WO2019187240A1 - Method for rapid identification of candida in which incomplete match probes are used - Google Patents

Abstract

[Problem] To develop a method with which it is possible to rapidly identify Candida species that cause candidiasis, which is a type of fungal infection. [Solution] Eight Candida species can be identified by carrying out real time PCR using one universal primer set and three incomplete match probes, generating three Tm values, and comparing the values with data registered in a database. Therefore, this method is useful as a method for the rapid identification of Candida species in candidiasis.

Description

Rapid identification of Candida using an incomplete match probe

The present invention relates to a rapid identification method for Candida using an imperfect-match probe (IM probe).

Candida species are the most common organisms of invasive fungal infections, causing widespread dissemination from local mucosal infections to multisystem organ failure.
Recently, due to medical advances such as major surgery, especially abdominal surgery, dialysis and immunosuppressants, invasive candidiasis is increasing and is the fourth leading cause of nosocomial bloodstream infection in the United States.
Candidaemia has an associated mortality rate of over 25%, and early appropriate antifungal therapy significantly reduces crude mortality.
However, since blood cultures take 2-5 days to be confirmed, treatment is greatly delayed.
In addition, for Candida species, rapid and accurate information is required in order to target treatment according to sensitivity.

In fungal infections, some studies report rapid identification of Candida species using melting Tm values (melting temperature values at which 50% of double-stranded DNA dissociates into single-stranded DNA). (Non-Patent Documents 1 and 2).
In these reports, the analysis of a single primer set was used to evaluate a clinical sample of candidemia.
In addition, using bacterial universal primers designed with a base sequence region common to all bacteria as targets, Tm values of the seven PCR amplification products obtained were measured, and combinations of the seven Tm values (= bases by bacterial species) There has been reported a method (Tm mapping method) for identifying a fingerprint as a fingerprint of a fungus (reflecting the difference in sequence) by collation with a database. (Patent Documents 1 and 2)

WO2007 / 097323 Publication Pamphlet WO2010 / 082640 Publication Pamphlet

Decat E et.al. Res Microbiol. 2013; 164: 110-117. Nemcova E et.al. PLoS One. 2015; 10: e0116940. Neely LA et.al. Sci Transi Med. 2013; 24: 182ra54. Mylonakis E et.al. Clin Infect Dis. 2015; 60: 892-899. Chakravorty S et. Al. J Clini Microbiol. 2010; 48: 258-267.

The Tm mapping method can identify more than 100 bacterial species by using the melting temperature (Tm) of 7 primer sets, and results are obtained within 3 hours.
However, because Candida species are eukaryotic species such as humans, it is more difficult to design fungal-specific primers than bacteria.
Moreover, it is cumbersome to prepare primers specific to each Candida species due to complexity, high cost, and the like.

Rapid identification of bacterial isolates using Sloppy Molecular Beacon Melting Temperature Signature (Sloppy Molecular Beacon; SMB), a type of DNA probe, has been reported (Non-patented) Reference 5).
This report attempts to identify bacterial isolates using 6 Tm values as signatures of bacterial isolates and using the Tm value as the distance between two points in 6-dimensional space.
The variation in Tm in the SMB melting temperature signature technique depends on the number and location of probe-target mismatches in irregular molecular beacon hybridization.

The inventors focused on molecular beacon technology, which is a kind of DNA probe, and tried to adapt a quenching probe that does not hydrolyze during PCR extension.
The quenching probe was fully evaluated in the Candida species reported in the Japanese DNA data bank, and was created corresponding to the place where there was no mutation (= same base sequence) between multiple mutants of the same Candida species. .
Moreover, since the quenching probe tends to self-quenze by forming a secondary structure, an optimal probe that does not take a secondary structure with a delta G value was selected.
Further research has been conducted, and it has been found that Candida species can be identified by a combination of Tm values obtained by three quenching probes, and the present invention has been completed.
Hereinafter, the present invention will be described in detail.

The rapid identification method of Candida using the incomplete match probe of the present invention is a rapid identification method of Candida that is performed in the following steps (FIG. 1).
(1) A process of extracting Candida DNA from a sample such as blood.
(2) A step of performing PCR using the extracted Candida DNA as a template and using a Candida universal primer set and three incomplete match probes.
(3) A step of obtaining an incomplete match probe melting temperature (Tm) value.
(4) A step of checking the Tm value with a database.
To identify Candida by:

Step (1): a step of extracting Candida DNA from a sample such as blood.
Candida DNA is directly extracted from a clinical sample (such as a 2 mL whole blood sample) as a PCR template.
The extraction method is not particularly limited, and known methods may be used for collecting clinical samples, extracting DNA, and the like.
Here, it is preferable to use in combination with a bead method that enhances the extraction efficiency of bacterial nucleic acids and the like.

Step (2): A step of performing PCR using the extracted Candida DNA as a template and using a Candida universal primer set and three incomplete match probes.
The Candida universal primer set used in this step may be one set. For example, 20 to 25 base oligonucleotides that hybridize to the conserved region of Candida 18S ribosomal RNA (18S rRNA) are a set. Primers.
The incomplete match probe is a 20-35 base oligonucleotide having three quenching functions for PCR apricon amplified from a conserved region of Candida 18S ribosomal RNA (18S rRNA).
In three probes, the number of mismatches of one probe is 1 to 12. However, in the present invention, it is not necessary that all three probes have a mismatch. For example, if one probe has three mismatches, This includes cases where there is no need to mismatch the other two probes.

The thermostable DNA polymerase used for PCR in this step is preferably, for example, a thermostable DNA polymerase (KOD DNA polymerase) derived from the hyperthermophilic bacterium Thermococcus kodakarensis KOD1 strain in order to prevent the PCR efficiency from being lowered by a PCR inhibitor.
PCR is performed using, for example, a mixture containing polymerase, dNTPs, forward primer, reverse primer and each probe, denaturing at 98 ° C. for 10 seconds, annealing at 57 ° C. for 30 seconds, extending at 72 ° C. for 20 seconds, and at 82 ° C. for 2 seconds. It is sufficient to perform a cycle such as fluorescence capture of about 50 cycles.

Step (3): A step of obtaining (analyzing) a melting temperature (Tm) value of an incomplete match probe.
For example, the Tm value analysis after PCR is performed at 40 ° C. to 95 ° C. with an increase of 1 ° C./step. Subsequently, the Tm value may be obtained by analyzing the data profile using an analysis program (for example, Rotor-Gene Q software program, etc.) attached to the used Tm value analyzer.

Step (4): A step of checking the Tm value with the database.
Candida species are identified by comparing each Tm value to a database.
Specifically, a preliminary database of eight Candida species representing the average value of three measurements of Tm values (FIG. 8) is created, and the target Candida species has the closest match to the known Candida species in the database. Identify by finding.
Here, the identification program is created in order to express each Tm value as a single point in the three-dimensional space.
The distance between that point and the point determined for each of the known Candida species is calculated to generate a series of distance indicators, or difference values (D values) (FIG. 3).

The Candida universal primer used in step (2) of the rapid identification method of Candida of the present invention may be designed so as to universally amplify the 18S ribosomal RNA gene of Candida. For example, it may be designed and chemically synthesized using a multi-alignment software program (Clustal X).
The Candida universal primer set used in this step may be one set. For example, a primer composed of 20 to 25 nucleotide oligonucleotides corresponding to the conserved region of Candida 18S ribosomal RNA (18S rRNA) is used. Can be mentioned.
Specific primers are as follows.
-Forward direction: 5'-CTTTCGATGGTAGGATAGTGG-3 ': SEQ ID NO: 1
[21 bp, nucleotides 210-230 from C. albicans 18S rRNA (accession number AF114470) gene]
-Reverse direction: 5'-GCTTTCGCAGTAGTTAGTCTTC-3 ': SEQ ID NO: 2
[22 bp, nucleotides 802-823 from C. albicans 18S rRNA (accession number AF114470) gene]

The “quenching probe” of an imperfect-match quenching probe (IMQ probe) used for the incomplete match probe method has a function of “quenching” the fluorescence emitted from the excited fluorescent dye. Means ~ 35 base oligonucleotide.
In the three types of probes, the number of mismatches of one probe is 1 to 12. However, in the present invention, it is not necessary that all three types of probes have a mismatch. For example, if there is a mismatch of three probes. This includes the case where there is no need to match the other two probes.
The IMQ probe may be designed, for example, by selecting multiple sites of Candida 18S rRNA (accession number AF114470), designing using a multi-alignment software program (Clustal X), and chemically synthesizing.

Specific IMQ probes are as follows.
<Probe 1>
-5'-CTTTCCTTCTGGGTAGCCATTT--3 ': SEQ ID NO: 3
[22bp, binds to nucleotides 585-607 of C.albicans 18S rRNA (accession number AF114470) gene (probe is incompletely matched nucleotide sequence)]
<Probe 2>
-5'-TGGAATAATAGAATAGGACGTTATGGTTC-3 ': SEQ ID NO: 4
[29bp, binds to nucleotides 679-708 of C.albicans 18S rRNA (accession number AF114470) gene (probe is incompletely matched nucleotide sequence)]
<Probe 3>
-5'-GCATCAGTAATCAGTTGTCAGAGGAGAAATTC-3 ': SEQ ID NO: 5
[32bp, binds to nucleotides 758-790 of C. albicans 18S rRNA (accession number AF114470) gene (probe is incompletely matched nucleotide sequence)]

The detection / identification sensitivity of the Candida identification method of the present invention is 1.2 CFU C. albicans / PCR Tube (FIG. 9).
A Candida blood pseudo blood sample was prepared by mixing 8 strains of Candida strains registered in the database into the blood of healthy subjects, and Candida species were detected and identified by the method of the present invention (FIG. 10).
As a result, it was possible to accurately identify all 8 Candida species.
Candida species were rapidly identified by the method of the present invention using 15 blood samples (3 species) of actual Candidaemia patients (FIG. 11).
As a result, it was possible to accurately detect and identify Candida species in about 15 hours after blood collection in all 15 specimens.

It is a schematic diagram of the identification method of Candida of this invention. It is a schematic diagram concerning the design of a universal primer and probe of Candida spp. In the method for identifying Candida of the present invention. It is a figure explaining D value. The three Tm values are defined as the position of one point in the three dimensions, and the distance between the two points with a known Candida bacterium in the database is represented as “Difference Value”. The closer this value is to zero, the more closely the detected bacteria match the Candida species in the database. It is a figure which shows the difference in Tm value of three probes in eight types of Candida species. It is a figure which shows that the combination of Tm value which is respectively different in eight Candida species is possible. It is a table | surface (Table 1) explaining a specific probe and probe arrangement | sequence. It is a table | surface (Table 2) explaining the position and number of the base sequence mismatch of each of three probes in 8 types of Candida species. It is a table | surface (Table 3) of Tm value in each probe of 8 types of Candida species in a database. It is a table | surface (Table 7) which shows the confirmation experiment of the detection and identification sensitivity of the identification method of Candida of this invention. It is a table | surface (Table 8) which shows the result of having identified Candida 8 microbial species using the Candida blood pseudo blood sample. It is a table | surface (Table 9) which shows the result of having performed the rapid identification of the Candida species by the method of this invention using 15 blood samples of a Candidaemia patient.

Next, a method for identifying Candida using the incomplete match probe of the present invention will be described in Examples.
The probe according to the present invention was expressed as an IMQ probe (imperfect-match quenching probes). The overall flow (workflow) is shown in FIG.
Candida strains were identified at the species level by obtaining Candida strains from patient blood, sputum and urine culture isolates at Toyama University Hospital and sequencing them.
Blood samples were whole blood collected from patients with Candidaemia at Toyama University Hospital and Nagasugi Hospital, and all procedures in the following examples were approved by Toyama University Ethics Committee and Nagasugi Hospital Ethics Committee and all The procedure was performed with written consent from the patient and the methods of the examples were performed according to approved guidelines.

<Extraction of Candida DNA from the culture>
Candida colonies were collected in a sterile inoculation loop and suspended in 1 mL of molecular grade distilled water (deionized and sterilized distilled water for molecular biology, NACALAI TESQUE INC Kyoto Japan; hereinafter distilled water).
The sample was then centrifuged at 20,000 × g for 10 minutes and 900 μL of supernatant was carefully removed so as not to lose the pellet. DNA was isolated from the resulting pellet using a DNA extraction kit (QIAamp UCP Pathogen Mini Kit, Qiagen, Germany) and glass beads according to the instructions in the product instructions. Finally, Candida DNA was eluted with 100 μL of elution buffer.

<Extraction of Candida DNA from whole blood>
A total of 2 mL of venous blood was collected into an EDTA-2K tube (NIPRO Osaka Japan).
The blood sample was then centrifuged at 100 × g for 5 minutes to centrifuge blood cells, and the resulting supernatant fraction (1 mL) was used.
The supernatant was again centrifuged at 20,000 × g for 10 minutes, and 900 μL of the supernatant fraction was carefully removed to avoid disturbing the pellet.
Next, 1 mL of distilled water was added to the pellet and gently turned upside down, followed by centrifugation at 20,000 × g for 5 minutes.
Finally, 1 mL of the supernatant fraction was carefully removed so as not to resuspend the pellet.
DNA was isolated from the pellet using a DNA extraction kit and glass beads according to the instructions in the product instructions.
Finally, Candida DNA was eluted with 100 μL of elution buffer.

<PCR assay>
In the following, Rotor-Gene Q (Qiagen) was used for target DNA amplification, real-time detection, and Tm value analysis of incomplete match probes.
All PCR assays were performed as 1 PCR, 1 probe assay per tube.
An Eppendorf tube dedicated to 1.5 mL PCR, which is a 0.2 mL PCR tube (Qiagen) for PCR of RNase and DNase free (Eppendorf, Germany), was used.
A fungal universal primer targeting the known 18S ribosomal RNA gene (18S rDNA) was used (PLoS One. 2015; 10: e0129032).
Quenching probes (Q probes) were designed using software (Clustal X) for creating various base sequence alignments and synthesized at NIPPON STEEL SUMIKIN Eco-Tech Corporation (Ibaraki, Japan).

<Candida sp. Universal primer>
・ Forward: 5'-CTTTCGATGGTAGGATAGTGG-3 '
[21 bp, nucleotides 210-230 from C. albicans 18S rRNA (accession number AF114470) gene]
・ Reverse direction: 5'-GCTTTCGCAGTAGTTAGTCTTC-3 '
[22 bp, nucleotides 802-823 from C. albicans 18S rRNA (accession number AF114470) gene]

<Probe 1>
5'-CTTTCCTTCTGGGTAGCCATTT--3 '
[22bp, binds to nucleotides 585-607 of C.albicans 18S rRNA (accession number AF114470) gene (probe is incompletely matched nucleotide sequence)]
<Probe 2>
5'-TGGAATAATAGAATAGGACGTTATGGTTC-3 '
[29bp, binds to nucleotides 679-708 of C.albicans 18S rRNA (accession number AF114470) gene (probe is incompletely matched nucleotide sequence)]
<Probe 3>
5'-GCATCAGTAATCAGTTGTCAGAGGAGAAATTC-3 '
[32bp, binds to nucleotides 758-790 of C. albicans 18S rRNA (accession number AF114470) gene (probe is incompletely matched nucleotide sequence)]

<PCR procedure>
KOD FX Neo (Toyobo) was used as the PCR enzyme.
The PCR reaction mixture (50 μL) consists of KOD FX Neo, 0.4 mM dNTP, and 0.02 U 1 × PCR buffer (included with KOD FX Neo: Toyobo), 0.2 μM forward primer, 0.6 μM reverse primer And 0.02 μM of each probe.
Each sample was incubated at 95 ° C. for 5 minutes, then heat denatured at 98 ° C. for 10 seconds, annealed at 57 ° C. for 30 seconds, extended at 72 ° C. for 20 seconds, and subjected to fluorescence capture at 82 ° C. for 2 seconds.
50 cycles were carried out under the above PCR conditions.

<Melting point (Tm) value analysis>
For Tm value analysis, the PCR tube containing each probe in the resulting PCR amplicon was heated at 95 ° C. for 10 seconds and then cooled at 40 ° C. for 90 seconds.
Tm value analysis after PCR was performed at 40 ° C. to 95 ° C. with an increase of 1 ° C./step.
The data profile was then analyzed using the Rotor-Gene Q software program to identify Tm values.

<Analysis sensitivity test>
One colony of C. albicans was cultured on Sabouraud dextrose agar for 48 hours, then scraped off the surface, suspended in phosphate buffered saline (PBS), and seeded again in the medium.
To ensure pure culture of only one strain of Candida albicans, one colony was scraped again from the second medium after 48 hours, suspended in PBS and diluted 10-fold.
Each dilution was divided into several aliquots.
In order to evaluate the number of colonies (equivalent CFU / ml), the cells were plated on 3 sheets of Sabouraud dextrose agar.
The detection limit (LOD) was determined by serially diluting DNA based on a known number of Candida albicans in PBS and measuring the presence or absence of PCR detection in each diluted sample.

<Analysis based on nucleotide sequence of fungal genomic DNA>
Fungal primers targeting the known D1-D2 region and the internal transcription spacer (ITS) region were purified (QIAquick PCR Purification Kit; QIAGEN) and then sequenced (3500 Genetic Analyzer; Applied Biosystems).
Bacterial species identification is performed using the BLAST nucleotide database tool of DNA Data Bank (http://www.ddbj.nig.ac.jp/index-j.html) and online nucleotide homology for bacterial species identification I did a search.

<Biochemical identification based on Candida species culture>
Whole blood samples (one aerobic blood culture bottle and one anaerobic blood culture bottle) were collected simultaneously with the blood sample for Tm analysis of the same specimen taken from the same puncture site.
The whole blood sample was analyzed according to the standard method of the Clinical Laboratory Center of Toyama University Hospital (ISO 15189 certification).
Blood culture procedures were performed using the BacT / ALERT 3D system (bioMerieux, Inc., Mercy-l'Etoile, France).
Fungal identification was performed by CHROMager Candida (Becton Dickinson and Company, USA) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Bruker, USA).

The method for identifying Candida of the present invention uses one PCR primer set and three incomplete match probes to generate three Tm values and compare them with data registered in a database. Candidiasis (8 species) with candidiasis can be rapidly identified.
The time required for identification is within 3.5 hours after blood collection.
Since the new genetic testing method of the present invention can be carried out quickly and easily and inexpensively, it can be kitted as an in-vitro diagnostic agent and is medically useful.

Claims (5)

A method for identifying Candida, comprising the following steps (1) to (4):
(1) A step of extracting Candida DNA from a sample such as blood.
(2) A step of performing PCR using the extracted Candida bacteria DNA as a template and using one set of Candida universal primers and three incomplete match probes.
(3) A step of obtaining a melting temperature (Tm) value of an incomplete match probe.
(4) A step of checking the Tm value with a database. The universal primer of the above step (2) is designed in the conserved region of 18S ribosomal RNA of Candida, and three incomplete match probes are designed for the PCR apricot amplified with the universal primer The method for identifying Candida spp. According to claim 1. The method for identifying Candida spp. According to claim 2, wherein the incomplete match probe in the step (2) has 20 to 35 bases and the number of mismatches is 15 or less. 20-35 bases designed for targeting PCR apricots amplified with universal primers designed for the conserved region of 18S ribosomal RNA of Candida, and incomplete matches with 15 or fewer mismatches probe. An incomplete match probe whose base sequence is any one of SEQ ID NO: 2 to SEQ ID NO: 4.

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