KR20230100894A - Composition and method for the detection of Coxiella burnetii IS1111 - Google Patents

Abstract

The present invention relates to a primer set capable of detecting Q fever bacteria ( Coxiella burnetii ) and a composition or method using the same. The present invention provides a sensitive, specific and rapid process for diagnosing Q fever and can improve the clinical treatment potential of patients through early and robust detection.

Description

Composition and method for detecting the IS1111 gene of Q fever {Composition and method for the detection of Coxiella burnetii IS1111}

The present invention relates to a primer set capable of detecting IS1111 of Coxiella burnetii and a composition or detection method comprising the same.

Q fever was designated as a statutory infectious disease in 2006, and cases are reported every year. In particular, the number of reported cases tends to increase significantly after 2015. Of the 679 cases of Q fever reported by 2019 after designation as a legal infectious disease, 211 cases were reached only from January to November 2019. Among many reported cases of Q fever in Europe, the Netherlands showed a rapid increase in Q fever patients, showing about 2,300 cases of Q fever in 2009, and France showed more than 1,500 cases of Q fever every year. In Japan, as a result of analysis of the Q fever seropositivity rate and bacterial isolation (1990-2008) in livestock and humans, seropositivity rates of more than 20% were shown in cattle, sheep, and goats, and a number of bacterial isolation reports were reported in raw milk and patient samples. .

Q fever has very diverse clinical symptoms, so diagnosis cannot be made based on symptoms alone. Pathogen isolation, genetic testing, and serological testing are required to confirm the diagnosis. Although it is important to treat the disease by treatment, there is a lack of basic research on acute and chronic infection of Q fever in human and veterinary fields.

The causative agent of Q fever is Coxiella burnetii, and in order to respond to the occurrence of Q fever, various genetic characteristics of this pathogen are intertwined with the function, regulation, evaluation of pathogenic factors, quantitative measurement of metabolic effects, and nutrition of order Rickettsia and Legionella. It is necessary to study the characteristics of a wide range of pathogens, such as understanding the evolutionary trend of genetics and antibiotic resistance.

In the future, it is predicted that the environmental regions (humidity and temperature) that are suitable for pathogen propagation due to climate change such as eco-friendly leisure activities, increase in wildlife habitat, and warming on the Korean Peninsula will increase, and the United States, United Kingdom, Spain, Australia, China, Brazil, and Algeria As cases of Q fever infection continue to be reported in companion animals such as cats and dogs, it is believed that similar cases of transmission of infection by companion animals will occur in Korea.

For the management of Q fever, it is necessary to expand the research field to the genetic diversity of domestic isolated Q fever bacteria, clinical adaptation mechanisms, and pathological mechanisms in host cells. Basic research is urgently needed.

Korean Patent Registration No. 2200940 discloses a real-time genetic test method for rapidly detecting 11 high-risk pathogens or toxins, including Q. A method for detecting Coxiella verneti using this is disclosed.

However, a primer set for detecting Q. fever having a specific sequence and a composition for diagnosing Q. fever have not yet been disclosed.

Korean Registered Patent No. 2200940 Korea Patent No. 2044683

The present invention was derived from the above needs, and the present inventors completed the present invention by confirming a primer set capable of detecting Q fever bacteria, a composition for diagnosing Q fever, and a method for detecting Q fever bacteria using the same.

In order to solve the above problems, the present invention is a primer of SEQ ID NO: 1 and SEQ ID NO: 2; And a probe of any one of SEQ ID NOs: 3 to 5; provides a primer set for detecting Q.

In one example of the present invention, the primer set is real-time polymerase chain reaction (real-time PCR), nested polymerase chain reaction (nested PCR), droplet digital polymerase chain reaction (droplet digital PCR) or recombinase polymerase amplification (recombinase polymerase amplification).

In another example of the present invention, the probe is labeled with a fluorophore at the 5' end and a quencher at the 3' end, and the fluorophore is fluorescein, 6-carboxyfluorescein (FAM, 6-carboxyfluorescein), hexachloro-6-carboxyfluorescein (HEX, hexachloro-6-carboxyfluorescein), tetrachloro-6-carboxyfluorescein (TET, tetrachloro-6-carboxyfluorescein), 2-chloro-7-phenyl -1,4-dichloro-6-carboxyfluorescein (VIC, 2-chloro-7-phenyl-1,4-dichloro-6-carboxyfluorescein), 2,7-dimethoxy-4,5-dichloro-6- Carboxyfluorescein (JOE, 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein), 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid (5-((2-aminoethyl)amino )naphthalene-1-sulfonic acid), coumarin and coumarin derivatives, cyanine-5 (Cy5, Cyanine-5), lucifer yellow, texas red, tetramethylrhodamine, At least one selected from the group consisting of Yakima Yellow (YG) and Cal Fluor Red 610 (CFR), wherein the quencher is tetramethylrhodamine (TAMRA), 4-( 4-(4-dimethylaminophenylazo)benzoic acid, 4-dimethylaminophenylazophenyl-4-maleimide, carboxytetramethylrhodamine And it may be at least one selected from the group consisting of BHQ dyes.

The concentration of the primer or probe may be variously selected and used by a person skilled in the art according to experimental conditions, preferably 1 to 1000 nM each, more preferably 100 to 500 nM each, but limited thereto. it is not going to be

The step of performing the real-time polymerase chain reaction is 30 to 50 cycles, 30 to 46 cycles, 30 to 44 cycles, 33 to 50 cycles, 33 to 46 cycles, 33 to 44 cycles, 36 to 50 cycles, 36 to 46 cycles , 36 to 44 cycles, 38 to 50 cycles or 38 to 46 cycles, for example, it may be performed under conditions of 38 to 44 cycles, but is not limited thereto.

The present invention provides a composition and method for detecting fever bacillus Q, including the primer set.

In one example of the present invention, the sample for detecting Q. fever may be separated from feces, blood, serum, urine, milk, lactic acid secretion or biological tissue.

In addition, the present invention provides a composition for diagnosing Q fever comprising the primer set.

In another example of the present invention, the Q fever may be at least one selected from the group consisting of chills, sweat, boredom, diarrhea, pneumonia, and endocarditis.

The present invention comprises the steps of preparing an isolated DNA sample; Amplifying by polymerase chain reaction using the primer set; And obtaining an amplification result; provides a method for detecting Q fever bacteria including.

The primer set capable of detecting Q fever and the composition or method using the same of the present invention provide a sensitive, specific, and rapid process for diagnosing Q fever, and can contribute to improving the clinical treatment potential of patients through early and powerful detection.

1 shows the results of quantitative real time (qRT)-PCR for confirming queue sequence according to an embodiment of the present invention.
Figure 2 shows the results of culturing the standard strain ACCM-D (-glucose) agar according to an embodiment of the present invention. (A) 100ul (B) 300ul
3 shows 16s rRNA and IS1111 nested-PCR results according to an embodiment of the present invention (left -16S rRNA, right -IS1111).
4 shows the result of indirect immunofluorescence antibody method (×400) according to an embodiment of the present invention. (A) 10 ^-1 (B) 10 ^-2
Figure 5 shows the result of vero cell infection (×400) using the standard strain ACCM-D (-glucose) broth culture according to one embodiment of the present invention. (A) 14 dpi, high (B) 28 dpi, high (C) 35 dpi, high (D) 14 dpi, low (E) 28 dpi, low (F) 35 dpi, low
Figure 6 shows the results of ACCM-D (-glucose) broth culture (dpi 28) according to an embodiment of the present invention. (A) Standard strain (B) KZ-Q2 (C) KZ-Q3 (D) Negative control

Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, in the following description, many specific details such as specific components are shown, which are provided to help a more general understanding of the present invention, and it is common in the art that the present invention can be practiced without these specific details. It will be self-evident to those who have the knowledge of And, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted.

<Example 1> Q fever strain

In the present invention, KZ-Q2 and KZ-Q3 were used as domestic clinical isolates along with C. burnetii ATCC VR615 as a standard strain.

<Example 2> Cultivation of Q fever bacteria using host cells (Vero cells)

① The standard strain cultured for 2 weeks in 10 ml of ACCM-D broth was washed with 10 ml of ACCM-D broth (3,000g, 4℃, 10 minutes, swing bucket).

② After removing the supernatant by centrifugation, prepare bacterial solutions with 1 ml of RPMI medium for infected cells (FBS 2% + L-glutamine 0.2 mM + Daunorubicin hydrochloride 0.4 μg), *800 μl (high concentration), *200 μl (low concentration), respectively. ) was inoculated into vero cells cultured in a T-25 flask.

* Concentration applied by quantification by the qRT-PCR method established in the present invention (high concentration = 1.24 × 10 ⁷ GE, low concentration = 2.56 × 10 ⁶ GE)

③ The RPMI medium for infected cells was replaced at intervals of one week and the presence or absence of vacule formation was checked under a microscope.

<Example 3> Cultivation of Q fever bacteria using mice (Balb/c, female)

① One day before Q. fever inoculation, cyclophosphamide (CPA) 4 mg/200 μl was intraperitoneally administered to mice.

② The sample to be inoculated was intraperitoneally administered to mice. In the case of cells, 10 ⁶ GE (copies) were inoculated according to the concentration, and in the case of a stock vial sample or buffy coat, 200 μl was inoculated.

③ At the second week of infection, spleens, livers, and plasma were obtained from infected individuals. In the case of tissue samples, put 1 ml/well of 1X ACCM-D broth and the sample in a 24-well plate, mince using the back side of a 5 ml syringe piston, and then remove the remaining 1 ml of 1X ACCM-D broth from the piston. The waters were harvested.

④ The sample was transferred to a 15 ml tube, being careful not to contain impurities (e.g., tissue outer membrane, fat component, etc.), and then centrifuged at 1,000g, 4℃, 5 minutes, and only the supernatant was harvested.

⑤ Mix 1 ml of sample to be inoculated with 9 ml of 1X ACCM-D broth, transfer to a T25 flask, and incubate in an incubator under 5% CO ₂ , 2.5% O ₂ , 37°C, humid conditions with the container upright. When changing the culture vessel, the culture scale was set to 20 ml for the T75 flask and 110 ml for the 500 ml cell culture flask.

<Example 4> Establishment of cell-free culture method for pure culture of Q.

- Establishment of culture method under 5% CO ₂ , 2.5% O ₂ conditions in ACCM-D agar and broth medium

- Cell-free culture (ACCM-D agar, semi-solid)

① 2X ACCM-D (-Glucose) Powder 1 pouch (Sunrise Science Product, USA) was added to 500 ml of distilled water (DW) and mixed well using a stirrer.

② After adjusting the pH to 4.75 using 6N NaOH, it was filtered with a 0.45um pore filter system.

③ Mix with 0.5% ultrapure agarose in DW in a 1:1 ratio, pour 20 ml into a 90 mm petridish, and solidify for 30 minutes (semi-solid).

④ 100 μl of sample to be inoculated such as bacterial solution, 1.25 ml of 2X ACCM-D, 0.75 ml of DW, and 0.5 ml of 0.5% agarose were mixed together and smeared on a semi-solid medium.

* The amount of inoculation is adjusted according to the condition of the sample

⑤ After hardening for 20 minutes to 1 hour in a refrigerated state (2~8℃), it was cultured in an incubator under 5% CO ₂ , 2.5% O ₂ , 37℃, humid conditions.

- Cell-free culture (ACCM-D broth)

① Add 1 pouch of 2X ACCM-D (-Glucose) Powder to 1 L of distilled water (DW) and mix well using a stirrer.

② After adjusting the pH to 4.75 using 6N NaOH, it was filtered with a 0.45um pore filter system.

③ 100 μl of sample to be inoculated, such as bacterial solution, and 9.9 ml of 1X ACCM-D broth were mixed and transferred to a T25 flask, and cultured in an incubator under 5% CO2, 2.5% O2, 37°C, humid conditions with the container upright.

* Since the culture speed differs depending on the amount of culture, the T75 flask is cultured at 20 ml and the 500 ml cell culture flask is cultured at 110 ml.

- Stock vial production (based on ACCM-D broth)

① After centrifuging the culture sample at 2,000×g, 4℃, 10mins, discard the supernatant.

② Float the bacterial pellet with stock reagent (10% glycerol in 270 mM sucrose solution) to homogenize it, and dispense 1 ml each into a cryovial.

③ Store the manufactured stock vial at -80℃.

<Example 5> Confirmation of Q fever culture

- Nested-PCR (16S rRNA, IS1111)

① DNA extracted from 200 μl of Q. fever culture sample using Genomic DNA Prep Kit (QIAGEN, Germany) was used.

② For 16s rRNA and IS1111, nested-PCR was performed under the following conditions. A total of 20 μl was reacted by mixing 10 μl of Quick Taq HS DyeMix (TOYOBO, Japan), 1 μl of each primer (10pmole/μl), 6 μl of DW, and 2 μl of DNA (Table 1).

Targets Primers Amplicon
size (bp) 16S rRNA 16S_6 F1
16S_6 R1 CGT AGG AAT CTA CCT TRT AGW GG
GCC TAC CCG CTT CTG GTA CAA TT 1416 16S_6 F2
16S_6 R2 TGA GAA CTA GCT GTT GGR RAG T
GCC TAC CCG CTT CTG GTA CAA TT 624 IS1111 IS1111-1F
IS1111-1R TAT GTA TCC ACC GTA GCC AGT C
CCC AAC AAC AAC CTC CTT ATT C 685 IS1111-2F
IS1111-2R GAG CGA ACC ATT GGT ATC G
CTT TAA CAG CGC TTG AAC GT 202

③ For 16s rRNA, the first and second amplification conditions were reacted at 93℃ for 3 minutes, repeated 30 times at 93℃ for 30 seconds, 56℃ for 30 seconds, and 72℃ for 1 minute, then reacted at 72℃ for 5 minutes at 4℃. kept.

④ IS1111 was reacted at 94℃ for 2 minutes as the first amplification condition, followed by 35 cycles of 94℃ for 20 seconds, 54℃ for 20 seconds, and 72℃ for 30 seconds, followed by a reaction at 72℃ for 5 minutes. The secondary amplification conditions were reacted at 94°C for 2 minutes, repeated 35 times at 94°C for 20 seconds, 52°C for 20 seconds, and 72°C for 30 seconds, then reacted at 72°C for 5 minutes and stored at 4°C.

⑤ The PCR product was purified and the nucleotide sequence was obtained through Sanger sequencing, which was analyzed using Nucleotide BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi) web.

- Indirect immunofluorescent antibody (IFA)

Experiments were conducted using the Q Fever IFA IgG kit (FOCUS, USA).

① After placing 10 μl of the culture sample on a well in a 24-well slide glass (for IFA) (three wells per sample), dry it using a slide warmer.

② The slide was placed in a coplinza containing acetone (iced) and allowed to stand at -20℃ for 20 minutes.

③ After naturally drying the remaining acetone, drop 10 μl of positive or negative control serum per well (2 of 3 wells were made positive, 1 was made negative), and left in a 37℃ humid incubator for 30 minutes. made it

④ After gently washing the solution on the slide with PBS (preventing direct contact with the sample), the slide was placed in a coplinza containing fresh PBS and allowed to stand on a stirrer at an appropriate speed for 10 minutes.

⑤ Slides were put in distilled water for a while, removed, and then dried naturally. 10 μl of IgG conjugate was dropped per well, and left in a 37°C humid incubator for 30 minutes.

⑥ The distilled water process of process ④ and process ⑤ was repeated once again.

⑦ After dropping an appropriate amount of mounting medium on the slide, it was covered with a cover slide, and residual solution or air bubbles were removed using a paper towel.

⑧ Filming and analysis were conducted through a fluorescence microscope (×200, ×400).

<Example 5> Confirmation of isolate-specific gene expression pattern using qRT-PCR

- Establish qRT-PCR targeting specific genes

① DNA extracted from 200 μl of the culture medium using the Genomic DNA Prep Kit (QIAGEN, Germany) was used.

② About 100 C. burnetii IS1111 gene sequences were collected from NCBI, and 3 sets of primer and probe sequences were constructed for simultaneous detection using the Primer3 program (Table 2).

Name 5'-Sequence-3' Primer Q-IS1111-F
Q-IS1111-R AAC CAT TGG TAT CGG ACG TT (SEQ ID NO: 1)
GTT GTT CCA CCG TTT TAC CC (SEQ ID NO: 2) Probe Q-IS1111-1
Q-IS1111-3
Q-IS1111-4 FAM-TAT CCC AAC GCA GTT GAT CA (SEQ ID NO: 3)-BHQ1
HEX-TAT CTC AAC GCA GTT GAT CA (SEQ ID NO: 4)-BHQ1
Cy5-TAT CTC AAC GCA GTG GAT CA (SEQ ID NO: 5)-BHQ2

③ The reaction mix for IS1111 qRT-PCR is as follows.

Triplex: TaqPathTM qPCR Master Mix (Thermo, USA) 10 μl, each primer (10 pmole/μl) 1 μl, probe (10 pmole/μl) 1 μl, DW 5 μl, DNA 2 μl mixed to make a total of 20 μl reacted.

Duplex (Q-IS1111-1, Q-IS1111-3): 10 μl of TaqPathTM qPCR Master Mix (Thermo, USA), 1 μl of each primer (10 pmole/μl), 1 μl of probe (10 pmole/μl), A total of 20 μl was reacted by mixing 4 μl of DW and 2 μl of DNA.

④ 7500 fast (Applied biosystems) and LC480 cycler (Roche) equipment were used, and the qRT-PCR conditions were 50℃ for 2 minutes, 95℃ for 20 seconds, 95℃ for 15 seconds, 56℃ for 1 minute, repeated 40 times, It was maintained at 10°C.

⑤ After the reaction was completed, the calculated graph and quantitative values were used for analysis.

<Test Example 1> Using qRT-PCR C. burnetii Establishment of quantitative analysis method of

In order to culture the culture medium of Q fever, mice or host cells must be pretreated and inoculated. However, qRT-PCR was established to measure the amount of C. burnetii in host cells because the appropriate inoculum was unknown when culturing Q fever in mice or host cells.

As the target gene, IS1111, a specific gene used for laboratory diagnosis of Q fever, was used. As a result, it was found that the standard strain VR615 was amplified in FAM, KZQ2 and KZQ3 in HEX, and there was a part with specific gene mutation (FIG. 1).

<Test Example 2> Establishment of Q fever culture method (standard strain)

- White colonies were confirmed on the 16th day after inoculating 100 μl and 300 μl, respectively, of the standard strain stored in cell culture on ACCM-D (-glucose) agar. In ACCM-D (-glucose) broth, the growth of bacteria was also confirmed in a scattered form (Figs. 2 and 6).

- The results of 16s rRNA, IS1111 nested-PCR, and indirect immunofluorescence antibody method were confirmed for colonies cultured in the medium as follows (Figs. 3 and 4).

- As a result of experimenting with vero cells to confirm cell adaptation of Q fever, it was confirmed that the number of vacuoles rapidly increased on the 28th day after infection in the high-dose inoculated group and on the 35th day after infection in the low-dose inoculated group (Fig. 5).

<Test Example 3> Establishment of Q fever culture method (clinical isolate)

- Unlike standard strains, clinical strains stored in cell culture were not cultured in ACCM-D (-glucose) broth. Based on the fact that the culture period was shortened when animal passage was performed in experiments using standard strains, non-cell culture was performed for strains that had passed through animal passage.

- Mice were inoculated with clinical isolates KZ-Q2 and KZ-Q3, and then spleen tissues were processed and inoculated into ACCM-D (-glucose) broth. As a result, on the 28th day after inoculation, the growth of bacteria in the form of white powder was confirmed on the bottom surface of the culture container, as in the case of the standard strain suspected to be Q fever bacteria (FIG. 6).

- 16s rRNA and IS1111 nested-PCR and qRT-PCR established in the present invention confirmed whether the cultured strain was C. burnetii , and both 16s rRNA and IS1111 genes were positive (FIG. 7).

<110> KDCA <120> Primer set for the detection of Coxiella burnetii <130> M21-0000 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> artificial sequence <220> <223> Q-IS1111-F <400> 1 aaccattggt atcggacgtt 20 <210> 2 <211> 20 <212> DNA <213> artificial sequence <220> <223> Q-IS1111-R <400> 2 gttgttccac cgttttaccc 20 <210> 3 <211> 20 <212> DNA <213> artificial sequence <220> <223> Q-IS1111-1 <400> 3 tatcccaacg cagttgatca 20 <210> 4 <211> 20 <212> DNA <213> artificial sequence <220> <223> Q-IS1111-3 <400> 4 tatctcaacg cagttgatca 20 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> Q-IS1111-4 <400> 5 tatctcaacg cagtggatca 20

Claims (12)

Primers of SEQ ID NO: 1 and SEQ ID NO: 2; And a probe of any one of SEQ ID NOs: 3 to 5; a primer set for detecting Q. The method of claim 1, wherein the primer set is real-time polymerase chain reaction (real-time PCR), nested polymerase chain reaction (nested PCR), droplet digital polymerase chain reaction (droplet digital PCR) or recombinase polymerase amplification ( Recombinase polymerase amplification), a set of primers for detecting Q. The primer set for detecting Q. fever according to claim 1, wherein the probe is labeled with a fluorophore at the 5' end and a quencher at the 3' end. The method of claim 3, wherein the fluorophore is fluorescein, 6-carboxyfluorescein (FAM, 6-carboxyfluorescein), hexachloro-6-carboxyfluorescein (HEX, hexachloro-6-carboxyfluorescein), Tetrachloro-6-carboxyfluorescein (TET, tetrachloro-6-carboxyfluorescein), 2-chloro-7-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC, 2-chloro-7-phenyl- 1,4-dichloro-6-carboxyfluorescein), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE, 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein), 5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid), coumarin and coumarin derivatives, cyanine-5 (Cy5, Cyanine -5), lucifer yellow, texas red, tetramethylrhodamine, Yakima Yellow (YG), and Cal Fluor Red 610 (CFR). Primer set for detecting at least one selected from the group consisting of Q. According to claim 3, wherein the quencher is tetramethylrhodamine (TAMRA), 4- (4-dimethylaminophenylazo) benzoic acid (4- (4-dimethylaminophenylazo) benzoic acid), 4-dimethylaminophenyl azophenyl -A set of primers for detecting Q. fever bacillus, which is at least one selected from the group consisting of 4-dimethylaminophenylazophenyl-4-maleimide, carboxytetramethylrhodamine, and BHQ dyes A composition for detecting Q. fever bacteria comprising the primer set of any one of claims 1 to 5 The composition for detecting Q. fever according to claim 6, wherein the sample for detecting Q. fever is separated from feces, blood, serum, urine, milk, lactic acid secretion or biological tissue. A composition for diagnosing Q fever comprising the primer set of any one of claims 1 to 5 The composition for diagnosis of Q fever according to claim 8, wherein the Q fever is at least one selected from the group consisting of chills, sweat, boredom, diarrhea, pneumonia, and endocarditis. The composition for diagnosing Q fever according to claim 8, wherein the sample for diagnosing Q fever is isolated from feces, blood, serum, urine, milk, lactic acid secretion or biological tissue. preparing an isolated DNA sample;
Amplifying by polymerase chain reaction using the primer set of any one of claims 1 to 5; and
A method for detecting Q. fever bacteria comprising the step of obtaining an amplification result; The method of claim 11, wherein the sample for detecting Q fever bacteria is isolated from feces, blood, serum, urine, milk, lactic acid secretion or living tissue.

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