Patent Application: US-91484506-A

Abstract:
the present invention describes a novel molecular method based on the polymerase chain reaction in a multiplex variant in order to detect and identify candida species with clinical relevance , namely c . albicans , c . glabrata , c . krusei , c . parapsilosis , c . tropicalis , c . guilliermondii , c . lusitaniae e c . dubliniensis . the strategy uses the existence of sequences , whether conserved or variable , in fungal ribosomal genes and in the use of a combination of universal primers , specific for fungi , and internal primers , specific for each one of the candida species . in this sense , two fragments from the internal transcribed spacer regions of the ribosomal rna are amplified by multiplex pcr , allowing the easy identification of the candida species in question . this methodology allows a rapid , effective and low - cost identification of candida species with clinical relevance , bearing several advantages over the currently available diagnostic methods .

Description:
the present invention provides in its most general form , a method to detect and identify in a rapid and precise manner , candida species with clinical importance in a determined sample , comprising the following steps : 1 . ( i ) release , isolation and / or concentration of the nucleic acids of the fungi possibly present in the sample , 1 . ( ii ) amplification by multiplex pcr of specific fragments of the its regions in the nucleic acids of step ( i ), according to the candida species considered , 1 . ( iii ) visualization of the products of the amplification of step ( ii ) by agarose gel electrophoresis . the amplification of nucleic acids is performed by a pcr reaction ( saiki et al ., science 239 : 487 - 91 ( 1988 )). in the amplification of the step ( ii ), the following primers are used in the pcr reaction : gtcaaacttggtcattta ( uni1 — seq id no . 1 ), ttcttttcctccgcttattga ( uni2 — seq id no . 2 ), agctgccgccagaggtctaa ( calb — seq id no . 3 ), gatttgcttaattgccccac ( ctro — seq id no . 4 ), gtcaaccgattatttaatag ( cpar — seq id no . 5 ), ctggccgagcgaactagact ( ckru — seq id no . 6 ), ttcggagcaacgcctaaccg ( clus — seq id no . 7 ), ttggcctagagataggttgg ( cgui — seq id no . 8 ), ctcaaacccctagggtttgg ( cdub — seq id no . 9 ). the sequences presented as seq id no . 1 and 2 represent the universal primers and their target is the terminal region of the 18s unit and the initial region of the 25s unit , respectively , of all the fungi belonging to the candida genera . the expression ‘ universal primers ’ means that this primer set amplifies the its - 1 and its - 2 regions in the big majority , or even in the entirety , of fungal species . the sequences of the universal primers are phylogenetically conserved in a way that allows the dna amplification of different genera of fungi and their targets are the rrna genes that flank the its regions , i . e . the rrna genes 18 s and 25 s . the universal primers used in the method reported in the present invention were described by trost et al . ( trost et al ., j . microbiol . methods 56 : 201 - 11 ( 2004 )). the pcr products resulting from the amplification using the universal primers ( seq id no 1 , 2 ) present sizes ranging from 433 bp ( c . lusitaniae ) to 929 bp ( c . glabrata ). however , in this way , the majority of candida species is not easily discriminated having only with base on these fragments . therefore , exclusive variations in the its - 1 and its - 2 regions of each candida species were used to design primers with the intent of amplifying a second fragment of minor size , which would facilitate the discrimination of the considered candida species . in this sense , specific variations in the its - 1 and its - 2 regions from the type strains and all the clinical strains available in the database of embl / genbank were analyzed through interspecies alignment to find blocks of conserved regions among different strains . next , these sequences were compared among species in order to find variable regions that would allow designing specific primers for each candida species . this in silico study concluded with the design of primers in variable regions among species , but at the same time , conserved among strains ( seq id no : 3 - 9 ), excluding by that the possibility of occurring interspecies variability . in the particular case of c . glabrata , a strategy based on a single fragment was designed , since the size of the obtained fragment by amplification with the universal primers ( 929 bp ) allowed the easy discrimination of this species . the sequences presented as seq id no . 3 , 4 , 5 , 6 , 7 , 8 and 9 represent the specific primers for each one of the candida species , and that have as target , as illustrated next in the examples section ( see table i ): the its - 1 region from c . albicans ( seq id no . 3 ), the its - 1 region from c . tropicalis ( seq id no . 4 ), the its - 1 region from c . parapsilosis ( seq id no . 5 ), the its - 2 region from c . krusei ( seq id no . 6 ), the its - 2 region from c . lusitaniae ( seq id no . 7 ), the its - 1 region from c . guilliermondii ( seq id no . 8 ), and the its - 2 region from c . dubliniensis ( seq id no . 9 ). it is important to note that all the specific primers amplify in the opposite direction of the uni2 primer , except the clus primer ( seq id no . 7 ), which amplifies in the opposite direction of the uni1 primer . a single base pair difference is sufficient to design a discriminatory primer . the primers used in this invention and its sequence , and the sizes of the pcr products to obtain are described in table i . the methodology herein presented was optimized using dna from candida type strains and tested in various strains isolated from clinical specimens . thus , the candida strains used to validate the method were isolated from clinical specimens in two hospitals in portugal , one in the north ( hospital de sao joao , porto ) and other in the south of the country ( hospital de santa maria , lisboa ), in a total of 386 clinical isolates ( 245 c . albicans , 61 c . parapsilosis , 25 c . tropicalis , 19 c . krusei , 18 c . glabrata , 13 c . guilliermondii and 5 c . lusitaniae ) and 8 type strains ( c . albicans atcc 18804 , c . glabrata atcc 2001 , c . tropicalis atcc 750 , c . parapsilosis atcc 22019 , c . krusei atcc 6258 , c . lusitaniae atcc 34449 , c . guilliermondii atcc 6260 and c . dubliniensis atcc mya - 646 ). the primary identification of the clinical isolates was carried out in the hospitals using commercial identification systems based on biochemical characteristics and afterwards confirmed using the multiplex pcr strategy according to the presented invention . furthermore , all the identifications which results were different from those provided by the reference institutions were confirmed using molecular fingerprinting ( correia et al ., j . clin . microbiol . 42 : 5899 - 903 ( 2004 )). the amplification of nucleic acids to its detection has as an obvious advantage the fact that the detection sensitivity is increased . furthermore , using universal primers , its regions from several candida species can be amplified conjointly , while the posterior amplification using specific primers allows the identification of multiple species simultaneously present in the same sample . it is important to note that the its regions , from which the specific primers were designed , have no dna sequence match in mammals , bacteria or virus . this is important since the falsely positive amplification of mammal dna that would be present in the clinical samples is avoided . all the primers used were synthesized by mwg biotech . the present invention has further the obvious possibility of preparation of kits containing the necessary elements in order to perform the process . the kit must comprise a compartmentalized transport system in small cells in order to receive in a tight confinement , the necessary reagents . the reagents used in the invention must be provided in the kit in predetermined amounts use in the process of candida species identification . one or more cells must contain the primer mixture and the remaining reagents , including the taq polymerase enzyme to be used in the pcr reactions , in the lyophilized form or in an appropriate buffer solution . candida cells were grown overnight in liquid yepd medium at 26 ° c . with aeration on a mechanical stirrer ( 150 rpm ). the cells were collected by centrifugation and the sediment was suspended in 200 μl of lysis buffer ( 2 % triton x - 100 , 1 % sds , 100 mm nacl , 10 mm tris - hcl e 1 mm edta , ph 8 . 0 ). for cellular disrupture , 200 μl of glass beads with a 0 . 5 mm diameter and 200 μl of phenol / chloroform ( 1 : 1 ) were added and the tubes agitated during three intervals of 60s intercalated with periods of cooling on ice . after the removal of cellular debris by a centrifugation of 5 minutes at 18 . 000 × g , the supernatant was collected and 1 ml of cold absolute alcohol was added before mixing by inversion . the tubes were centrifuged at 18 . 000 × g during 3 minutes and the sediment was suspended in 400 μl of te buffer ( 100 mm tris - hcl , 1 mm edta , ph 8 . 0 ). a 5 minutes treatment with rnase a ( 1 mg / ml ) at 37 ° c . was carried out before the addition of 10 μl of sodium acetate 3 m . the dna was again precipitated by the addition of 1 ml of cold absolute alcohol , mixed by inversion and once again centrifuged . finally , the dna was dried at air temperature and suspended in 50 μl of sterilized water . the dna content and its purity were determined by spectrophotometry at 260 and 280 nm and diluted to a final concentration of 100 ng / μl to be used according to example 2 . multiplex pcr amplification was performed in a 20 μl volume consisting of 0 . 8 × pcr buffer [ 160 mm ( nh 4 ) 2 so 4 , 670 mm tris - hcl ( ph 8 . 8 )], 3 . 5 mm mgcl 2 , dntps mixture ( 200 μm each ), primer mix ( seq id no 1 and 2 , 0 . 55 μm each ; seq id 3 and 6 , 0 . 15 μm each ; seq id no 4 and 7 , 0 . 2 μm ; seq id no 5 , 0 . 3 μm ; seq id no 8 , 0 . 05 μm ; seq id no 9 , 0 . 4 μm ), 1 u taq polymerase dna and 100 ng of genomic dna , with the remaining volume consisting of sterilized water . to perform multiplex pcr amplification using whole cells , part of an isolated colony was directly suspended in the reaction tube . the reaction was carried out as usual in a thermal cycler biometra tpersonal ( whatman biometra ) under the following conditions : 40 cycles of 15 s at 94 ° c ., 30 s at 55 ° c ., and 45 s at 65 ° c ., after an initial period of 10 minutes for denaturation and enzyme activation at 94 ° c . negative control reactions were performed simultaneously with each test replacing the dna by sterilized water in the pcr mixture . a 10 μl aliquot from each of the amplification products was separated by electrophoresis in a 2 % agarose gel . the use of ethidium bromide ( 0 . 5 μg ) allowed the visualization of the dna fragments with a digital imaging system ( alpha innotech corporation ) and the identification of the candida species in question was possible by comparison with a 100 bp dna ladder ( fermentas ). in order to determine the detection limit of the method , human peripheral blood was seeded separately with cells from several candida species , amongst which c . albicans , c . glabrata , c . krusei , c . parapsilosis and c . tropicalis , until concentration of 2 . 5 × 10 5 cfu / ml was obtained . the cell number ( cfu / ml ) was estimated by hemacytometer counting and confirmed by plating serial dilutions of seeded blood with candida onto solid medium sabouraud plates and colony counting forming units after 2 days of incubation at 30 ° c . the seeded blood was then diluted several times with unseeded blood ( concentrations in the range from 2 . 5 × 10 5 to 1 . 25 × 10 3 cfu / ml ) and 200 μl of the diluted samples were exposed to dna isolation , according to example 4 . in order to isolate dna from candida present in peripheral blood , a method based on heat , detergent and mechanical disruption of candida cells was used , according to shin et al . ( 1997 ). thus , to 200 μl of the sample obtained as described in example 3 were added 800 μl of txte buffer ( 10 mm tris - hcl , 1 mm edta , 1 % triton x - 100 , ph 8 . 0 ) in a sterile centrifuge tube of 1 . 5 ml . the mixture was then incubated for 10 min at 25 ° c . to originate lysis of blood cells . debris resulting from the blood cell reupture was collected by centrifugation at 18 , 000 × g for 8 min . next , the debris was washed three times with txte buffer and the candida cells were suspended in 300 μl of txte buffer and 200 μl of glass beads with a diameter of were added . after boiling for 15 minutes in a water bath , the mixture was agitated for 20 minutes using a vortex . finally , the tubes were centrifuged at 18 . 000 × g for 20 s and the supernatant was used in pcr amplification , as described in example 2 . 1 . bikandi , j ., r . s . millan , m . d . moragues , g . cebas , m . clarke , d . c . coleman , d . j . sullivan , g . quindos , and j . ponton . 1998 . rapid identification of candida dubliniensis by indirect immunofluorescence based on differential localization of antigens on c . dubliniensis blastospores and candida albicans germ tubes . j . clin . microbiol . 36 : 2428 - 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