WO2005052155A1

WO2005052155A1 - Nucleic acid primers for detecting serpula lacrymans dry rot fungus, and methods and test kit in which they are used

Info

Publication number: WO2005052155A1
Application number: PCT/FI2004/000724
Authority: WO
Inventors: Jaan Palgi; Simo Nikkari
Original assignee: Mobidiag Oy
Priority date: 2003-11-28
Filing date: 2004-11-26
Publication date: 2005-06-09
Also published as: EP1687425A1; FI20031748A0; FI114552B

Abstract

The present invention relates to specific nucleic acid primers that are useful in the molecular detection of the Serpula lacrymans dry rot fungus and can be used for identifying and detecting the fungus. The invention also relates to methods for identifying and detecting the S. lacrymans dry rot fungus, in which methods said specific nucleic acid primers are used, and to test kit useful in such methods. In addition, the invention relates to the use of said primers for identifying and detecting the Serpula lacrymans dry rot fungus.

Description

NUCLEIC ACID PRIMERS FOR DETECTING SERPULA LACRYMANS DRY ROT FUNGUS, AND METHODS AND TEST KIT IN WHICH THEY ARE USED

FIELD OF THE INVENTION [0001] The present invention relates to specific nucleic acid primers useful in the molecular detection of the Serpula lacrymans dry rot fungus. The invention also relates to methods for identifying and detecting the Serpula lacrymans dry rot fungus, in which methods said specific nucleic acid primers are used, and to test kit useful in such methods. In addition, the invention relates to the use of said primers for identifying and detecting the Serpula lacry- mans dry rot fungus.

BACKGROUND OF THE INVENTION [0002] The Serpula lacrymans dry rot fungus is one of the best- known and most harmful decaying agents of man-made environments, and it causes significant financial damage. It has also been suspected that S. lacry- mans could cause clinical pulmonary symptoms in subjects exposed to the fungus. [0003] Dry rot fungus often exists in old and renovated buildings. It develops in wet and poorly ventilated spaces and destroys wood fairly quickly under favourable conditions, even in four months. Rotting wood softens and crumbles. In some cases, when the growth of the dry rot fungus has started for instance from water damage the fungus may stay viable even after the water damage has been repaired and the structures dried. Once started, the dry rot fungus is even capable of spreading to dry wood, and it may also spread through other porous materials. The destruction of the wooden parts of a build- ing or structure caused by the dry rot fungus may make the building or structure completely unusable. [0004] The detection of the S. lacrymans dry rot fungus and its distribution in the structures must be thoroughly investigated, and its prevention should be initiated immediately. The fruit body of the dry rot fungus may be easily detectable by visual inspection. In some cases, however, a typical fruit body and a distinct mycelium may not exist, and the existence of the dry rot fungus may go visually undetected. Therefore, efficient and accurate methods are needed to detect and identify the dry rot fungus. [0005] The most common and important method in fungus diagnos- tics has classically been the culture method, i.e. growing fungi on suitable nu- trient media. The culture method is generally used in laboratories, but it has several drawbacks. Firstly, the culture method is slow and, depending on the fungus species, it may take weeks to get results. Secondly, finding suitable growth conditions for each species may be difficult and, thus, one of the fungus species may remain undetected. Thirdly, rapidly growing fungus species may reproduce quickly and cover any slower growing species under them, in which case the slower growing species remain unidentified. [0006] Attempts have been made to solve the above-mentioned problems related to fungus culture with molecular methods based on amplifica- tion and hybridisation of nucleic acids. A fungus species is then detected and identified simultaneously, which speeds up diagnostics and a time-consuming culture is not required. In general, in an in vitro DNA amplification method, known DNA sequences are amplified from DNA isolated from a sample by using DNA primers designed species-specifically for the microbe in question. Moreth and Schmidt [Holzforschung 54 (2000) 1-8] have studied the possibilities to specifically detect the S. lacrymans dry rot fungus by means of the DNA amplification method and attempted to find specific primers for specifically amplifying the target DNA. The use of known primers in a PCR amplification reaction did not, however, produce a reliable result, because the method was rela- tively insensitive. [0007] Taking the above into consideration, new and efficient methods are required to specifically detect fungi species, especially the S. lacrymans dry rot fungus.

BRIEF DESCRIPTION OF THE INVENTION [0008] The purpose of the present invention is to provide means and methods that are useful in the diagnostics of the S. lacrymans dry rot fungus but do not have the drawbacks described above. [0009] More precisely, the purpose of the present invention is to provide means and methods to identify and detect the S. lacrymans dry rot fungus quickly, specifically and reliably without the time-consuming culture. [0010] In particular, the purpose of the invention is to provide new means and methods that are useful in the S. lacrymans fungus diagnostics based on molecular methods and that are sensitive, efficient, and species- specific. [0011] In addition, the purpose of the invention is to provide means and methods to verify results obtained by unspecific diagnostics analyses of S. lacrymans dry rot fungus. [0012] The present invention provides specific nucleic acid primers, especially DNA primers that are useful in the determination, identification and detection of nucleic acids, especially DNA, originating from the S. lacrymans dry rot fungus by means of different DNA or RNA amplification methods. [0013] The present invention relates to nucleic acid sequences that specifically hybridize with the ITS-rDNA-region sequences of the S. lacrymans dry rot fungus. More precisely, the present invention relates to a nucleic acid sequence that is a sequence containing 19 to 30, preferably 19 to 25, and most preferably 19 to 22, nucleotide units and hybridizes specifically with the rDNA of the Serpula lacrymans dry rot fungus, said nucleic acid sequence comprising a nucleic acid sequence selected from a group comprising a DNA sequence identified by SEQ. ID. NO. 1 , a DNA sequence identified by SEQ. ID. NO. 2, a DNA sequence identified by SEQ. ID. NO. 3, and DNA sequences complementary to said sequences, or corresponding RNA sequences. These sequences are useful in various nucleic acid amplification methods both as primers and as probes. [0014] The present invention also relates to a primer pair comprising 1 ) a first nucleic acid sequence that is a sequence containing 19 to 30, preferably 19 to 25, and most preferably 19 to 22, nucleotide units and hybridizes specifically with the rDNA of the Serpula lacrymans dry rot fungus, said nucleic acid sequence comprising a DNA sequence identified by SEQ. ID. NO. 1 that originates from the rDNA ITS1 region of the Serpula lacrymans dry rot fungus, or a DNA sequence complementary with it, or a corresponding RNA sequence, and 2) a second nucleic acid sequence that is a sequence containing 19 to 30, preferably 19 to 25, and most preferably 19 to 22, nucleotide units and hybridizes specifically with the rDNA of the Serpula lacrymans dry rot fungus, the nucleic acid sequence comprising a DNA sequence that originates from the rDNA ITS2 region of Serpula lacrymans dry rot fungus and is selected from a group comprising a DNA sequence identified by SEQ. ID. NO. 2 and a DNA sequence identified by SEQ. ID. NO. 3, or DNA sequences complementary to said sequences, or corresponding RNA sequences. The present invention also relates to a method for identifying and detecting the S. lacrymans dry rot fungus from a sample by using these specific primer pairs, the method comprising a) amplifying the nucleic acid in the sample being examined by using one of the above-mentioned primer pairs, and b) detecting the amplification. [0015] In one embodiment of the invention, the method optionally after step b) comprises a further step comprising c) verifying the presence of the dry rot fungus by amplifying the nucleic acid in the sample being examined by using another suitable primer pair selected from the ones listed above. [0016] In one preferred embodiment, the invention relates to a method for identifying and detecting the S. lacrymans dry rot fungus from a sample by using these spebific primer pairs and nucleic acid sequences, the method comprising a) amplifying the nucleic acid in the sample being examined by using a primer pair that comprises a DNA sequence identified by SEQ. ID. NO. 1 and a DNA sequence identified by SEQ. ID. NO. 2, b) detecting the amplification, and optionally d ) verifying the presence of the dry rot fungus by using a primer pair that comprises a DNA sequence identified by SEQ. ID. NO. 1 and a DNA sequence identified by SEQ. ID. NO. 3, which is possibly labelled, and/or c2) verifying the presence of the dry rot fungus is verified through a hybridization reaction by using a nucleic acid identified by sequence number 3. [0017] The present invention also relates to test kit comprising a desired combination of the above-mentioned primer pairs, and possibly a probe, and other reagents, such as buffers, positive controls, a dNTP mixture, an enzyme, and other corresponding reagents, required in the amplification or detection of amplification. [0018] By means of the nucleic acid sequences, nucleic acid primer pairs and methods of the invention, a superior - even 1 ,000 times higher - detection sensitivity, and thus also improved reliability in detecting the S. lacrymans dry rot fungus, is achieved in the amplification reactions in comparison with the ones used earlier. This makes it possible to quickly and effortlessly detect the dry rot fungus from sample amounts that are smaller than before and even when visual examination of the location does not reveal spore growth. BRIEF DESCRIPTION OF THE FIGURES [0019] Figure 1 is a schematic view of the rDNA region ITS1 - 5,8S - ITS2 of the S. lacrymans dry rot fungus. The nucleic acid primers SL1 F, SL2R, and SL3R are marked with arrows in Figure 1. [0020] Figure 2 shows an agarose gel electrophoresis analysis of fungus DNA samples amplified by a DNA polymerase chain reaction for evaluating the specificity of the nucleic acid primers. The examined samples were: Serpula lacrymans (SL), Serpula himantioides (SH), Merulius tremellosus (MT, a positive control (+) (i.e. an environmental sample containing S. lacrymans DNA), a water control (-) (i.e. a negative control), and a DNA molecular weight standard (M) of 100 base pairs. [0021] Figure 3 shows an agarose gel electrophoresis analysis of the sensitivity analysis of nucleic acid primers described in example 3, in which the primer pairs of the invention were compared with known (Moreth and Smith, supra) primers at a DNA content of 10 pf to 1 fg. In Figure 3, SL1 F/SL2R and SL1 F/SL3R are the primer pairs of the invention, X is a known primer pair, (-) depicts the water control (i.e. negative control), and M depicts the DNA molecular weight standard of 100 base pairs.

DETAILED DESCRIPTION OF THE INVENTION [0022] The present invention is based on studies that aimed to find more specific and sensitive methods for identifying and detecting the S. lacrymans dry rot fungus by means of different DNA amplification methods. The study was directed to the ITS region of the ribosomal DNA (rDNA) of the S. lacrymans dry rot fungus. The ITS regions are the internally transcripted DNA regions on both sides of the 5,8S-rDNA gene (Figure 1 ). The primers of the invention were designed so as to make the selected nucleic acid sequences as species-specific as possible and to make them attach only to the corresponding rDNA sequences of their host organisms. In the design of the primers of the invention, a planning strategy based on alignment, the BioEdit program, and the ClustalW alignment algorithm were used. The sequences used in the alignment (Example 1) were obtained from the EMBL/GenBank sequence database. The DNA sequences of S. lacrymans that are S. /ac/ymans-specific, i.e. the ones that differ most from other base sequences used in the alignment, were selected as the design objects of the S. lacrymans amplification primers. The base sequences of the selected primers were also compared with the EMBL/GenBank sequence database using the BLAST algorithm. [0023] The designed primers are shown in Table 1.

Table 1. Primers for specifically determining the DNA of S. lacrymans dry rot fungus.

* The location of the primer in the S. lacrymans sequence (GenBank AJ437601. SEQ. ID. NO. 4)

[0024] The primers of the present invention identify the ribosomal DNA (rDNA) of the S. lacrymans dry rot fungus. Of the designed primers, SLF1 (SEQ. ID. NO. 1 ) is used as the first primer. As the second primer in the primer pair of the invention, the primer comprising the SEQ. ID. NO. 2 or 3 is used. The pair-wise use of the primers (SLF1/SLF2 or SLF1/SLF3) in the amplification reaction ensures a specific amplification of the S. lacrymans DNA. It is apparent to a person skilled in the art that said sequences and the DNA sequences complementary with them and the corresponding RNA sequences could equally well work as primers and primer pairs, and they can also, according to the invention, be used to identify the S. lacrymans dry rot fungus. [0025] The pair-wise use of the primers of the invention in the amplification reaction produces as the result DNA fragments whose lengths are 443 (SL1 F/SL2R) and 399 (SL1 F/SL3R) base pairs, respectively (Table 1 , Figure 2), and the primer pair specifically identifies the S. lacrymans rDNA. In addition, the primer SL3R, for instance, can be used to control the amplification reactions in such a manner that a PCR product amplified with the SL1 F/SL2R primer pair is used as a sample in a new reaction that uses the SL1 F/SL3R primer pair. The primer SL3R can also be used as a verifying probe for the SL1 F/SL2R amplification product in a hybridization reaction. [0026] According to the invention, the specific primers and primer pairs can be used to identify and detect the S. lacrymans dry rot fungus in any method employing DNA amplification that is known to a person skilled in the art. Examples of such methods include detection method containing PCR or TMA steps, such as real-time PCR and quantitative PCR. Various PCR methods are described for instance in publication PCR Primer: A Laboratory Man- ual, ed. Dieffenbach C, Dveksler G.S. 1995, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. A preferred method is the conventional PCR method that can be implemented in any commercially available equipment, such as GenAmp PCR System 2700 (Applied Biosystems, Foster City, CA, USA) or Mastercycler (Eppendorf Scientific, Inc. New York, USA). [0027] The amplification can also be performed manually by transferring the reaction tubes from one temperature to another. [0028] The amplification reaction is detected in a manner known per se. The synthesized fragments can thus be visualized by using agarose gel electrophoresis or any other method known to a person skilled in the art that allows the detection of PCR products synthesized in an amplification reaction. Such methods include for instance real-time PCR and other methods based on a labelled probe. [0029] The primers of the invention can also be used as oligonu- cleotide probes in methods based on hybridization, for instance in Southern blot, Northern blot and colony hybridization methods. Such methods are known per se to a person skilled in the art and can be implemented both in a solution and on a solid support binding DNA, such as nitro-cellulose, nylon membrane, or glass. The primers can also be used in DNA chip technology, in which case they are attached to a chip. [0030] The nucleic acid sequences of the invention can be used as labelled or unlabelled primers depending on the method by which the amplification is detected, or when they are used as probes, depending on the method by which the hybridization is detected. Suitable labels include fluorescent labels (e.g. Cy5, Cy3, Cy2, TexasRed, FITC, Alexa 488, TMR, FluorX, ROX, TET, HEX), radioactive labels (e.g. ³²P, ³³P, ³²S), and chemiluminescent labels (e.g., HiLight Single-Color Kit Plus (Qiagen GmbH, Hilden, Germany). In the present invention the Cy5-dCTP fluorescent label (Amersham Pharmacia Biotech UK Limited, England) is especially preferred. The invention also comprises applications in which no label is needed, such as those in which the de- tection is based on an electric impulse (e.g. eSensor™ DNA Detection System, Motorola Life Science, Pasadena, CA, USA). [0031] In the method of the present invention, the sample to be analyzed may be a fungus culture, material sample, or an air sample collected from the site to be examined, or any other sample from which nucleic acid can be isolated. Because the primer pair used in the method is very specific, the analysis only requires small amounts of samples, and the sample can also be unprocessed without any preceding nucleic acid purification and isolation steps. [0032] ln one specific embodiment of the present invention, the sample to be analyzed can also be from a subject whose pulmonary and respi- ratory passage symptoms are suspected to be caused by the S. lacrymans dry rot fungus. This sample may be a sputum sample or any other sample that may contain the rDNA of the S. lacrymans dry rot fungus. [0033] The nucleic acid in the sample - which may be any of the above samples - to be analyzed in the method of the present invention is am- plified using one of the primer pairs of the invention, for example the SL1 F/SL2R or SL1 F/SL3R primer pair, after which the amplification of the nucleic acid is detected by one of the above-mentioned methods, preferably elec- trophoretically using agarose gel. If desired, the result obtained from the amplification by means of the method of the invention can be verified by using the amplified nucleic acid as a sample in a new amplification reaction that uses the primer pair SL1 F/SL3R, when the first amplification used the primer pair SL1 F/SL2R, and/or the presence of the dry rot fungus can be verified from the SL1 F/SL2R amplification product by contacting it with the SL3R probe in a hybridization reaction. [0034] The isolated specific nucleotide sequences of the invention, especially the sequences having the SEQ. ID. NOS. 1 , 2, and 3, differ in their base sequence from the sequences described earlier, and they also bind to different DNA sequences than for instance the primers that are described in publication Moreth and Schmidt, supra. When the nucleic acid sequences of the invention identified by SEQ. ID. NOS. 1 , 2, and 3 were used as primers in a test evaluating the specificity and sensitivity of the primers in the PCR amplification of the S. lacrymans dry rot fungus, they specifically identified the S. lacrymans rDNA and did not identify the closely related fungus species, Serpula himantioides (SH), or Merulius tremellosus (MT) (Figure 2). Similarly, when examining the sensitivity of the primers of the invention in PCR amplification, they were clearly more sensitive in the amplification reaction in compari- son with known primers specific to the S. lacrymans dry rot fungus (Figure 3) and could amplify the rDNA of the dry rot fungus up to 10 fg per amplified sample, i.e. the sensitivity was approximately 1 ,000 times higher than when using known primers. [0035] Next, the invention will be described in more detail by means of examples; the invention and its embodiments are, however, not limited to the examples described below.

EXAMPLE 1

The designing of the PCR primers of the invention [0036] The primers of the invention were designed using ITS region alignment so as to make them as species-specific as possible and to make them attach only to rDNA sequences corresponding to their host organisms. [0037] The BioEdit program and ClustalW alignment algorithm were used in designing the primers. In the alignment, the ITS-rDNA-region se- quence of the Serpula lacrymans (AJ437601 ) taken from the EMBL/GenBank sequence database was compared with the ITS-rDNA-region sequence of Serpula himantioides (AJ245949), which is a close relative belonging to the same Serpula family. In addition, more distant species, such as Coniophora puteana (AJ249500) and Donkioporia expansa (AJ249500) were used in the alignment. On the basis of the alignment, DNA sequences were found, whose base sequences had variations between the close relatives S. lacrymans and S. himantioides belonging to the Serpula group. The base sequence of the corresponding ITS-rDNA sequences in the more distant species, C. puteana and D. expansa, differs completely from the corresponding DNA sequences of the fungi belonging to the Serpula group. Therefore, the ITS-DNA sequences whose base sequences differed as much as possible from the other sequences in the comparison were selected as the designing objects of the amplification primers. The S. /acrymans-specific primers (Table 1) were ordered through an order service (www.siqma-genosys.co.uk, Sigma Genosys, Eng- land). The specificity and sensitivity of these primers were tested in the manner described in Examples 2 and 3. EXAMPLE 2

The use of primers of the invention in a DNA polymerase chain reaction [0038] A sample DNA was amplified using the general method described below. [0039] The DNA was purified from the wood material sample being analyzed and the desired DNA was amplified using a polymerase chain reaction (PCR). The reaction mixture was prepared by mixing the primers SL1 F/SL2R (SEQ. ID. NOS. 1 and 2) or SL1 F/SL3R (SEQ. ID. NOS. 1 and 3) (cf. Table 1 ) with other required components. The reaction mixture (50 μ\) con- tained 20 pmol SL1 F primer mixture and 20 pmol SL2R primer mixture (or 20 pmol SL3R primer mixture instead of SL2R), 200 μU of each of the following: dATP, dGTP, dCTP, and dTTP (Sigma, USA), 1 x HotStarTaq PCR buffer (Qiagen, Germany) that contains MgCI₂ (final concentration 1.5 mM), 2.5 U HotStarTaq DNA polymerase (Qiagen, Germany), and 5 μ\ DNA. The tempera- ture program was as follows: polymerase activation 15 min 95°C, and after this, 38 cycles as follows: 35 s 94°C, 35 s 54°C, 35 s 72°C, and a final extension 7 min 72°C. The success of the amplification was checked with gel electrophoresis in a 2-percent agarose gel that contained ethidiumbromide. The results are presented in Figure 2. [0040] Figure 2 shows that the primers of the invention (SEQ. ID.

NOS. 1 to 3) as primer pairs work and are specific in comparison with primers published in the literature. The results show that the primers in question specifically identify the rDNA of S. lacrymans.

EXAMPLE 3

The analysis of the dry rot fungus samples [0041] The specificity and sensitiveness of the primers of the invention in the amplification of the S. lacrymans dry rot fungus were evaluated using a 2-percent agarose gel electrophoresis. The specificity was examined by comparing PCR products obtained from DNA clean cultured samples of Ser- pula lacrymans (SL), Serpula himantioides (SH), Merulius tremellosus (MT) by using the method described in Example 2. The primers worked in pairs (SL1 F/SL2R and SL1 F/SL3R) in the amplification reaction and produced DNA fragments having a length of 443 and 399 base pairs respectively (Table 1 , Figure 2), and specifically identified the rDNA of S. lacrymans. As can be seen from Figure 2, the primer pairs did not amplify the DNA of S. himantioides that is closely related to S. lacrymans. [0042] The sensitivity of the primers of the invention in PCR amplification was examined by an agarose gel electrophoresis that compared PCR products amplified by the method described in Example 2. A dilution set of 10 pg - 1 fg DNA per reaction in sterile water was made of the DNA samples. The PCR products obtained with the primer pairs of the invention were compared with primers described in the literature. In the amplification reactions, the primer mixtures of the invention were clearly more sensitive than the S. lacry- maπs-specific primers published in the literature (Moreth and Schmidt, supra) (Figure 3), and they were capable of amplifying the rDNA of the dry rot fungus up to 10 fg per PCR reaction, i.e. the sensitivity was approximately 1 ,000 times higher than that of the published primers (primers X).

Claims

CLAIMS 1. An isolated nucleic acid sequence, characterized in that it is a sequence containing 19 to 30 nucleotide units and hybridizes specifically with the rDNA of the Serpula lacrymans dry rot fungus, said nucleic acid se- quence comprising a DNA sequence selected from a group comprising a DNA sequence identified by SEQ. ID. NO.1, a DNA sequence identified by SEQ. ID. NO.2, a DNA sequence identified by SEQ. ID. NO.3, and DNA sequences complementary to said sequences or corresponding RNA sequences. 2. A nucleic acid sequence as claimed in claim 1, character- ize d in that it is labelled. 3. A primer pair, characterized in that it comprises 1) a first nucleic acid sequence that is a sequence containing 19 to 30 nucleotide units and hybridizes specifically with the rDNA of the Serpula lacrymans dry rot fungus, said nucleic acid sequence comprising a DNA se- quence identified by SEQ. ID. NO.1 that originates from the rDNA ITS1 region of the Serpula lacrymans dry rot fungus, or a DNA sequence complementary with it, or a corresponding RNA sequence, and 2) a second nucleic acid sequence that is a sequence containing 19 to 30 nucleotide units and hybridizes specifically with the rDNA of the Serpula lacrymans dry rot fungus, the nucleic acid sequence comprising a DNA sequence that originates from the rDNA ITS2 region of Serpula lacrymans dry rot fungus and is selected from a group comprising a DNA sequence identified by SEQ. ID. NO.2 and a DNA sequence identified by SEQ. ID. NO.3, or DNA sequences complementary to said sequences, or corresponding RNA se- quences. 4. A primer pair as claimed in claim 3, characterized in that the first nucleic acid sequence is a DNA sequence identified by SEQ. ID. NO. 1, or a DNA sequence complementary to it, or a corresponding RNA sequence, and the second nucleic acid sequence is a DNA sequence identified by SEQ. ID. NO. 2 or 3, or a DNA sequence complementary to said sequences, or a corresponding RNA sequence. 5. A method for identifying and detecting the S. lacrymans dry rot fungus in a sample being analyzed, characterized by a) amplifying the nucleic acid in the sample being examined by us- ing one of the primer pairs according to claim 3 or 4, and b) detecting the amplification. 6. A method as claimed in claim 5, characterized by a further step after step b) comprising: d) verifying the presence of the dry rot fungus by amplifying the nucleic acid in the sample being examined by using another suitable primer pair selected from the ones listed above, and/or c2) verifying the presence of the dry rot fungus in the amplification product through a hybridization reaction by using the nucleic acid identified by SEQ. ID. NO.3 as a probe, when nucleic acids identified by SEQ. ID. NOS.1 and 2 have been used as primers in the amplification reaction. 7. Test kit, characterized by containing a desired combination of primer pairs according to claim 3 or 4, or suitable corresponding hybridization probes, and optionally other required reagents. 8. The use of the nucleic acid sequences according to claim 1 or 2 or the primer pairs according to claim 3 or 4 in fungus diagnostics as primers and/or hybridization probes.