Spreading of ESFY Phytoplasmas in Stone Fruit in Catalonia (Spain)

A survey was carried out in nine stone fruit commercial orchards located in Barcelona province where plum and apricot trees of different cultivars showing European stone fruit yellows (ESFY) symptoms were present. A 4‐year survey with visual inspection of symptoms in one apricot orchard showed a rather high ESFY disease spread, also in a Japanese plum plantation newly infected plants were detected every year in a similar rate (about 2%). All the inspected symptomatic trees were polymerase chain reaction (PCR) tested and ESFY phytoplasma identity was confirmed by restriction fragment length polymorphism analyses and sequencing of ribosomal DNA amplification products. In apricot plantation the detection of ESFY phytoplasma was also tested on 69 asymptomatic trees sampled in summer 2002. The nested PCR with 16SrX group‐specific primers allowed detection of ESFY phytoplasmas in 50% of the trees that indeed showed symptoms by the next winter (2003). The molecular detection of ESFY phytoplasma in asymptomatic apricot trees indicates the risk of maintaining phytoplasma foci in the fields where eradication is based only on visual inspection.     

Susceptibility and Tolerance of New French Prunus domestica Cultivars to European Stone Fruit Yellows Phytoplasmas

Five new cultivars of French prune (Prune d'Ente) and 11 cultivars or new clones of greengage (Reine Claude) were bud-inoculated with European stone fruit yellows (ESFY) phytoplasmas and evaluated for their host response in the nursery over a period of 6 years. The ESFY infection was confirmed by specific polymerase chain reaction (PCR) detection and 4',6-diamidino-2-phenylindole (DAPI) staining in all Prunus domestica cultivars tested which were, thus, all susceptible to ESFY phytoplasmas. The new Prune d'Ente cultivars were more sensitive as they generally developed ESFY symptoms and showed a mortality of 19%. The fruit weight was significantly reduced. No mortality was observed with Reine Claude genotypes and only two cultivars exhibited weak ESFY symptoms. A new sensitive method was developed to measure the impact of ESFY phytoplasmas on the shoot growth. A significant growth reduction was found in all Prune d'Ente cultivars but only in four out of 11 Reine Claude genotypes. Therefore, the latter were found to be tolerant of ESFY infection.     

Seasonal Colonization Pattern of European Stone Fruit Yellows Phytoplasmas in Different Prunus Species Detected by Specific PCR

The seasonal variation in the colonization of two Japanese plum trees by European stone fruit yellows (ESFY) phytoplasmas was followed by polymerase chain reaction (PCR) detection for 2 years. Samples were obtained monthly from nine above-ground sampling sites and one root. The colonization of the trees was systemic from July until leaf fall. The ESFY phytoplasmas were also detected in off-season grown leaves during winter until March. In contrast, almost no phytoplasmas could be detected in normally grown leaves in April and May. Similar results have been obtained for European plum cultivars using 4′,6-diamidino-2-phenylindole (DAPI) staining and PCR and for apricot cultivars tested by PCR. A possible explanation of this phenomenon is discussed. The root system of the corresponding Prunus marianna GF 8–1 rootstocks remained infected throughout the year and the phytoplasmas were equally distributed within the roots as determined when the trees were uprooted. In vitro culture was used to demonstrate that ESFY phytoplasmas detected by PCR in winter in aerial parts of the tree were viable. Nine ESFY-diseased shoot cultures were obtained at four different time points during winter. Sampling protocols for ESFY phytoplasma detection by PCR in routine diagnosis are discussed.     

Universal and group-specific real-time PCR diagnosis of flavescence dorée (16Sr-V), bois noir (16Sr-XII) and apple proliferation (16Sr-X) phytoplasmas from field-collected plant hosts and insect vectors

Three real‐time PCR–based assays for the specific diagnosis of flavescence dorée (FD), bois noir (BN) and apple proliferation (AP) phytoplasmas and a universal one for the detection of phytoplasmas belonging to groups 16Sr‐V, 16Sr‐X and 16Sr‐XII have been developed. Ribosomal‐based primers CYS2Fw/Rv and TaqMan probe CYS2 were used for universal diagnosis in real‐time PCR. For group‐specific detection of FD phytoplasma, ribosomal‐based primers fAY/rEY, specific for 16Sr‐V phytoplasmas, were chosen. For diagnosis of BN and AP phytoplasmas, specific primers were designed on non‐ribosomal and nitroreductase DNA sequences, respectively. SYBR^® Green I detection coupled with melting curve analysis was used in each group‐specific protocol. Field‐collected grapevines infected with FD and BN phytoplasmas and apple trees infected with AP phytoplasma, together with Scaphoideus titanus, Hyalesthes obsoletus and Cacopsylla melanoneura adults, captured in the same vineyards and orchards, were used as templates in real‐time PCR assays. The diagnostic efficiency of each group‐specific protocol was compared with well‐established detection procedures, based on conventional nested PCR. Universal amplification was obtained in real‐time PCR from DNAs of European aster yellows (16Sr‐I), elm yellows (16Sr‐V), stolbur (16Sr‐XII) and AP phytoplasma reference isolates maintained in periwinkles. The same assay detected phytoplasma DNA in all test plants and test insect vectors infected with FD, BN and AP phytoplasmas. Our group‐specific assays detected FD, BN, and AP phytoplasmas with high efficiencies, similar to those obtained with nested PCR and did not amplify phytoplasma DNA of other taxonomic groups. Melting curve analysis was necessary for the correct identification of the specific amplicons generated in the presence of very low target concentrations. Our work shows that real‐time PCR methods can sensitively and rapidly detect phytoplasmas at the universal or group‐specific level. This should be useful in developing defence strategies and for quantitative studies of phytoplasma–plant–vector interactions.     

A new approach to apple proliferation detection: a highly sensitive real-time PCR assay

The present paper describes a new approach for diagnosis of apple proliferation (AP) phytoplasma in plant material using a multiplex real-time PCR assay simultaneously amplifying a fragment of the pathogen 16S rRNA gene and the host, Malus domestica, chloroplast gene coding for tRNA leucine. For the first time, such an approach, with an internal analytical control, is described in a diagnostic procedure for plant pathogenic phytoplasmas enabling distinction between uninfected plant material and false-negative results caused by PCR inhibition. Pathogen detection is based on the highly conserved 16S rRNA gene to ensure amplification of different AP phytoplasma strains. The newly designed primer/probe set allows specific detection of all examined AP strains, without amplifying other fruit tree phytoplasmas or more distantly related phytoplasma strains. Apart from its specificity, real-time PCR with serial dilutions of initial template DNA ranging over almost five orders of magnitude (undiluted to 80,000-fold diluted) demonstrated linear amplification over the whole range, while conventional PCR showed a reliable detection only up to 500-fold or 10,000-fold dilutions, respectively. Compared to existing analytical diagnostic procedures for phytoplasmas, a rapid, highly specific and highly sensitive diagnostic method becomes now available.     

Sensitive detection of mycoplasmalike organisms in field-collected and in vitro propagated plants of Brassica, Hydrangea and Chrysanthemum by polymerase chain reaction

A polymerase chain reaction (PCR) protocol, previously designed for amplification of a DNA fragment from aster yellows mycoplasmalike organism (MLO), was employed to investigate the detection of MLO DNA in field-collected and in vitro micropropagated plants. PCR with template DNA extracted from symptomatic, naturally-infected samples of Brassica, Chrysanthemum and Hydrangea, each yielded a DNA band corresponding to 1.0 Kbp. However, no DNA product was observed when either infected Ranunculus (with phyllody disease) or Gladiolus with (symptoms of ‘germs fins’) was used as source of template nucleic acid for PCR; further experiments indicated absence of target DNA in the case of Ranunculus and the presence of substances in Gladiolus which inhibited the PCR. The MLO-specific DNA was detected by PCR using less than 95 pg of total nucleic acid (equivalent to total nucleic acid from 1.9, ug tissue) in the case of field-collected Hydrangea and less than 11.4 pg of nucleic acid (equivalent to total nucleic acid from 19 ng of tissue) in the case of field-collected Brassica. The findings illustrate highly sensitive detection of MLOs in both field-grown and in vitro micropropagated infected plants.     

Novel lysine biosynthetic gene sequences (LYS1 and LYS5) used as PCR targets for the detection of the pathogenic Candida yeast

We report here a sensitive and specific polymerase chain reaction (PCR) detection assay for the pathogenic Candida yeast based on the novel LYS1 [encoding saccharopine dehydrogenase (SDH)] and LYS5 [encoding phosphopantetheinyl transferase (PPTase)] gene sequences of the fungal unique lysine biosynthetic pathway. Both LYS1 and LYS5 DNA-specific PCR primers SG1, SG2 and SG3, SG4, respectively, amplified predicted 483 and 648-bp fragments from Candida albicans genomic DNA but not from other selected fungal, bacterial, or human DNA. The 18S rDNA control primers exhibited positive amplifications in all PCR assays. The LYS1-and LYS5-specific primers strongly amplified C. albicans and Candida tropicalis target sequences; however, the LYS1 primers also weakly amplified fragments from Candida kefyr and Candida lusitaniae DNA. Both sets of primers amplified target sequences from less than 10 pg of serially diluted C. albicans DNA, and the LYS1 specific primers also detected DNA isolated from serially diluted 50 C. albicans cells. The PCR primers reported here are sufficiently sensitive and specific for the potential early detection of Candida infections with no possibility of false positive results from cross-contamination with bacterial or human DNA.     

Detection of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> by novel chromosomal polymerase chain reaction primers

A new sensitive and specific method for the detection of Erwinia amylovora was developed. The method is based on the detection of a chromosomal DNA sequence specific for this bacterial species and enables detection of E. amylovora pathogenic strains, including recent isolates that lack plasmid pEA29 and thus cannot be detected by the previously popular PCR methods based on the detection of this plasmid. A species-specific random amplified polymorphic DNA (RAPD) marker was identified, cloned, and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed. The E. amylovora specific sequence, 1269 bp long, was amplified in polymerase chain reaction and detected with electrophoresis in agarose gel stained with ethidium bromide. Amplification with other bacterial species did not produce any PCR product detectable by electrophoresis. Matching of the E. amylovora specific sequence to chromosomal DNA was confirmed by computer analysis of the E. amylovora genome. A consistent sensitivity limit of the method was 3 CFU/reaction, and in some cases it was possible to detect 0.6 CFU/reaction. Due to its high sensitivity and specificity, our method of E. amylovora detection is currently the most reliable, taking into account that the reliability of PCR methods based on plasmid pEA29 has been compromised by the isolation of pathogenic E. amylovora strains that lack this plasmid.     

Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of canine parvovirus DNA directly in faecal specimens

Aims: To develop a specific and highly sensitive loop-mediated isothermal amplification (LAMP) technique for the rapid detection of canine parvovirus (CPV) DNA directly in suspected faecal samples of dogs by employing a simple method of template preparation. Methods and Results: LAMP reaction was developed by designing two sets of outer and inner primers, which target a total of six distinct regions on VP2 gene of CPV. The template DNA was prepared by a simple boiling and chilling method. Of the 140 faecal samples screened by the developed LAMP and the conventional PCR assays, 104 samples (74·28%) were found positive by LAMP, whereas 81 samples (57·85%) were found positive by PCR. The specificity of the LAMP assay was tested by cross-examination of common pathogens of dogs and further confirmed by sequencing. The detection limit of the LAMP was 0·0001 TCID(50) ml(-1) , whereas the detection limit of the PCR was 1000 TCID(50) ml(-1) . Conclusions: The developed LAMP assay detects CPV DNA in faecal specimens directly within an hour by following a simple and rapid boiling and chilling method of template preparation. The result also shows that the developed LAMP assay is specific and highly sensitive in detecting CPV. Significance and Impact of the Study: The result indicates the potential usefulness of LAMP which is a simple, rapid, specific, highly sensitive and cost-effective field-based method for direct detection of CPV from the suspected faecal samples of dogs.     

Detection and identification of Trypanosoma of African livestock through a single PCR based on internal transcribed spacer 1 of rDNA

Primers hybridising with the rDNA cistron have previously been evaluated for PCR diagnosis specific for kinetoplastids, and shown to detect and differentiate the Trypanosoma brucei complex and Trypanosoma cruzi. Kin1 and Kin2 primers, amplifying internal transcribed spacer 1, were subsequently evaluated for the diagnosis of African livestock trypanosomosis. Based on the size of the PCR products obtained, Kin primers allowed detection and identification of three Trypanosoma congolense types (savannah, forest and Kenya Coast), with distinction among themselves and from the subgenus Trypanozoon (T. brucei spp., Trypanosoma evansi and Trypanosoma equiperdum), Trypanosoma vivax, Trypanosoma simiae and Trypanosoma theileri. These primers were shown to be suitable for the sensitive and type-specific diagnosis of African livestock trypanosome isolates through a single PCR even in the case of multi-taxa samples. With field samples (buffy-coat from cattle blood) sensitivity was close to the sensitivity observed in single reactions with the classical specific primers for the Trypanozoon subgenus and T. congolense-type savannah, but was lower for detection of T. vivax. Additional reaction, improvement of DNA preparation, and/or new primers design are necessary to improve the sensitivity for detection of T. vivax in field samples. However, these primers are suitable for isolate typing through a single PCR.     

Detection of mycoplasma contaminations by the polymerase chain reaction

The polymerase chain reaction (PCR) has been used for the general detection ofMollicutes. 25Mycoplasma andAcholeplasma species were detected including important contaminants of cell cultures such asM. orale, M. arginini, M. hyorhinis, M. fermentans, A. laidlawii and additional human and animal mycoplasmas. PCR reactions were performed using a set of nested primers defined from conserved regions of the 16S rRNA gene. The detection limit was determined to be 1 fg mycoplasma DNA, which is equivalent to 1–2 genome copies of the 16S rRNA coding region. The identity of the amplification products was confirmed by agarose gel electrophoresis and restriction enzyme analysis. DNA from closely and distantly related micro-organisms did not give rise to specific amplification products. The method presented here offers a much more sensitive, specific and rapid assay for the detection of mycoplasmas than the existing ones.     

Nested PCR Assays for Detection of Monilinia fructicola in Stone Fruit Orchards and Botryosphaeria dothidea from Pistachios in California

Nested polymerase chain reaction (PCR) assays were developed based on microsatellite regions for detection of Monilinia fructicola, the causal agent of brown rot of stone fruits, and Botryosphaeria dothidea, the causal agent of panicle and shoot blight of pistachio. The nested PCR primers specific to M. fructicola were developed based upon the sequence of a species‐specific DNA fragment amplified by microsatellite primer M13. The external and internal primer pairs EMfF + EMfR and IMfF + IMfR amplified a 571‐ and a 468‐bp fragment, respectively, from M. fructicola, but not from any other fungal species present in stone fruit orchards. The nested PCR primer pairs specific to B. dothidea were developed based upon the sequence of a species‐specific 1330‐bp DNA fragment amplified by microsatellite primer T3B. The external and internal primer pairs EBdF + EBdR and IBdF + IBdR amplified a 701‐ and a 627‐bp fragment, respectively, from B. dothidea, but not from any other fungal species associated with pistachio. The nested PCR assays were sensitive enough to detect the specific fragments in 1 fg of M. fructicola or B. dothidea DNA or in the DNA from only two conidia of M. fructicola or B. dothidea. The nested PCR assays could detect small numbers of M. fructicola conidia caught on spore‐trap tapes and detect visible infections of B. dothidea in pistachio tissues. Microsatellite regions with high numbers of copies are widely dispersed in eukaryotic genomes. The results of this study indicate that microsatellite regions could be useful in developing highly sensitive PCR detection systems for phytopathogenic fungi.     

Detection and quantitative analysis of zoospores of Pythium porphyrae, causative organism of red rot disease in Porphyra, by competitive PCR

The detection and quantitative analysis of Pythium porphyraezoospores was performed by PCR using PP-1 and PP-2 primers specific tothe internal transcribed spacer region of P. porphyrae. To estimatethe amount of fungal zoospores of P. porphyrae, an internal standardplasmid (pPPISC) containing a modified DNA fragment was constructed. Both ends of this fragment were complementary to the PCR primers. Amplification using primers PP-1 and PP-2 produced DNA fragments ofapproximately 700 and 400 bp from the target DNA of P. porphyraezoospores and from the pPPISC, respectively. To perform quantitativePCR, known quantities of pPPISC were added to reaction mixturescontaining the experimental DNAs extracted from zoospores. After aco-amplification reaction, the two different sized PCR products wereseparated by agarose gel electrophoresis and visualized by ethidium bromidestaining. The number of zoospores was estimated by comparing thefluorescence intensities of the PCR products using a charge-coupled deviceimage analyzer. The results show that competitive PCR using P.porphyrae specific primers and competitor pPPISC are useful tools for thequantitative analysis of P. porphyrae zoospores in seawater from Porphyra cultivation farms.     

Hypervariable spacer regions are good sites for developing specific PCR-RFLP markers and PCR primers for screening actinorhizal symbionts

While the ribosomal RNA like highly conserved genes are good molecular chronometers for establishing phylogenetic relationships, they can also be useful in securing the amplification of adjoining hyper-variable regions. These regions can then be used for developing specific PCR primers or PCR-RFL profiles to be used as molecular markers. We report here the use of ITS region ofrrn operon ofFrankia for developing PCR-RFL profiles capable of discriminating between closely related frankiae. We have also made use of the ITS 1 region of the nuclearrrn operon ofAlnus nepalensis (D Don) for designing a PCR primer for specific amplification of nuclear DNA of this tree.     

A型肉毒神经毒素基因的PCR检测

目的:建立快速筛查A型肉毒毒素的PCR方法。方法:根据GenBank中报道的肉毒毒素基因序列,综合应用多种生物软件分析设计特异的检测引物,从提取的基因组DNA、热裂解产物和菌液等不同形式的模板中扩增大小为457bp的A型肉毒毒素特异基因片段,以肉毒梭菌其他血清型及破伤风梭菌为对照。结果:检测方法无交叉反应,灵敏度可达10pgDNA,3×103个菌。结论:建立的检测方法特异性强、灵敏度高,可以用于A型肉毒毒素基因的快速筛查。     

A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.)

A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V‐C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA‐A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl₂, Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low‐cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V‐C.

Significance and Impact of the Study

Incidence of phytoplasma diseases is increasing worldwide and particularly in the tropical and subtropical world. Co‐infection of phytoplasma and virus(s) is also common. Therefore, use of single primer PCR in detecting these pathogens would require more time and effort, whereas multiplex PCR involving several pairs of primers saves time and reduces cost. In this study, we have developed a multiplex nested PCR assay that provides more sensitive and specific detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma in white jute simultaneously. It is the first report of simultaneous detection of CoGMV and a phytoplasma in Corchorus capsularis by multiplex nested PCR.     

Specific detection of an oil-degrading bacterium, Corynebacterium sp. IC10, in sand microcosms by PCR using species-specific primers based on 16S rRNA gene sequences

A species-specific PCR technique to detect an oil-degrading bacterium, Corynebacterium sp. IC10, released into sand microcosms is described. PCR primers, specific to strain IC10, were designed based on 16S rRNA gene sequences and tested against both closely and distantly related bacterial strains using four primer combinations involving two forward and two reverse primers. Two sets of them were specific to the strain IC10 and Corynebacterium variabilis and one set was selected for further analysis. The PCR amplification was able to detect 1 pg template DNA of strain IC10 and 1.2×10⁴ c.f.u. of IC10 ml wet sand^–1 in the presence of 3×10⁸ Escherichia coli cells. In non-sterile sand microcosms seeded with the strain IC10, the sensitivity of detection decreased to 9.6×10⁵ c.f.u. ml wet sand^–1. The detection sensitivity thus depends on the complexity of background heterogeneous DNA of environmental samples. The assay is suitable for detection of Corynebacterium sp. IC10 in laboratory microcosms, however, cross reaction with non-oil degrading coryneforms may prohibit its use in uncharacterized systems.     

A duplex-PCR method for species- and pathovar-level identification and detection of the quarantine plant pathogen Xanthomonas arboricola pv. pruni

A PCR-based method was developed for the stone fruit quarantine pathogen Xanthomonas arboricola pv. pruni (Xap), which provides rapid, sensitive and specific in planta detection and isolate identification. Primers specific for Xap were identified using random amplified polymorphic DNA (RAPD). Simplex PCR with these primers had a limit of detection per PCR reaction of approximately 10 CFU for isolate cultures and 50 CFU for plant material when used on tenfold dilutions of isolate culture or genomic DNA extracted from spiked samples, respectively. The primers were adapted as a high-throughput single-step screening based on a digoxigenin-labeled DNA probe assay with a detection limit of 4 × 10² CFU from isolate cultures. A duplex-PCR method was designed that includes the pathovar-level with species-level primers based on species-specific regions of the quinate metabolic gene qumA, increasing diagnostic confidence and offering the first molecular test for all X. arboricola pathovars.     

DNA extraction and PCR detection of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis> spores from naturally contaminated honey and bees using spore-decoating and freeze-thawing techniques

Summary Paenibacillus larvae causes American foulbrood (AFB), a severe disease that affects the brood of honey bee Apis mellifera. AFB is worldwide distributed and causes great economic losses to beekeepers, but in many cases early diagnosis could help in its prevention and control. The aim of the present work was to design a reliable protocol for DNA extraction of P. larvae spores from naturally contaminated honey and adult bees. A novel method that includes a step of spore-decoating followed by an enzymatic spore disruption and DNA purification was developed. Also a freeze-thaw cycle protocol was tested and the results were compared. The DNA extracted was used as template for specific bacterial detection by amplification of a 16S rDNA fragment. Both methods allowed the direct detection by polymerase chain reaction (PCR) of P. larvae spores present in naturally contaminated material. The spore-decoating strategy was the most successful method for DNA extraction from spores, allowing specific and remarkably sensitive PCR detection of spores in all honey and bees tested samples. On the other hand freeze-thawing was only effective for detection of spores recovered from bees, and extensive damage to DNA affected detection by PCR. This work provides new strategies for spore DNA extraction and detection by PCR with high sensitivity, and brings an alternative tool for P. larvae detection in natural samples.     

Identification of pathogenic yeast species by polymerase chain reaction amplification of the RPS0 gene intron fragment

Aims: This work focuses on the development of a method for the identification of pathogenic yeast. With this aim, we target the nucleotide sequence of the RPS0 gene of pathogenic yeast species with specific PCR primers. PCR analysis was performed with both the genomic DNA, whole cells of clinical isolates of Candida species and clinical samples. Methods and Results: A single pairs of primers, deduced from the nucleotide sequence of the RPS0 gene from pathogenic yeast, were used in PCR analysis performed with both the genomic DNA and whole cells of clinical isolates of Candida species and clinical samples. The primers designed are highly specific for their respective species and produce amplicons of the expected sizes and fail to amplify any DNA fragment from the other species tested. The set of primers was tested successfully for the identification of yeast from colonies, blood cultures and clinical samples. These results indicate that genes containing intron sequences may be useful for designing species‐specific primers for the identification of fungal strains by PCR. The sensitivity of the method with genomic DNA was evaluated with decreasing DNA concentrations (200 ng to 1 pg) and different cell amounts (10⁷–10⁵ cells). Conclusion: The results obtained show that the amplification of RPS0 sequences may be suitable for the identification of pathogenic and other yeast species. Significance and Impact of the Study: Identification of Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for the interruption of transmission of this yeast. The approach described in this work is based on standard technology, and it is specific, sensitive and does not involve complex and expensive equipment. Furthermore, the method developed in this work not only can be used in eight yeast species, but also provides the basis to design primers for other fungi species of clinical, industrial or environmental interest.