 2 to 14 is 1 to 3. mu. mol, respectively, and the content of the probes represented by SEQ ID nos. 17 to 26 is 0.5 to 1. mu. mol, respectively, relative to 1. mu. mol of the primer represented by SEQ ID No. 1.

3. The nucleic acid reagent of claim 1, wherein the nucleic acid reagent further comprises a positive internal quality control;

4. the nucleic acid reagent of claim 3, wherein the nucleic acid reagent comprises tubes A, B, and C; the tube A contains a primer shown in SEQ ID NO.1-2 and a probe shown in SEQ ID NO. 17-20; the tube B contains primers shown in SEQ ID NO.3-4 and 7-12 and probes shown in SEQ ID NO.21 and 23-25; the C tube contains primers shown in SEQ ID NO.5-6 and 13-16 and probes shown in SEQ ID NO.22 and 26-27.

5. The nucleic acid reagent according to claim 4, wherein the probes represented by SEQ ID No.17, 21, 22 have a first fluorescent label; the probes shown in SEQ ID NO.18, 23 and 26 have a second fluorescent label; the probes shown in SEQ ID NO.19 and 24 have a third fluorescent label; the probes shown in SEQ ID NO.20, 25 and 27 have a fourth fluorescent label; the first fluorescent label, the second fluorescent label, the third fluorescent label and the fourth fluorescent label are different from each other and are each independently selected from one of a FAM fluorescent label, a VIC fluorescent label, a CY5 fluorescent label and a ROX fluorescent label.

6. The nucleic acid agent of any one of claims 1 to 5, wherein the invasive fungus comprises at least one of Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Mucor, Aspergillus, Cryptococcus neoformans, Pneumocystis yeri, and Candida auriculata.

7. a kit for detecting invasive fungi, comprising the nucleic acid reagent according to any one of claims 1 to 6, and optionally further comprising at least one of a reaction system buffer, a template DNA and water.

8. Use of the nucleic acid reagent of any one of claims 1 to 6 in the preparation of a kit for detecting invasive fungi.

9. A system for detecting invasive fungi, the system comprising a PCR instrument having an a-tube detector, a B-tube detector, and a C-tube detector, a computing device, and an output device; the tube A detector, the tube B detector and the tube C detector are respectively a nucleic acid reagent storage container loaded with the nucleic acid reagent according to any one of claims 4 to 6; the PCR instrument comprises a first fluorescence channel, a second fluorescence channel, a third fluorescence channel and a fourth fluorescence channel; the first, second, third and fourth fluorescent channels are different from each other and each independently selected from one of FAM, VIC, CY5 and ROX fluorescent channels; the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the discrimination as follows:

10. A method for detecting an invasive fungus, wherein the method comprises: performing PCR amplification on a nucleic acid sequence of a sample to be detected by using the nucleic acid reagent according to any one of claims 4 to 6; the PCR instrument for carrying out the PCR amplification comprises a first fluorescence channel, a second fluorescence channel, a third fluorescence channel and a fourth fluorescence channel; the first, second, third and fourth fluorescent channels are different from each other and each independently selected from one of FAM, VIC, CY5 or ROX fluorescent channels; and the following discrimination is made: