KR100406064B1 - Multiplex primers for Candida albicans and Candida dubliniensis - Google Patents

Abstract

The present invention relates to a primer pair for diagnosing infection of Candida albicans and Candida dubliniensis and a method of using the same.

Description

Candida albicans and Candida dubliniensis probe {Multiplex primers for Candida albicans and Candida dubliniensis}

The present invention relates to a probe for detecting Candida albicans and Candida dubuliniensis, a detection method thereof, and a detection kit thereby.

Candida albicans and Candida dubliniensis cause cardiac fungal infections and superficial fungi but can cause systemic fungal infections in patients with impaired cellular immune function through the use of anticancer agents and immunosuppressants. Candida albicans and Candida dubuliniensis cause complications from opportunistic infections such as AIDS patients, or pulmonary candidiasis, and mortality from secondary infections caused by Candida albicans and candida dubuliniensis is also increasing.

When infections caused by Candida albicans and Candida dubuliniensis occur on the mucous membranes or skin, it is easy to detect the bacteria, but it is difficult to diagnose when it occurs in the heart. Especially in sepsis with high mortality, only 50% of bacteria are detected from blood. Compared with the polymerase chain reaction (PCR) using the 18S rRNA gene, the beta-glucan test and the antigen detection method by Cand-Tek, 75% for the polymerase chain reaction method, It is reported that the detection rate is 54% for the beta-glucan test method and 21% for Cand-Tek. The detection rate of about 50% is also shown by the metabolite D-arabitol detection method. Polymerase chain reaction has been reported as the most sensitive method and diagnostic kits have been developed. However, it has not been used as an established method in clinical laboratories. In addition, the recent separation of Candida albicans and Candida dubuliniensis using CHROM agar medium can be used only as a primary preliminary experimental step for Candida species and to provide accurate diagnosis. It has been reported that there are disadvantages such as germination tube test, thick film spore formation observation, and definitive test such as sugar fermentation test. Furthermore, the two strains, Candida albicans and Candida dubuliniensis, show not only biochemical similarity but also 95% similarity in terms of 16S rRNA gene composition. Therefore, a sensitive and rapid and accurate method for distinguishing and identifying the two strains can be obtained. need. In order to solve these problems, there is a need for the development of a method capable of quickly and accurately detecting Candida albicans and Candida dubulinensis without culturing from blood, biopsy or urine. It is thought that the development of diagnostic methods at the genetic level, which is not influenced by environmental changes, rather than the classic method, which mainly depends on the difference of metabolites, can reduce various problems.

SUMMARY OF THE INVENTION An object of the present invention is to provide a probe capable of specifically detecting Candida albicans and Candida dubuliniensis, and furthermore, when the infection occurs not only in the mucous membrane or skin, but also in the heart. The present invention provides a method capable of accurately diagnosing a candidiasis caused by Candida albicans or Candida dubuliniensis, and if the infection is caused by any of the two bacteria.

Figure 1 is a result of confirming the strain specificity by separating the electrophoresis after polymerase reaction with the primer pair of SEQ ID NO: 1 and: 2 prepared in Example 1-a for each of the various strains. (M, DNA size standard; 1, negative control; 2, Candida albicans ; 3, C. dubliniensis ; 4, C. krusei ; 5, C. famata ; 6, C. kefyr ; 7, C. glabrata ; 8, C. parapsilosis ; 9, C. intermedia ; 10, C. tropicalis ; 11, C. guilliermondii ; 12, Saccharomyces cerevisiae ; 13, Cryptococcus neoforman ; 14, Malassezia pachydermatis ; 15, Rhodotorula species ; 16, Hansenula species ; 17, Trichosporon beigelii ; 18, Geotrichum candidum ; 19, human cell (MC-IXC) ATCC CRL-2270)

Figure 2 is a result of confirming the sensitivity by separating the electrophoresis after polymerase reaction with the primer pair prepared in Example 1-a by varying the concentration of Candida albicans cells in the sample. (M, DNA size standard; 1, negative control; 2, 0 cells / ml; 3, 10 cells / ml; 4, 10²Cells / ml; And 5, 10³Cells / ml)

Figure 3 is a result of confirming the strain specificity by separating the electrophoresis after polymerase reaction with the primer pair of SEQ ID NO: 3 and: 4 prepared in Example 1-b for various strains, respectively. (M, DNA size standard; 1, negative control; 2, Candida albicans ; 3, C. dubliniensis ; 4, C. krusei ; 5, C. Famata ; 6, C. kefyr ; 7, C. glabrata ; 8, C. parapsilosis ; 9, C. intermedia ; 10, C. tropicalis ; 11, C. guilliermondii ; 12, Saccharomyces cerevisiae ; 13, Cryptococcus neoforman ; 14, Malassezia pachydermatis ; 15, Rhodotorula species ; 16, Hansenula species ; 17, Trichosporon beigelii ; 18, Geotrichum candidum ; 19, human cell (MC-IXC) ATCC CRL-2270)

4 is a result of confirming the sensitivity by polymerase reaction with the primer pairs of SEQ ID NO: 3 and: 4 prepared in Example 1-b by varying the concentration of Candida Dubuliniensis cells in the sample and electrophoresis to be. (M, DNA size standard; 1, negative control; 2, 10 cells / ml; 3, 10 ² cells / ml; and 4, 10 ³ cells / ml; 5, 10 ⁴ cells / ml; 6, 0 cells / Ml)

FIG. 5 shows SEQID NOS: 1 and 2, SEQ ID NOs: 3 and 4: 4, respectively, prepared in Examples 1-a, b, and c for Candida albicans, Candida dubuliniensis, and Candida intermedial strains. The result of confirming the strain specificity by the electrophoresis after the polymerase reaction with the primer pair of the number: 5 and: 6. (M, DNA size standard; 1, negative control; 2, Candida albicans ; 3, C. dubliniensis ; 4, C. intermedia ; 5, 1, negative control; 6, Candida albicans ; 7, C. dubliniensis ; 8, C. intermedia ; 9, negative control group; 10, Candida albicans ; 11, C. dubliniensis ; 12, C. intermedia ; 13, negative control group; 14, Candida albicans ; 15, C. dubliniensis ; 16, C. intermedia )

The present invention relates to a primer pair for diagnosing infection of Candida albicans and Candida dubliniensis and a method of using the same. More specifically, it specifically amplifies an integrin-like protein (INT-1) expressing gene of Candida albicans, and oligonucleotide having a nucleotide sequence of SEQ ID NO: 1 and a base sequence of SEQ ID NO: 2 A pair of primers consisting of oligonucleotides; Oligonucleotides having a nucleotide sequence of SEQ ID NO: 3 and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 4 specifically amplify the agglutinin-like protein (ALSD1) expressing gene of Candida dubuliniensis Primer pairs consisting of nucleotides; An oligonucleotide having a nucleotide sequence of SEQ ID NO: 5, which specifically amplifies a type II DNA topoisomerase gene that is common to both Candida albicans and Candida dubuliniensis A primer pair consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: 6; Candida albicans detection method comprising the step of performing a polymerase chain reaction with the primer pairs according to SEQ ID NO: 1 and: 2, and kitda albicans detection kit comprising the same; Candida dubulinensis detection method comprising the step of performing a polymerase chain reaction with the primer pairs according to SEQ ID NO: 3 and: 4 and kit for detecting candida dubulinensis comprising the same; And a polymerase chain reaction further having a primer pair according to SEQ ID NO: 1 and: 2 and a primer pair according to SEQ ID NO: 3 and: 4, and optionally further having a primer pair according to SEQ ID NO: 5 and: 6 It relates to a simultaneous detection method of the Candida albicans and Candida dubuliniensis comprising the step of performing, and a kit for simultaneous detection of Candida albicans and Candida dubuliniensis comprising the same.

Primer pairs for Candida albicans, the first primer pair according to the present invention, are characterized by encoding and arranging a similar integrin protein (INT-1) gene of Candida albicans as a template. It was originally designed by the present inventors to be able to specifically bind to each noncoding strand. An oligonucleotide having a nucleotide sequence of SEQ ID NO: 1 is a sense primer and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 2 is an anti-sense primer.

The primer pair for Candida dubulinensis, the second primer pair according to the present invention, is used as a template for the coding and non-coding strands by using the pseudo-agglutinin protein (ALSD1) gene of Candida dubulinensis, in which the nucleotide sequence of the gene is known. It was originally designed by the present inventors to specifically bind to each other. An oligonucleotide having a nucleotide sequence of SEQ ID NO: 3 is a sense primer and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 4 is an antisense primer.

Primer pairs for two common pairs of Candida albicans and Candida dubuliniensis according to the present invention are type II DNA which is commonly present in both Candida albicans and Candida dubuliniensis which have known nucleotide sequences. It was originally designed by the present inventors to specifically bind to coding and non-coding strands by using the topoisomerase gene as a template. An oligonucleotide having a nucleotide sequence of SEQ ID NO: 5 is a sense primer and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 6 is an antisense primer.

Primer pairs of the present invention can be chemically synthesized using common gene synthesis methods.

When the first primer pair (SEQ ID NO: 1 and: 2) of the present invention is mixed with Candida albicans and subjected to polymerase chain reaction (PCR), a similar integrin protein (INT-), which is a specific gene unique to Candida albicans, is carried out. 1) The oligonucleotides specifically bind to the expression genes to act as primers of the polymerase. That is, a part of the gene (about 239 base pairs) is amplified, and the amplified gene (polymerase chain reaction material) is confirmed through electrophoresis and the like, and thus, the presence of Candida albicans can be confirmed.

When the second primer pair (SEQ ID NO: 3 and: 4) of the present invention is mixed with Candida dubuliniensis and subjected to a polymerase chain reaction, a pseudo agglutinin protein which is a specific gene possessed only by Candida dubuliniensis The oligonucleotide is specifically bound to the (ALSD1) expression gene to act as a primer of the polymerase. That is, a portion of the gene (about 175 base pairs) is amplified, and the amplified gene (polymerase chain reaction material) is confirmed through electrophoresis and the like, and thus can be confirmed the presence of Candida dubuliniensis.

When the third primer pair (SEQ ID NO: 5 and: 6) of the present invention is mixed with Candida albicans or Candida dubuliniensis, or Candida albicans and Candida dubuliniensis, the polymerase chain reaction is performed. The oligonucleotides specifically bind to the type II DNA topoisomerase expression gene, which is a specific gene commonly present in both Su and Candida dubuliniensis, to act as a primer of polymerase. That is, a portion of the gene (about 516 base pairs) is amplified, and the amplified gene (polymerase chain reaction material) is confirmed through electrophoresis and the like, thus confirming that the test strain belongs to the genus Candida. This primer pair confirmed the presence of a common nucleotide sequence specific to several kinds of Candida species, so it is possible to identify the genus belonging to the genus Candida before distinguishing between albicans and dubuliniensis. That is, by using the primer pairs together, it is possible to confirm whether the experiment was performed normally.

Another aspect of the present invention relates to a kit for detecting Candida albicans, a kit for detecting Candida dubulinensis, or a kit for detecting Candida albicans and Candida dubulinensis, optionally comprising primer pairs according to the present invention. will be. Ie, a Candida albicans detection kit comprising the first primer pair (SEQ ID NOs: 1 and: 2) according to the present invention; A kit for detecting candida dubliniensis comprising a second primer pair (SEQ ID NO: 3 and: 4) according to the present invention; And a first primer pair (SEQ ID NO: 1 and: 2) and a second primer pair (SEQ ID NO: 3 and: 4) according to the present invention, optionally adding a third primer pair (SEQ ID NO: 5 and: 6). It relates to a kit for simultaneous detection of Candida albicans and Candida dubuliniensis comprising. The construction is in accordance with the construction of a conventional detection kit and is characterized in that it comprises a primer pair according to the invention. For example, the first primer pair of the present invention is a similar integrin protein (INT-1) expressing gene of Candida albicans, and the similar agglutinin protein, which is a specific gene possessed only by Candida dubuliniensis, of the second primer pair of the present invention. (ALSD1) The expression gene and the type II DNA topoisomerase expression gene (top2) and polymerase chain reaction (PCR), in which the third primer pair of the present invention is common to Candida albicans and Candida dubulinensis, are performed. The kit may be prepared so that color detection can be easily read for infection by Candida albicans and Candida dubuliniensis. More specifically, for example, the primer pair according to the present invention is labeled with Biotin and is commonly used in liquid (25 pmol / μl) or lyophilized powder, Candida albicans, Candida dubuliniensis, and both bacteria. A container filled with an applied detection probe; And kits can be prepared consisting of a container filled with a control template DNA in liquid form (1 ng / μl) or lyophilized powder comprising a complementary array of the primer pair.

Hereinafter, a diagnosis method of candidiasis due to Candida albicans and Candida dubuliniensis will be described in more detail.

Example 1-a. Preparation of Primer Pairs

A primer pair was prepared by chemically synthesizing an oligonucleotide having a nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 2 using a DNA synthesizer.

Synthesized oligonucleotide primers were dissolved in distilled water and quantified using UV spectrophotometer, and the synthesized oligonucleotide primers were named “CAL primers”.

Example 1-b. Preparation of Primer Pairs

A primer pair was prepared by chemically synthesizing an oligonucleotide having a nucleotide sequence of SEQ ID NO: 3 and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 4 using a DNA synthesizer.

Synthesized oligonucleotide primers were dissolved in distilled water and quantified using UV spectrophotometer, and the synthesized oligonucleotide primers were named “CDL primers”.

Example 1-c. Preparation of Primer Pairs

A primer pair was prepared by chemically synthesizing an oligonucleotide having a nucleotide sequence of SEQ ID NO: 5 and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 6 using a DNA synthesizer.

Synthesized oligonucleotide primers were dissolved in distilled water and quantified using UV spectrophotometer, and the synthesized oligonucleotide primers were named “CCL primers”. In addition, the combination consisting of CAL primers, CDL primers and CCL primers prepared in Examples 1-a, b and c, respectively, was named “CAN multiplex primer”.

Example 2 Preparation of Specimens for Polymerase Chain Reaction Test

Candida species, yeast and human cells were incubated in Sabouraud Dextrose agar medium, respectively, suspended in 1 ml distilled water and centrifuged at 12,000 g for 2 minutes to obtain bacteria, which were obtained by 1M sorbitol ( Sorbitol), 0.1 M EDTA (pH 7.5), 2% mercaptoethanol solution was suspended and then lyticase (Lyticase) was mixed and incubated for 15 minutes at 37 ℃. After centrifugation at 12,000 g for 2 minutes, washed twice with 1M sorbitol, 0.1 M EDTA (pH 7.5) solution, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2, 0.1% Nonidet P-40 solution After suspension, proteinase (Proteinase) K was added thereto, and then cultured at 55 ° C. for 30 minutes. After heating at 95 ° C. for 10 minutes, centrifugation at 12,000 g for 5 minutes, the supernatant was used as a sample, namely, target DNA, used in the polymerase chain reaction method.

Example 3-a. Polymerase Chain Reaction

To a separate tube containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 mixed solution, 5 μl of the sample prepared in Example 2 was added and 100 pmol of CAL prepared in Example 1-a. After the primer was added, the reaction was performed by adding 20 nmol of dNTP and 1 unit of Taq DNA polymerase. For the amplification of the gene, the reaction cycle controller was used once for 3 minutes at 92 ° C, for 1 minute at 92 ° C, for 1 minute at 65 ° C, for 30 minutes for 1 minute at 72 ° C, and for 5 minutes at 72 ° C. The reaction was performed twice. Fragments of genes obtained from polymerase chain reaction were identified after electrophoresis using 2% agarose.

Example 3-b. Polymerase Chain Reaction

Polymerase chain reaction was carried out in the same manner as in Example 3-a, except that the CDL primer was used instead of the CAL primer.

Example 3-c. Polymerase Chain Reaction

Polymerase chain reaction was carried out in the same manner as in Example 3-a, except that CAL primer, CDL primer and CCL primer were simultaneously used instead of CAL primer, that is, CAN multiplex primer.

Example 4-a. Preparation of Candida Albicans Detection Kit

One vessel was filled with the primer pair prepared in Example 1-a at a concentration of 25 pmol / μl, and the other vessel contained the control template DNA containing an arrangement complementary to the primer pair at a concentration of 1 ng / μl. Each was lyophilized to prepare a kit for Candida albicans detection.

Example 4-b. Preparation of Candida Dubliniensis Detection Kit

One container was filled with the primer pair prepared in Example 1-b at a concentration of 25 pmol / μl, and the other container contained the control template DNA containing the arrangement complementary to the primer pair at a concentration of 1 ng / μl. Each of them was lyophilized to prepare a kit for detection of Candida dubulinensis.

Example 4-c. Preparation of Candida Albicans and Candida Dubuliniensis Detection Kits

One vessel was filled with the primer pairs prepared in Examples 1-a and b at a concentration of 25 pmol / μl, and the other vessel contained 1 ng / μl of the control template DNA comprising an arrangement complementary to the primer pairs. Each lyophilized to a concentration of to prepare a kit for the detection of Candida albicans and Candida dubulinensis.

Example 4-d. Preparation of Candida Albicans and Candida Dubuliniensis Detection Kits

One container was filled with the primer pairs prepared in Examples 1-a, b and c at a concentration of 25 pmol / μl, and the other container was 1 ng of control template DNA containing an array complementary to the primer pairs. Each lyophilized to a concentration of / μl to prepare kits for detection of Candida albicans and Candida dubulinensis.

Example 5-a. Preparation of Candida Albicans Detection Kit

The primer pair prepared in Example 1-a was labeled with biotin and filled in one container at a concentration of 25 pmol / μl, and the other container contained 1 ng / of control template DNA containing an arrangement complementary to the primer pair. Each was lyophilized to a concentration of μl to prepare a Candida albicans detection kit.

Example 5-b. Preparation of Candida Dubliniensis Detection Kit

The primer pair prepared in Example 1-b was labeled with biotin and filled in one container at a concentration of 25 pmol / μl, and the other container contained 1 ng / of control template DNA comprising an arrangement complementary to the primer pair. Each was lyophilized to a concentration of μl to prepare a kit for detection of Candida dubulinensis.

Example 5-c. Preparation of Candida Albicans and Candida Dubuliniensis Detection Kits

The primer pairs prepared in Examples 1-a and b were labeled with biotin to fill one container with a concentration of 25 pmol / μl, and the other container with control template DNA comprising an arrangement complementary to the primer pairs. Each kit was lyophilized to a concentration of 1 ng / μl to prepare a kit for detecting Candida albitans and Candida dubuliniensis.

Example 5-d. Preparation of Candida Albicans and Candida Dubuliniensis Detection Kits

The primer pairs prepared in Examples 1-a, b and c were labeled with biotin and filled in one container at a concentration of 25 pmol / μl, and in the other container a control template comprising an arrangement complementary to the primer pairs. Candida albicans and Candida dubulinensis detection kits were prepared by lyophilizing each of the DNA at a concentration of 1 ng / μl.

Experimental Example 1. Specificity test for Candida albicans

Ten similar strains (2, Candida albicans ; 3, C. dubliniensis ; 4, C. krusei) in the genus Candida to confirm the specificity of Candida albicans of the primer pair of the present invention prepared in Example 1-a ; 5, C. Famata ; 6, C. kefyr ; 7, C. glabrata ; 8, C. parapsilosis ; 9, C. intermedia ; 10, C. tropicalis ; and 11, C. guilliermondii ), seven other yeasts 12 , Saccharomyces cerevisiae ; 13, Cryptococcus neoforman ; 14, Malassezia pachydermatis ; 15, Rhodotorula species ; 16, Hansenula species ; 17, Trichosporon beigelii ; And 18, Geotrichum candidum and polymerase chain reaction was carried out in the same manner as in Example 3-a for human cells (19, human cell (MC-IXC) ATCC CRL-2270).

Figure 1 shows the electrophoresis picture showing the result. M is electrophoresis on DNA size standard and 1 is electrophoresis on negative control without DNA polymerase. As expected, the primer pairs of the present invention showed positive reaction only against Candida albicans and did not show positive reaction against all other bacteria, thus confirming specificity for Candida albicans. In particular, Candida albicans and Candida dubliniensis are classified as having very high taxonomic similarities. The results of this example suggest that the primer pairs of the present invention show only high specificity because only the Candida albicans is positive. Doing.

Experimental Example 2. Susceptibility Test on Candida albicans

Example 3 after preparing a sample as in Example 2 after adjusting the Candida albicans cells to sterile saline to 0, 10, 10 ² and 10 ³ cells / ml in order to determine the sensitivity to the Candida albicans of the probe of the present invention The reaction was carried out in the same manner as -a.

The results after electrophoresis are shown in FIG. 2. The detection sensitivity of the primer probe of the present invention was found to be excellent at 10 cells / ml.

Experimental Example 3. Specificity Test for Candida Dubuliniensis

Example 3-b against 10 similar strains and human cells in the same Candida species as in Experiment 1 to confirm the specificity of Candida dubuliniensis of the primer pair of the present invention prepared in Example 1-b Polymerase chain reaction was carried out in the same manner.

3 shows an electrophoretic picture showing the result. M is electrophoresis on DNA size standard and 1 is electrophoresis on negative control without DNA polymerase. As expected, the probe of the present invention showed positive reaction only against Candida dubuliniensis and did not show positive reaction against all other bacteria, thus confirming the specificity with respect to Candida dubuliniensis.

Experimental Example 4. Susceptibility Test for Candida Dubuliniensis

In order to determine the sensitivity of the probe of the present invention to Candida dubulinensis, after adjusting the Candida dubulinensis cells in sterile saline to 0, 10, 10 ² , 10 ³ and 10 ⁴ cells / ml as in Example 2 After the specimen was prepared, the reaction was carried out in the same manner as in Example 3-b.

The results after electrophoresis are shown in FIG. 4. The detection sensitivity of the primer probe of the present invention was found to be excellent at 10 cells / ml.

Experimental Example 5 Differentiation of Candida albicans, Candida Dubuliniensis and Candida Intermediate Strains Using CAN Multiplex Primer

Candida albicans , Candida dubuliniensis, C. dubliniensis , in order to confirm the specificity of the CAN multiplex primer consisting of CAL, CDL and CCL primers of the present invention for Candida albicans and Candida dubliniensis. ), Candida intermedia ( C. intermedia ) was carried out in the same manner as in Example 3-c polymerase chain reaction.

The results after electrophoresis are shown in FIG. 5. M is electrophoresis on DNA size standard and 1, 5, 9 are electrophoresis results on negative control without DNA polymerase. 2, 6, 10, 14 are electrophoresis for Candida albicans, 3, 7, 11, and 15 are electrophoresis for Candida dubulinensis, and 4, 8, 12, 16 are electrophoresis for Candida intermediary. Will be shown. 1 to 4 is the case using only the primer pair of SEQ ID NO: 1 and: 2, 5 to 8 is the case using only the primer pair of SEQ ID NO: 3 and: 4, 9 to 12 is the case of SEQ ID NO: 5 and: 6 Only the primer pairs are used, and 13 to 16 are the results when the primer pairs of SEQ ID NO: 1 and: 2, the primer pairs of SEQ ID NO: 3 and: 4, and the primer pairs of SEQ ID NO: 5 and: 6 are used simultaneously. .

As expected, the probe of the present invention showed that the primer pairs of SEQ ID NO: 1 and: 2 were specific for Candida albicans, and the primer pairs of SEQ ID NO: 3 and: 4 showed positive reaction only against Candida dubuliniensis. Could confirm.

Experimental Example 6. Detection of Candida albicans by kit

Experiment using the kit prepared in Example 5-a as follows.

① Lysis of Candida albicans was performed as follows.

The bacteria were suspended in 1M sorbitol, 0.1 M EDTA (pH 7.5), and 2% mercaptoethanol solution, and then mixed with Lyticase and incubated at 37 ° C for 15 minutes. Centrifugation at 12,000 g for 2 minutes, suspended in 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ , 0.1% Nonidet P-40 solution, and then added Proteinase K to 55 ° C. Incubated for 30 minutes to prepare a lysate.

② Nylon membrane (Nylon membrane; or Nitrocellulose membrane) was equilibrated by pre-soaking in 2 X SSC.

③ The lysate solution was fixed to the equilibrated membrane.

④ Prehybridization of the stomach membrane was performed at 68 ° C. for 3 hours using pre-hybridization buffer.

(5) The membrane was hybridized overnight at 68 ° C. using a hybridization buffer containing a probe labeled with Biotin.

(6) The membrane was washed twice with membrane cleaning solution 1 and twice with washing solution 2.

⑦ Blocking was performed at 68 ° C. for 1 hour using a blocking solution.

(8) The membrane was washed three times with phosphate buffered saline (pH 7.5) containing 0.1% Tween 20.

⑨ The membrane was conjugated for 30 minutes using 0.1 M Tris-HCl (pH 7.5) and 0.15 M sodium chloride solution containing streptoavidin-alkaline phosphatase conjugate (1 μg / ml). Was carried out.

⑩ The membrane was washed three times with phosphate buffered saline (pH 7.5) containing 0.1% Tween 20.

반응 a reaction solution containing an alkaline phosphatase substrate [nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphatate (BCIP)] in 0.1 M Tris-HCl (pH 7.5) and 0.15 M NaCl; Incubate the membrane together and react.

칸 The reaction stop was added to stop the reaction, and then Candida albicans was detected by color development.

Solution used in the reaction

1. Prehybridization buffer

50% formamide

5 X SSC

5 X Denhardt's solution

25 mM sodium phosphate (pH 6.5)

0.5 mg / ml freshly denatured salmon sperm DNA

2. Hybridization buffer

45% formamide

5 X SSC

1 X Denhar Liquid

20 mM sodium phosphate (pH 6.5)

0.2 mg / ml freshly denatured salmon sperm DNA

5% dextran sulfate

0.2 μg / ml freshly denatured probe DNA

3. Cleaning solution 1: 2 X SSC / 0.1% (w / v) SDS

4. Cleaning solution 2: 0.2 X SSC / 0.1% (w / v) SDS

5. 20 X SSC (pH 7.0)

3 M sodium chloride

0.3 M sodium citrate

6. 50 X Denhar Liquid

1% (w / v) Ficoll

1% (w / v) polyvinylpyrrolidone

1% (w / v) bovine serum albumin (fraction V)

7. Blocking solution: 3% (w / v) bovine serum albumin (Fraction V) in 0.1 M Tris-HCl (pH 7.5) and 0.15 M NaCl

8.Stop Solution: 20 mM Tris (pH 7.5) / 0.5 mM Na ₂ EDTA (ethylenediaminetetraacetic acid)

As a result, Candida albicans could be detected.

Experimental Example 7 Detection of Candida Dubuliniensis Using Kit

Using the kit prepared in Example 5-b, and instead of using Candida albicans, the same experiment as in Experiment 6 was carried out except that the lysate of Candida dubuliniensis was used.

As a result, Candida Dubuliniensis could be detected.

Experimental Example 8. Detection of Candida albicans and Candida dubuliniensis using the kit

Using the kit prepared in Example 5-c, the same experiment as in Example 6 was carried out except that only the Candida albicans and the lysate of Candida albicans and Candida dubuliniensis were used.

As a result, it was possible to detect Candida albicans and Candida dubuliniensis at the same time.

Experimental Example 9. Detection of Candida albicans and Candida dubuliniensis using the kit

Using the kit prepared in Example 5-d, and instead of using only Candida albicans, the same experiment as in Experiment 6 was carried out except that lysates of Candida albicans and Candida dubuliniensis were used.

As a result, it was possible to detect Candida albicans and Candida dubuliniensis at the same time.

Since the primer pair of the present invention has very high detection sensitivity and specificity for Candida albicans and Candida dubuliniensis, respectively, the primer pair of the present invention and the detection method using the same are Candida albicans or Candidiasis caused by Candida dubuliniensis infection can be diagnosed very quickly and individually, and at the same time, it provides a diagnostic method that is very reliable in the results.

Claims (11)

A primer pair specifically amplifying a similar integrin protein expressing gene of Candida albicans, comprising an oligonucleotide having a nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 2. A pair of primers specifically amplifying the similar agglutinin protein expressing gene of Candida dubliniensis, comprising an oligonucleotide having a nucleotide sequence of SEQ ID NO: 3 and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 4. Specific amplification of the type II DNA topoisomerase expressing genes common to both Candida albicans and Candida dubuliniensis, and oligonucleotide having the nucleotide sequence of SEQ ID NO: 5 and the base of SEQ ID NO: 6 Primer pair consisting of an oligonucleotide having a sequence. Candida albicans detection method comprising the step of performing a polymerase chain reaction with the primer pair of claim 1. Candida albicans detection kit comprising the primer pair of claim 1. A candida dubliniensis detection method comprising performing a polymerase chain reaction with the primer pair of claim 2. A kit for detecting candida dublininisis comprising the primer pair of claim 2. A method for the simultaneous detection of Candida albicans and Candida dubulinensis comprising the step of performing a polymerase chain reaction with the primer pair of claim 1 and the primer pair of claim 2 together. A kit for simultaneous detection of Candida albicans and Candida dubulinensis comprising a primer pair according to claim 1 and a primer pair according to claim 2. 10. The method of claim 8, further comprising performing a polymerase chain reaction with the primer pair of claim 3 further comprising the step of detecting candida albicans and candida dubuliniensis. The kit for simultaneous detection of Candida albicans and Candida dubulinensis according to claim 9, further comprising a primer pair according to claim 3.