CN117144038A - Composition, method and application for detecting echinocandin and triazole drug-resistant candida - Google Patents

Composition, method and application for detecting echinocandin and triazole drug-resistant candida Download PDF

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CN117144038A
CN117144038A CN202311135618.4A CN202311135618A CN117144038A CN 117144038 A CN117144038 A CN 117144038A CN 202311135618 A CN202311135618 A CN 202311135618A CN 117144038 A CN117144038 A CN 117144038A
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王琦琦
刘伟
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Peking University First Hospital
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Abstract

The invention belongs to the technical field of fungus detection, and particularly relates to a composition, a method and application for detecting echinocandin and triazole drug-resistant candida. The detection method comprises the following steps: extracting fungus DNA in a sample, adding a certain proportion of primer and probe into a PCR system, detecting by Real-time PCR, and carrying out qualitative and quantitative analysis according to the measured Ct value and an amplification curve. In the invention, a blocking oligonucleotide capable of specifically blocking wild sites is added in the design of a primer probe. The 3' -end phosphorylated modified oligonucleotide can effectively prevent the extension of a DNA chain catalyzed by DNA polymerase, effectively block a wild site and inhibit the amplification of a wild template. The detection method provided by the invention has low detection limit and high sensitivity, and can also effectively detect the drug-resistant strain when the sensitive strain and the drug-resistant strain are mixed for infection.

Description

Composition, method and application for detecting echinocandin and triazole drug-resistant candida
Technical Field
The invention belongs to the technical field of fungus detection, and particularly relates to a composition, a method and application for detecting echinocandin and triazole drug-resistant candida.
Background
Invasive mycoses (invasive fungal disease, IFD) refer to pathological changes and pathophysiological processes in which fungi invade human tissue, blood, and grow and reproduce therein leading to tissue damage, organ dysfunction and inflammatory responses. In recent years, with the increasing number of immunocompromised individuals, the incidence of invasive mycoses has risen year by year, with invasive candidiasis being the most common. Common drugs for treating invasive candidiasis are echinocandins, triazole antifungal drugs such as itraconazole, fluconazole, posaconazole and the like, and antifungal drugs for treating candida are very limited. And with the continuous appearance of drug-resistant bacteria, the death rate is gradually increased.
In order to better select an effective therapeutic agent, rapid detection of resistant bacteria is required. However, the molecular detection method of the drug-resistant strain is limited, and the main method in the laboratory at present is to perform in-vitro drug sensitivity test after obtaining the strain based on culture positive, determine the drug-resistant strain, further extract DNA and perform PCR amplification on the drug-resistant related genes and combine with sanger sequencing. However, the problems of low positive rate of culture, long time consumption of in-vitro drug sensitivity test, complicated detection method and the like limit quick and accurate detection of drug-resistant bacteria, and even delay of correct treatment. Therefore, there is a need to establish an accurate, rapid and sensitive detection method for drug-resistant fungi molecules.
The mechanism of resistance to various antifungal agents for the treatment of candida has been studied by those skilled in the art. The drug resistance mechanism of echinocandin is mainly mutation of FKS hot spot region of target enzyme coding gene. Studies have shown that 90% of echinocandin resistant Candida albicans are caused by site mutations at the S641 and S645 of FKS1, and 88% of Candida glabrata are caused by several hot spot mutations of FKS1, FKS 2; echinocandin resistance of candida otophylla is also mediated primarily by hot spot mutations of FKS 1. It can be seen that a method for detecting echinocandin resistant strains by detection of FKS hotspot mutations is feasible. Baslastov et al first detected the S645 mutation of Candida albicans FKS1 using multiplex Molecular Beacon (MB) Real-time PCR. Zhao et al utilized a multiplex MB and melting curve method to detect multiple hot spot mutations of candida glabrata FKS1 and FKS2, enabling detection to be completed in 3 hours with up to 100% accuracy. The mechanism of candida triazole drug resistance is more complex, and besides ERG11 point mutation, the mechanism mainly comprises ERG11 over-expression, efflux pump over-expression, transcription factor mutation and the like. The triazole drug resistance of the aspergillus fumigatus is mainly caused by the mutation of the target enzyme Cyp51A, so that the developed detection methods are relatively more. Baslastov et al first detected the mutation at position 54 of Cyp51A using multiplex MB Real-time PCR, and then multiplex MB and TaqMan probes were used sequentially for the detection of multiple mutation sites. The commercial detection kit AsperGenius (PathoNostics) based on the multiplex real-time PCR can effectively detect the TR34, L98H, Y121F and T289A mutation and is successfully used for directly detecting clinical samples such as BALF and the like. However, only AsperGenius in the above method evaluated the detection ability for detecting a mixture of a drug-resistant bacterium and a sensitive bacterium, and the result showed that the method was effective in the drug-resistant bacterium: the sensitive bacteria ratio is lower than 1:5, so that the detection is not carried out.
In recent years, along with the increasing of fungal infection, the use of broad-spectrum antibacterial agents and the progress of detection technology, mixed infection of different strains, even mixed infection of sensitive bacteria and drug-resistant bacteria, is increasing. However, the traditional drug sensitivity detection method needs to separate and purify different mixed strains on the basis of positive culture, and if the proportion of drug-resistant bacteria is low, the existence of drug-resistant bacteria is difficult to detect, so that insensitive drugs are used, the formation of dominant groups of the drug-resistant bacteria is accelerated, and the disease is delayed.
The site-specific closed Real-time PCR (ASB Real-time PCR) is a method capable of effectively detecting SNP and Indel, and has the characteristics of high sensitivity, small required template amount, high detection speed, capability of detecting mutant templates in wild templates, and the like. The method is firstly used for diagnosing tumors, can detect mutant tumor cells in a large number of wild tissues, has extremely high sensitivity, can be detected when the mutant cells only contain 0.1 percent, and is suitable for detecting formalin-embedded-paraffin fixed tumor tissue samples.
At present, less technical research is applied to detect drug-resistant fungi, and CN113249452A patent discloses a primer probe combination for detecting candida albicans echinocandin drug-resistant mutation targets and application thereof, mainly comprising primers and probes for detecting mutation sites F641S, S645P and R1361H, however, the method is difficult to realize detection of triazole drug-resistant candida. And the existing detection method is difficult to detect mixed infection of sensitive bacteria and drug-resistant bacteria. In the earlier study, we applied this method to the detection of triazole-resistant Aspergillus fumigatus and could effectively detect resistant strains in them when they are infected by a combination of sensitive and resistant strains. However, methods for detecting drug-resistant candida species, particularly candida glabrata and candida otophyllum, and the mutation sites that can be detected are relatively limited and more difficult.
Disclosure of Invention
Based on FKS mutation of the echinocandin drug-resistant candida and ERG11 and PDR1 mutation of triazole drug-resistant candida discovered in early fungus monitoring work, the invention designs a specific primer and a probe by utilizing an ASB Real-time PCR principle, and establishes a rapid molecular detection method capable of effectively detecting drug-resistant strains when sensitive and drug-resistant strains are mixed and infected, so as to solve the problems of low detection rate of drug-resistant bacteria, long detection time and difficult detection of mixed infection of sensitive bacteria and drug-resistant bacteria in clinic.
The invention provides a composition for detecting candida with echinocandin and/or triazole drug resistance, which designs a mutation site specific primer and a wild site specific blocking probe by using an ASB Real-time PCR technology and provides technical support for qualitative and quantitative detection of the candida with echinocandin and triazole drug resistance.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a composition for detecting candida that are echinocandin and/or triazole-based resistant, the candida comprising candida glabrata and/or candida otophylla; the composition is any one or more of the following primer probe combinations:
(1) Combining: comprising a primer pair a for detecting candida glabrata PDR1-Y682C locus, a probe a and a blocking oligonucleotide a; the primer pair a comprises a forward primer a with a nucleotide sequence shown as SEQ ID NO.1 and a reverse primer a with a nucleotide sequence shown as SEQ ID NO.2, the nucleotide sequence of the probe a is shown as SEQ ID NO.3, and the nucleotide sequence of the blocking oligonucleotide a is shown as SEQ ID NO. 4;
(2) And (2) combining two: comprising a primer pair b for detecting candida glabrata PDR1-I380L locus, a probe b and a blocking oligonucleotide b; the primer pair b comprises a forward primer b with a nucleotide sequence shown as SEQ ID NO.5 and a reverse primer b with a nucleotide sequence shown as SEQ ID NO.6, the nucleotide sequence of the probe b is shown as SEQ ID NO.7, and the nucleotide sequence of the blocking oligonucleotide b is shown as SEQ ID NO. 8;
(3) And (3) combining three: comprising a primer pair c for detecting the PDR1-G346D locus of Candida glabrata, a probe c and a blocking oligonucleotide c; the primer pair c comprises a forward primer c with a nucleotide sequence shown as SEQ ID NO.9 and a reverse primer c with a nucleotide sequence shown as SEQ ID NO.10, the nucleotide sequence of the probe c is shown as SEQ ID NO.11, and the nucleotide sequence of the blocking oligonucleotide c is shown as SEQ ID NO. 12;
(4) Combination four: comprising a primer pair D for detecting the PDR1-G1099D locus of Candida glabrata, a probe D and a blocking oligonucleotide D; the primer pair d comprises a forward primer d with a nucleotide sequence shown as SEQ ID NO.13 and a reverse primer d with a nucleotide sequence shown as SEQ ID NO.14, the nucleotide sequence of the probe d is shown as SEQ ID NO.15, and the nucleotide sequence of the blocking oligonucleotide d is shown as SEQ ID NO. 16;
(5) And (5) combining: comprising a primer pair e for detecting candida glabrata FKS2-S663P site, a probe e and a blocking oligonucleotide e; the primer pair e comprises a forward primer e with a nucleotide sequence shown as SEQ ID NO.17 and a reverse primer e with a nucleotide sequence shown as SEQ ID NO.18, the nucleotide sequence of the probe e is shown as SEQ ID NO.19, and the nucleotide sequence of the blocking oligonucleotide e is shown as SEQ ID NO. 20;
(6) And (3) combining six: a primer pair F for detecting candida otorhinoca ERG11-Y132F locus, a probe F and a blocking oligonucleotide F; the primer pair f comprises a forward primer f with a nucleotide sequence shown as SEQ ID NO.21 and a reverse primer f with a nucleotide sequence shown as SEQ ID NO.22, the nucleotide sequence of the probe f is shown as SEQ ID NO.23, and the nucleotide sequence of the blocking oligonucleotide f is shown as SEQ ID NO. 24;
(7) Combination seven: comprising a primer pair g for detecting candida otophylla FKS1-S639F site, a probe g and a blocking oligonucleotide g; the primer pair g comprises a forward primer g with a nucleotide sequence shown as SEQ ID NO.25 and a reverse primer g with a nucleotide sequence shown as SEQ ID NO.26, the nucleotide sequence of the probe g is shown as SEQ ID NO.27, and the nucleotide sequence of the blocking oligonucleotide g is shown as SEQ ID NO. 28.
Further, the probes a, b, c, d, e, f and g are 5 '-end-labeled FAM and 3' -end-labeled MBG.
Further, 3' -ends of the blocking oligonucleotides a, b, c, d, e, f and g are linked to PO4.
Further, the Tm value of the site-specific primer is 10℃lower than the PCR extension temperature.
Further, the blocking oligonucleotide is on the same strand as the site-specific primer; the 3' end of the blocking oligonucleotide is phosphorylated to prevent wild-type template amplification; the blocking oligonucleotide has a Tm value that is approximately equal to, but not exceeding, the PCR extension temperature.
The second object of the invention is to provide a method for qualitatively identifying candida in echinocandin and/or triazole drug resistance by using the composition, which can realize the qualitative identification of candida glabrata and candida otophyllum in echinocandin and triazole drug resistance, and especially can detect the drug-resistant candida in sensitive and drug-resistant strains during mixed infection.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for qualitatively identifying candida in echinocandin and/or triazole class resistance using the foregoing composition comprising the steps of:
s1: extracting fungus DNA in a sample;
s2: and (3) taking the fungus DNA obtained in the step (S1) as a template, carrying out real-time fluorescence quantitative PCR by using the composition to obtain an amplification curve, and judging whether a sample to be detected contains drug-resistant candida or not.
Further, in S2, the amplification reaction procedure is: the first stage: 2min at 50 ℃ and 10min at 95 ℃ for 1 cycle; and a second stage: 15s at 95℃and 1min at 60℃for 40 cycles; the reaction system is as follows: 1 μl of DNA template, taqPath ProAmp Master Mix μl, 1 μl of forward primer, 1 μl of reverse primer, 0.5 μl of probe, 4 μl of blocking oligonucleotide, and no nuclease water up to 20 μl.
Further, the concentration of the forward primer, the reverse primer, the probe and the blocking oligonucleotide is 10 mu M; the working concentration of the blocking oligonucleotide was 4 times that of the site-specific primer.
Further, if the real-time fluorescence quantitative PCR obtains an amplification curve and the Ct value is less than or equal to 35, judging that the drug-resistant candida is positive; if no amplification curve or Ct value is more than 35, the drug-resistant candida is judged to be negative.
The third object of the present invention is to provide a method for quantitatively determining the bacterial load of echinocandin and/or triazole-resistant candida based on the aforementioned method.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the method for quantitatively determining the bacterial load of the candida with echinocandin and/or triazole drug resistance based on the method comprises the steps that a sample to be detected is detected by the method to obtain a Ct value; substituting the Ct value into a linear equation of a standard curve corresponding to the target gene, and calculating the bacterial load of the drug-resistant bacteria.
Further, at 10-10 6 The drug resistant candida species include candida glabrata and/or candida otophylla within a linear range of individual spores/responses; the linear equation of the candida otorhinoceros ERG11-Y132F is Y= -3.3569X+37.962; the linear equation of candida glabrata PDR1-Y682C is Y= -3.6112X+36.902; the linear equation of candida glabrata PDR1-G1099D is Y= -3.1197X+35.782; the linear equation of candida glabrata PDR1-G346D is y= -2.6826x+34.402; the linear equation of candida glabrata PDR1-I380L is Y= -3.6439X+42.522; the linear equation of candida glabrata FKS2-S663P is Y= -3.1340X+40.116; the linear equation of candida otophylla FKS1-S639F is Y= -3.9855X+41.829; wherein Y is CT value, X is lg value of spore amount corresponding to each reaction hole.
It is a fourth object of the present invention to provide the use of the aforementioned composition for the preparation of a reagent or kit for detecting candida resistant in a mixed infection of candida resistant and candida susceptible.
The composition can effectively detect drug-resistant candida glabrata and drug-resistant candida otophylla in sensitive and drug-resistant strains when the strains are mixed for infection, and has high sensitivity and low detection limit.
Preferably, the drug-resistant candida is candida glabrata and/or candida otophyllum which are resistant to echinocandins and/or triazoles, and the sensitive candida is candida glabrata and/or candida otophyllum which are sensitive to echinocandins and/or triazoles.
The fifth object of the present invention is to provide a kit for detecting candida that are resistant to echinocandins and/or triazoles, said kit comprising the aforementioned composition.
Further, the drug-resistant candida includes candida glabrata and/or candida otophylla.
The invention has the beneficial effects that:
1. unlike the previous reported research, the invention does not use the DNA content as the evaluation of the detection method, but directly counts spores for detection, and the standard curve also uses the spore amount as the abscissa, so that the amount of spores contained in each reaction can be calculated according to the Ct value obtained by detection, thereby being more in line with the actual condition of clinical infection, and besides detecting the existence of drug-resistant bacteria, the bacterial load can be estimated, so that the treatment can be carried out more rapidly and accurately.
2. The standard curve of the detection method established by the invention is 10-10 6 There is a good linear range between individual spores/reactions, the lower limit of detection is typically 10 spores/reaction, and the lower limit of detection for certain primers and probes in the course of repeated validation sometimes reaches even 1 spore/reaction, with extremely high sensitivity.
3. The site-specific blocking real-time quantitative PCR established by the invention uses the specific blocking oligonucleotide peptide to block the amplification of a large number of wild templates, so that the sensitive strain can be detected when the proportion is as low as 1%, can be theoretically used for detecting mixed infection in clinical samples, can be detected even when the bacterial load in the samples is small, and does not need to rely on culture positivity.
Drawings
FIG. 1 is a schematic diagram of ASB Real-time PCR;
FIG. 2 is an amplification plot of CaueRG 11-Y132F;
FIG. 3 is a standard graph of Cauerg 11-Y132F;
FIG. 4 is an amplification plot of CgPDR 1-Y682C;
FIG. 5 is a standard graph of CgPDR 1-Y682C;
FIG. 6 is an amplification plot of CgPDR 1-G1099D;
FIG. 7 is a standard graph of CgPDR 1-G1099D;
FIG. 8 is an amplification plot of CgPDR 1-G346D;
FIG. 9 is a standard graph of CgPDR 1-G346D;
FIG. 10 is an amplification plot of CgPDR 1-I380L;
FIG. 11 is a standard graph of CgPDR 1-I380L;
FIG. 12 is a graph showing amplification of CgFKS 2-S663P;
FIG. 13 is a standard graph of CgFKS 2-S663P;
FIG. 14 is an amplification plot of CauFKS 1-S639F;
FIG. 15 is a standard graph of CauFKS 1-S639F;
FIGS. 16 to 22 show amplification curves of the drug-resistant bacteria at different ratios of the mixed strain, wherein FIG. 16 shows amplification curves of CauERG11-Y132F, FIG. 17 shows amplification curves of CgPDR1-Y682C, FIG. 18 shows amplification curves of CgPDR1-G1099D, FIG. 19 shows amplification curves of CgPDR1-G346D, FIG. 20 shows amplification curves of CgPDR1-I380L, FIG. 21 shows amplification curves of CgFKS2-S663P, and FIG. 22 shows amplification curves of CauFKS 1-S639F.
Detailed Description
The technical scheme of the present invention will be further clearly and completely described in connection with specific embodiments. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. Therefore, all other embodiments obtained by those skilled in the art without undue burden are within the scope of the invention based on the embodiments of the present invention.
In the embodiment of the invention, the tested strains are all preserved in the research center of fungus and mycosis of Beijing university and are identified to the seed level through molecules. The strain and the corresponding drug sensitive result and the drug resistance related gene mutation site are shown in Table 1. Primers and probes were designed for a total of 11 point mutations that have clearly led to drug resistance: the S663P mutation of echinocandin resistant Candida glabrata FKS2, the G346D, G1099D, I380L, Y682C mutation of triazole resistant Candida glabrata PDR 1; Y132F of echinocandin and triazole-resistant candida otorhinoceros ERG11 and S639F mutation sites of FKS 1.
TABLE 1 bacterial strains and corresponding drug-sensitive results and drug-resistant related gene mutation sites
In the embodiment of the invention, the schematic diagram of ASB Real-time PCR is shown in FIG. 1. Each reaction contains a site-specific forward primer, specifically amplifying mutation site A; an oligonucleotide with 3' -end modified by phosphorylation seals the wild type G site and inhibits the amplification of the wild type template; a specific Taqman probe for improving the specificity of amplification; a reverse primer. The principle of ASB Real-time PCR is that primer extension starts from the 3' end during PCR, and the 3' end base pair primer extension is critical because Taq DNA polymerase lacks 3' -5' exonuclease activity and cannot repair the base mismatch of the primer at the 3' end. If the primer 3' terminal base and template are complementary, the primer can be extended, whereas if not, it cannot be extended, resulting in a significant reduction or absence of amplification product. Therefore, the mutation site related to the drug resistance is designed at the 3' -end of one primer, and when the primer is used for PCR, the mutation site is contained in the detected gene if the detected gene can be amplified; if not amplified, this mutation site is indicated. However, for the detection of SNP-type drug-resistant mutation sites, this method can give rise to non-specific amplification in the presence of large amounts of wild-type templates. Therefore, in the research, a blocking oligonucleotide (blocking oligonucleotide) capable of specifically blocking a wild site is added, the 3' -end of the oligonucleotide modified by phosphorylation can prevent the extension of a DNA chain catalyzed by DNA polymerase, the wild site is effectively blocked, the amplification of a wild type template is inhibited, the Tm value can be increased by 10 ℃, and the specificity of the template is obviously improved, namely, only the template containing the mutation site can be amplified. In conclusion, ASB Real-time PCR performs fluorescent quantitative PCR by designing mutation site specific primers and a wild site specific blocking probe, and judges whether a gene template has mutation sites or not by judging whether an amplification curve exists or not.
Example 1
(1) Primer and probe design
The design principle of the primer and the probe of the invention is as follows:
site-specific primers: (1) may be on the forward or reverse primer; (2) the 3' end contains mutation site; (3) the Tm value was 10℃lower than the elongation temperature of PCR.
Blocking oligonucleotides: (1) on the same strand as the site-specific primer; (2) the wild type site requiring blocking is located in the middle of the oligonucleotide; (3) phosphorylation at the 3' end to prevent wild-type template amplification; (4) the Tm is about equal to, but not exceeding, the elongation temperature of the PCR; (5) the working concentration was 4 times that of the site-specific primer.
Primers and probes were further designed based on the above principle, and the information of the primers and probes is shown in Table 2.
TABLE 2 primer, probe sequences
In table 2, cg: candida glabra, candida glabrata; cau: candida auris, candida otophylla; f: forward Primer, forward Primer; r: reverse Primer, reverse Primer; p: probe, specific fluorescent Probe; ASB: allle-Specific Blocker, site-Specific blocking oligonucleotide.
(2) Reaction system and reaction conditions
The reaction system is shown in Table 3.
TABLE 3 reaction System
Reagent(s) Volume (mu L)
DNA template 1
TaqPath ProAmp Master Mix 10
Forward primer (10. Mu.M) 1
Reverse primer (10. Mu.M) 1
Probe (10 mu M) 0.5
Site-specific blocking oligonucleotides (10. Mu.M) 4
Nuclease-free water Is added to 20 mu L
The reaction conditions were as follows:
stage 1: (2 min at 50 ℃ C., 10min at 95 ℃ C.) for 1 cycle;
stage 2: (15 s at 95 ℃ C., 1min at 60 ℃ C.) for 40 cycles.
(3) Sample preparation and Standard Curve
Inoculating Candida to YPD solid medium, culturing at 35deg.C for 1-2 days, and counting 1×10 respectively 8 ，1*10 7 ，1*10 6 ，1*10 5 ，1*10 4 ，1*10 3 The spores were used to extract DNA, and the Biospin fungal genomic DNA extraction kit (BioFlux) was used to extract DNA. The specific operation is as follows:
1) The isolated Aspergillus strain was inoculated into PDA slant medium and cultured at 37℃for 2-4d. Adding 1mL of sterilized water for injection into a 1.5mL centrifuge tube, dipping a large number of spores on a culture medium after a sterile cotton swab is dipped, dissolving the spores in the centrifuge tube, centrifuging for 3min at 14000g, and discarding the upper liquid;
2) Adding 400 mu L LE Buffer and proper amount of acid-washed glass beads with the diameter of 425-600nm into a centrifuge tube, uniformly mixing, placing into a tissue homogenizer for cracking and shaking for 10min, and placing into a metal water bath instrument for warm bath for 15-30min;
3) 130 mu L of DA Buffer is added, and after uniform mixing, ice bath is carried out for 5min, and 14000g is centrifuged for 3min;
4) Transferring the supernatant of the centrifuge tube into a new 1.5mL centrifuge tube;
5) Adding the E Binding Buffer with the volume 1.5 times of that of the supernatant, and uniformly mixing;
6) Transferring the mixed liquid to Spin column, centrifuging 6000g for 1min, and discarding the liquid;
7) Adding 500 mu L G Binding Buffer to Spin column, centrifuging for 30s at 10000g, and discarding the liquid;
8) Adding 600 mu L of Wash Buffer into Spin column, centrifuging 10000g for 30s, discarding the liquid, and repeating the steps;
9) Adding 100 μl of the solution buffer into Spin column, incubating for 2min at room temperature, centrifuging for 2min with 10000g, discarding Spin column, storing genomic DNA in 1.5mL centrifuge tube, and storing at-20deg.C. Amplifying 1 μl as template, i.e. 1 spore amount corresponding to each reaction well*10 6 ，1*10 5 ，1*10 4 ，1*10 3 ，1*10 2 And (5) drawing a standard curve corresponding to the amplified Ct value and the spore amount by 10.
Results: the standard curve is shown in Table 4, and the spore amount corresponding to each reaction well is 1×10 6 ，1*10 5 ，1*10 4 ，1*10 3 ，1*10 2 And 10 times, the obtained Ct value has a better linear range, and the bacterial load of the drug-resistant bacteria can be estimated approximately according to the linear range of each gene. The lower limit of detection was about 10 spores of DNA per reaction well, see in particular FIGS. 2-15.
TABLE 4 Standard curve
Assay Equation R 2
CauERG11-Y132F Y＝-3.3569X+37.962 0.9983
CgPDR1-Y682C Y＝-3.6112X+36.902 0.9947
CgPDR1-G1099D Y＝-3.1197X+35.782 0.9970
CgPDR1-G346D Y＝-2.6826X+34.402 0.9803
CgPDR1-I380L Y＝-3.6439X+42.522 0.9842
CgFKS2-S663P Y＝-3.1340X+40.116 0.9925
CauFKS1-S639F Y＝-3.9855X+41.829 0.9719
Note that: y is CT value, X is lg value of spore amount corresponding to each reaction hole.
Example 2 detection efficacy against drug resistant strains in Mixed sensitive and drug resistant strains
Sensitive bacteria and drug-resistant bacteria are respectively taken 1 x 10 8 Extracting DNA from the spores, mixing the DNA of the trisensitive bacteria and the DNA of the drug-resistant bacteria according to different proportions to obtain templates with the drug-resistant strain DNA accounting for different proportions of the total DNA: 100%,50%,25%,10%,5%,1%,0% (resistance/total) of templates were amplified on a Applied Biosystems ViiA Real-Time PCR System according to the method and conditions of example 1, using 1. Mu.L of the templates in different proportions as reaction templates, and the ability of the method to detect drug-resistant strains in mixed infection was judged. The lowest proportion with obvious amplification curve is the lowest proportion capable of detecting drug-resistant bacteria.
Results: as shown in FIGS. 16-22, the detection methods for 7 mutation sites can successfully detect the drug-resistant strain when the DNA of the drug-resistant strain accounts for 1% of the total template, and the detection methods have no obvious amplification curve or large Ct value when the DNA of the drug-resistant strain accounts for the wild template.

Claims (10)

1. A composition for detecting candida that are echinocandin and/or triazole-based resistant, characterized in that the candida comprises candida glabrata and/or candida otophylla; the composition is any one or more of the following primer probe combinations:
(1) Combining: comprising a primer pair a for detecting candida glabrata PDR1-Y682C locus, a probe a and a blocking oligonucleotide a; the primer pair a comprises a forward primer a with a nucleotide sequence shown as SEQ ID NO.1 and a reverse primer a with a nucleotide sequence shown as SEQ ID NO.2, the nucleotide sequence of the probe a is shown as SEQ ID NO.3, and the nucleotide sequence of the blocking oligonucleotide a is shown as SEQ ID NO. 4;
(2) And (2) combining two: comprising a primer pair b for detecting candida glabrata PDR1-I380L locus, a probe b and a blocking oligonucleotide b; the primer pair b comprises a forward primer b with a nucleotide sequence shown as SEQ ID NO.5 and a reverse primer b with a nucleotide sequence shown as SEQ ID NO.6, the nucleotide sequence of the probe b is shown as SEQ ID NO.7, and the nucleotide sequence of the blocking oligonucleotide b is shown as SEQ ID NO. 8;
(3) And (3) combining three: comprising a primer pair c for detecting the PDR1-G346D locus of Candida glabrata, a probe c and a blocking oligonucleotide c; the primer pair c comprises a forward primer c with a nucleotide sequence shown as SEQ ID NO.9 and a reverse primer c with a nucleotide sequence shown as SEQ ID NO.10, the nucleotide sequence of the probe c is shown as SEQ ID NO.11, and the nucleotide sequence of the blocking oligonucleotide c is shown as SEQ ID NO. 12;
(4) Combination four: comprising a primer pair D for detecting the PDR1-G1099D locus of Candida glabrata, a probe D and a blocking oligonucleotide D; the primer pair d comprises a forward primer d with a nucleotide sequence shown as SEQ ID NO.13 and a reverse primer d with a nucleotide sequence shown as SEQ ID NO.14, the nucleotide sequence of the probe d is shown as SEQ ID NO.15, and the nucleotide sequence of the blocking oligonucleotide d is shown as SEQ ID NO. 16;
(5) And (5) combining: comprising a primer pair e for detecting candida glabrata FKS2-S663P site, a probe e and a blocking oligonucleotide e; the primer pair e comprises a forward primer e with a nucleotide sequence shown as SEQ ID NO.17 and a reverse primer e with a nucleotide sequence shown as SEQ ID NO.18, the nucleotide sequence of the probe e is shown as SEQ ID NO.19, and the nucleotide sequence of the blocking oligonucleotide e is shown as SEQ ID NO. 20;
(6) And (3) combining six: a primer pair F for detecting candida otorhinoca ERG11-Y132F locus, a probe F and a blocking oligonucleotide F; the primer pair f comprises a forward primer f with a nucleotide sequence shown as SEQ ID NO.21 and a reverse primer f with a nucleotide sequence shown as SEQ ID NO.22, the nucleotide sequence of the probe f is shown as SEQ ID NO.23, and the nucleotide sequence of the blocking oligonucleotide f is shown as SEQ ID NO. 24;
(7) Combination seven: comprising a primer pair g for detecting candida otophylla FKS1-S639F site, a probe g and a blocking oligonucleotide g; the primer pair g comprises a forward primer g with a nucleotide sequence shown as SEQ ID NO.25 and a reverse primer g with a nucleotide sequence shown as SEQ ID NO.26, the nucleotide sequence of the probe g is shown as SEQ ID NO.27, and the nucleotide sequence of the blocking oligonucleotide g is shown as SEQ ID NO. 28.
2. The composition of claim 1, wherein probes a, b, c, d, e, f and g are 5 'labeled FAM and 3' labeled MBG.
3. The composition of claim 1, wherein 3' ends of blocking oligonucleotide a, blocking oligonucleotide b, blocking oligonucleotide c, blocking oligonucleotide d, blocking oligonucleotide e, blocking oligonucleotide f, and blocking oligonucleotide g are linked to PO4.
4. A method for qualitatively identifying candida in echinocandin and/or triazole class resistance using the composition of any one of claims 1-3 comprising the steps of:
s1: extracting fungus DNA in a sample;
s2: performing real-time fluorescent quantitative PCR (polymerase chain reaction) by using the composition according to any one of claims 1-3 with the fungus DNA obtained in the step S1 as a template to obtain an amplification curve, and judging whether a sample to be detected contains drug-resistant candida or not.
5. The method of claim 4, wherein in S2, the amplification reaction procedure is: the first stage: 2min at 50 ℃ and 10min at 95 ℃ for 1 cycle; and a second stage: 15s at 95℃and 1min at 60℃for 40 cycles; the reaction system is as follows: 1 μl of DNA template, taqPath ProAmp Master Mix μl, 1 μl of forward primer, 1 μl of reverse primer, 0.5 μl of probe, 4 μl of blocking oligonucleotide, and no nuclease water up to 20 μl.
6. The method of claim 4, wherein if the real-time fluorescent quantitative PCR yields an amplification curve and a Ct value of 35 or less, determining that the drug-resistant candida is positive; if no amplification curve or Ct value is more than 35, the drug-resistant candida is judged to be negative.
7. A method for quantitatively determining the bacterial load of echinocandin and/or triazole-resistant candida based on the method of any one of claims 4-6, characterized in that a sample to be tested is detected by the method of any one of claims 4-6 to obtain a Ct value; substituting the Ct value into a linear equation of a standard curve corresponding to the target gene, and calculating the bacterial load of the drug-resistant bacteria.
8. The method according to claim 7, wherein the ratio is 10-10 6 The drug resistant candida species include candida glabrata and/or candida otophylla within a linear range of individual spores/responses; the linear equation of the candida otorhinoceros ERG11-Y132F is Y= -3.3569X+37.962; the linear equation of candida glabrata PDR1-Y682C is Y= -3.6112X+36.902; the linear equation of candida glabrata PDR1-G1099D is Y= -3.1197X+35.782; the linear equation of candida glabrata PDR1-G346D is y= -2.6826x+34.402; the linear equation of candida glabrata PDR1-I380L is Y= -3.6439X+42.522; the linear equation of candida glabrata FKS2-S663P is Y= -3.1340X+40.116; the linear equation of candida otophylla FKS1-S639F is Y= -3.9855X+41.829; wherein Y is CT value, X is lg value of spore amount corresponding to each reaction hole.
9. Use of a composition according to claim 1 for the preparation of a reagent or kit for detecting candida resistant in a mixed infection, wherein the mixed infection is a mixed infection of candida resistant and candida susceptible.
10. A kit for the detection of echinocandins and/or triazole-resistant candida, characterized in that it contains a composition according to any one of claims 1 to 3.
CN202311135618.4A 2023-09-05 2023-09-05 Composition, method and application for detecting echinocandin and triazole drug-resistant candida Pending CN117144038A (en)

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