Fast and reliable PCR/sequencing/RFLP assay for identification of fungi in onychomycoses

Fusarium spp. and other non-dermatophyte fungi are repeatedly isolated from abnormal nails. To investigate whether these fungi are the aetiological agents of infection or simply transient contaminants, a PCR/sequencing/RFLP assay was developed for direct and routine identification of the infecting fungi in onychomycosis. Fungal DNA was readily extracted using a commercial kit after dissolving nail fragments in a Na2S solution. Amplification of part of the 28S rDNA by PCR was performed with universal primers and the fungal species were identified by sequencing. The PCR/sequencing results were comparable with microbiological identification from the same nail sample. In addition to dermatophytes, Fusarium spp. and other less frequently isolated non-dermatophyte fungi were identified as single fungal agents in onychomycosis. Moreover, mixed infections were clearly demonstrated in 10 % of cases by RFLP analysis of PCR products. Identification of infectious agents could be obtained in 2 days, whilst results from fungal cultures take 1–3 weeks. Rapid and reliable molecular identification of the infectious fungus expedites the choice of appropriate antifungal therapy, thereby improving the cure rate of onychomycosis.

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A PCR Method That Can Be Further Developed into PCR-RFLP Assay for Eight Animal Species Identification

Journal of Analytical Methods in Chemistry ◽

10.1155/2018/5890140 ◽

2018 ◽

Vol 2018 ◽

pp. 1-6 ◽

Cited By ~ 9

Author(s):

Feng Guan ◽

Yu-Ting Jin ◽

Jin Zhao ◽

Ai-Chun Xu ◽

Yuan-Yuan Luo

Keyword(s):

Species Identification ◽

Animal Species ◽

Limit Of Detection ◽

Cost Effective ◽

Universal Primers ◽

Universal Primer ◽

Routine Identification ◽

Pcr Rflp ◽

Fast Processing ◽

Rflp Assay

There are many PCR-based methods for animal species identification; however, their detection numbers are limited or could not identify unknown species. We set out to solve this problem by developing a universal primer PCR assay for simultaneous identification of eight animal species, including goat, sheep, deer, buffalo, cattle, yak, pig, and camel. In this assay, the variable lengths of mitochondrial DNA were amplified using a pair of universal primers. PCR amplifications yielded 760 bp, 737 bp, 537 bp, 486 bp, 481 bp, 464 bp, 429 bp, and 359 bp length fragments for goat, sheep, deer, buffalo, cattle, yak, pig, and camel, respectively. This primer pair had no cross-reaction with other common domestic animals and fish. The limit of detection varied from 0.01 to 0.05 ng of genomic DNA for eight animal species in a 20 µl PCR mixture. Each PCR product could be further digested into fragments with variable sizes and qualitative analysis by SspI restriction enzyme. This developed PCR-RFLP assay was sufficient to distinguish all targeted species. Compared with the previous published related methods, this approach is simple, with high throughput, fast processing rates, and more cost-effective for routine identification of meat in foodstuffs.

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An Epidemic of Almond Witches'-broom in Lebanon: Classification and Phylogenetic Relationships of the Associated Phytoplasma

Plant Disease ◽

10.1094/pdis.2002.86.5.477 ◽

2002 ◽

Vol 86 (5) ◽

pp. 477-484 ◽

Cited By ~ 34

Author(s):

Yusuf Abou-Jawdah ◽

Armig Karakashian ◽

Hana Sobh ◽

Marta Martini ◽

Ing-Ming Lee

Keyword(s):

Rflp Analysis ◽

Pigeon Pea ◽

Universal Primers ◽

Rrna Gene ◽

Nested Polymerase Chain Reaction ◽

Main Trunk ◽

Stunted Growth ◽

Rdna Sequences ◽

Pcr Products ◽

Almond Trees

An epidemic of almond witches'-broom has devastated almond production in Lebanon. Thousands of almond trees have died over the past 10 years due to the rapid spread of the disease. The symptoms, which include early flowering, stunted growth, leaf rosetting, dieback, off-season growth, proliferation of slender shoots, and witches'-brooms arising mainly from the main trunk and roots, resemble those caused by phytoplasmal infections. For the detection of the putative causal agent, nested polymerase chain reaction (PCR) was performed using universal primers (P1/P7, R16mF2/R16mR1, and R16F2n/R16R2) commonly used for the specific diagnosis of plant pathogenic phytoplasmas. Phytoplasmas were readily detected from infected trees with witches'-broom symptoms collected from three major almond growing regions in Lebanon. Restriction fragment length polymorphism (RFLP) analysis of PCR products amplified by the primer pair R16F2n/R16R2 revealed that the phytoplasma associated with infected almonds is similar to, but distinct from, members of the pigeon pea witches'-broom phytoplasma group (16SrIX). A new subgroup, 16SrIX-B, was designated. Sequencing of the amplified products of the phytoplasma 16S rRNA gene indicated that almond witches'-broom (AlmWB) phytoplasma is most closely related to members of the pigeon pea witches'-broom phytoplasma group (with sequence homology ranging from 98.4 to 99.0%). Phylogenetic analysis of 16S rDNA sequences from AlmWB phytoplasma and from representative phytoplasmas from GenBank showed that the AlmWB phytoplasma represents a distinct lineage within the pigeon pea witches'-broom subclade. The same phytoplasma appears also to infect peach and nectarine seedlings.

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Molecular identification of fungi associated with avocado (Persea americana Mill.) fruits

Agro-Science ◽

10.4314/as.v20i1.13 ◽

2021 ◽

Vol 20 (1) ◽

pp. 80-86

Author(s):

N.G. Iyanyi ◽

A.E. Ataga ◽

I.S. Rotimi ◽

I. Blessing

Keyword(s):

Pcr Amplification ◽

Fungal Species ◽

Persea Americana ◽

Molecular Techniques ◽

Control Measures ◽

Universal Primer ◽

Identification Of Fungi ◽

Pcr Products ◽

Fungal Organisms ◽

Health Complications

Avocado (Persea americana Mill.) is grown for its nutritious fruit. However, the quantity and quality of these fruits are threatened by some fungal organisms which can cause health complications when it is consumed by man. DNA extraction provides a unique tool for identification of organisms. This study was conducted to isolate and identify fungal species associated with avocado fruit using both morphological and molecular techniques. Fungal species were isolated from Persea americana purchased from Choba market, Port Harcourt, Rivers State, Nigeria using Potato Dextrose Agar (PDA) as a growth medium. The morphology of isolated fungi on PDA were cotton-like blackish grey spots, white villous colonies, greyish powdery spores and black spores for isolates 1 to 4 respectively. Extraction of DNA from fungal isolates was carried out using Zymo Fungal/Bacteria DNA Miniprep Kit. PCR amplification of the ITS1-2 regions of isolates was carried out using fungal universal primer pair; ITS4 and ITS5.PCR amplification of the ITS1-2 gene sequences yielded amplicons between 537-580 base pairs. PCR products were sequenced and the sequencing result after BLAST search revealed the identity of the four fungal species as follows: Lasiodiplodia theobromae, Fusarium proliferatum, Penicillium sp. and Aspergillus niger. This study will promote the knowledge of specific fungal species associated with Persea americanna and help plant pathologists to proffer preventive and control measures to enhance fruit protection and yield quality.

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First Report of Syringa oblata and S. reticulata Leafroll Disease in China

Plant Disease ◽

10.1094/pdis-93-3-0322c ◽

2009 ◽

Vol 93 (3) ◽

pp. 322-322

Author(s):

Z. N. Li ◽

H. Min ◽

Y. Yan ◽

Z. Zhao ◽

W. J. Wu ◽

...

Keyword(s):

16S Rdna ◽

Rflp Analysis ◽

Universal Primers ◽

University Campus ◽

Sequence Comparisons ◽

16S Rdna Sequence ◽

First Report ◽

Pcr Products ◽

Leafroll Disease ◽

Total Dna

Syringa oblata is an important ornamental tree widely grown in China. In September of 2008, S. oblata plants exhibiting symptoms of leafroll and yellowing were found in a garden on the Northwest A&F University campus. Samples were collected from this site. Total DNA was extracted from 0.5 g of phloem tissue from leaf midribs and stems of each sample. DNA samples were analyzed with a nested PCR assay using phytoplasma 16S rDNA universal primers R16mF2/R16mR1 followed by specific primers R16F2n/R16R2 (1), which amplified a 1,452- and 1,246-bp product, respectively. We tested all 30 lilac samples, 20 of which had symptoms of leafroll and yellowing. These produced the expected 1,452- and 1,246-bp PCR products In contrast, the remaining 10 samples from symptomless trees yielded no PCR products. We also surveyed another lilac variety (Syringa reticulata), which is widely grown on the campus, and tested 50 samples with the above method. Again, 1.4- and 1.2-kb PCR products were amplified from all 30 trees displaying leafroll and yellowing symptoms, but not from the other 20 samples from symptomless trees. A comparative analysis of sequences derived from the two hosts showed that the phytoplasmas infecting them were most similar (>99%) to paulownia witches'-broom (PaWB) phytoplasma (GenBank Accession No. EF199937). Restriction fragment length polymorphism (RFLP) analysis of the nested 1.2-kb 16S rDNA products with endonucleases AluI and MseI indicated that all symptomatic plants were infected by the phytoplasmas belonging to aster yellow group (16SrI) subgroup D (16SrI-D) PaWB phytoplasma (2). 16S rDNA sequence comparisons and RFLP analysis of the cloned 16S rDNA from S. oblata (GenBank Accession No. FJ445224) and S. reticulate (GenBank Accession No. FJ445225) indicated that the phytoplasmas infecting them were nearly identical (99.8% identity). To our knowledge, this is the first report of the presence of the phytoplasma associated with a leafroll disease of S. oblata and S. reticulata in China. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

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Diagnostic Accuracy of a Direct Panfungal Polymerase Chain Reaction Assay Performed on Stained Cytology Slides

Veterinary Pathology ◽

10.1177/0300985821991562 ◽

2021 ◽

pp. 030098582199156

Author(s):

Alexandra N. Myers ◽

Unity Jeffery ◽

Zachary G. Seyler ◽

Sara D. Lawhon ◽

Aline Rodrigues Hoffmann

Keyword(s):

Polymerase Chain Reaction ◽

Diagnostic Accuracy ◽

Molecular Techniques ◽

Fungal Culture ◽

Chain Reaction ◽

Identification Of Fungi ◽

Fungal Dna ◽

Polymerase Chain ◽

Panfungal Pcr ◽

Pcr Targeting

Molecular techniques are increasingly being applied to stained cytology slides for the diagnosis of neoplastic and infectious diseases. Such techniques for the identification of fungi from stained cytology slides have not yet been evaluated. This study aimed to assess the diagnostic accuracy of direct (without nucleic acid isolation) panfungal polymerase chain reaction (PCR) followed by sequencing for identification of fungi and oomycetes on stained cytology slides from dogs, cats, horses, and other species. Thirty-six cases were identified with cytologically identifiable fungi/oomycetes and concurrent identification via fungal culture or immunoassay. Twenty-nine controls were identified with no cytologically or histologically visible organisms and a concurrent negative fungal culture. Direct PCR targeting the internal transcribed spacer region followed by sequencing was performed on one cytology slide from each case and control, and the sensitivity and specificity of the assay were calculated. The sensitivity of the panfungal PCR assay performed on stained cytology slides was 67% overall, 73% excluding cases with oomycetes, and 86% when considering only slides with abundant fungi. The specificity was 62%, which was attributed to amplification of fungal DNA from control slides with no visible fungus and negative culture results. Direct panfungal PCR is capable of providing genus- or species-level identification of fungi from stained cytology slides. Given the potential of panfungal PCR to amplify contaminant fungal DNA, this assay should be performed on slides with visible fungi and interpreted in conjunction with morphologic assessment by a clinical pathologist.

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Molecular Characterization and Classification of Phytoplasmas Associated with Canola Yellows and a New Phytoplasma Strain Associated with Dandelions

Plant Disease ◽

10.1094/pdis.2001.85.1.76 ◽

2001 ◽

Vol 85 (1) ◽

pp. 76-79 ◽

Cited By ~ 24

Author(s):

Keri Wang ◽

Chuji Hiruki

Keyword(s):

Fragment Length Polymorphism ◽

Rflp Analysis ◽

Universal Primers ◽

Dna Fragments ◽

Chain Reaction ◽

Aster Yellows ◽

Distinct Subgroup ◽

16S Ribosomal Dna ◽

Polymerase Chain

DNA isolated from symptomatic canola (Brassica napus, Brassica rapa) and dandelion (Taraxacum officinale) was used to amplify 16S ribosomal DNA fragments by polymerase chain reaction using two pairs of universal primers P1/P6 and R16F2n/R2. Restriction fragment length polymorphism (RFLP) analysis of the amplified DNA fragments using endonucleases AluI, HhaI, HpaII, MseI, RsaI, and Sau 3AI revealed two distinct types of phytoplasmas in canola with similar symptoms. One had the same RFLP profiles as the phytoplasmas in subgroup 16SrI-A, whereas the other one had RFLP profiles similar to those of phytoplasmas in subgroup 16SrI-B. Phytoplasmas were detected in symptomatic dandelion plants that were collected from canola and alfalfa fields where severe alfalfa witches'-broom occurred. Comparative studies indicated that two different phytoplasmas were associated with the dandelion plants. One was identified as a member of subgroup 16SrI-A, whereas another one was classified as a member of a distinct subgroup in the aster yellows group on the basis of the unique RFLP patterns.

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Rapid Detection of Phytophthora infestans in Late Blight-Infected Potato and Tomato Using PCR

Plant Disease ◽

10.1094/pdis.1997.81.9.1042 ◽

1997 ◽

Vol 81 (9) ◽

pp. 1042-1048 ◽

Cited By ~ 80

Author(s):

C. L. Trout ◽

J. B. Ristaino ◽

M. Madritch ◽

T. Wangsomboondee

Keyword(s):

Late Blight ◽

Phytophthora Infestans ◽

Pcr Amplification ◽

Accurate Method ◽

Phytophthora Species ◽

Universal Primers ◽

Direct Pcr ◽

Field Samples ◽

Pcr Products ◽

Oomycete Pathogen

Late blight caused by the oomycete pathogen Phytophthora infestans is a devastating disease of potato and tomato worldwide. A rapid and accurate method for specific detection of P. infestans is necessary for determination of late blight in infected fruit, leaves, and tubers. Ribosomal DNA (rDNA) from four isolates of P. infestans representing the four genotypes US1, US6, US7, and US8 was amplified using polymerase chain reaction (PCR) and the universal primers internal transcribed spacer (ITS) 4 and ITS5. PCR products were sequenced using an automated sequencer. Sequences were aligned with published sequences from 5 other Phytophthora species, and a region specific to P. infestans was used to construct a PCR primer (PINF). Over 140 isolates representing 14 species of Phytophthora and at least 13 other genera of fungi and bacteria were used to screen the PINF primer. PCR amplification with primers PINF and ITS5 results in amplification of an approximately 600 base pair product with only isolates of P. infestans from potato and tomato, as well as isolates of P. mirabilis and P. cactorum. P. mirabilis and P. cactorum are not pathogens of potato; however, P. cactorum is a pathogen of tomato. P. infestans and P. cactorum were differentiated by restriction digests of the amplified product. The PINF primer was used with a rapid NaOH lysis technique for direct PCR of P. infestans from infected tomato and potato field samples. The PINF primer will provide a valuable tool for detection of P. infestans in potatoes and tomatoes.

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The simplest and currently the most widely adopted method to obtain 16S rRNA genes from the environment is through the use of PCR. rRNA genes can be amplified directly from the total community DNA using rRNA-specific primers, and then cloned using standard methods (Giovannoni et al. 1990). By taking advantage of the highly conserved nature of rRNA, universal primers capable of annealing to rRNA genes from all three domains (Archaea, Bacteria, Eukarya) or primers designed to amplify rRNA genes from a particular group of organisms can be used. Following PCR amplification, the amplified products can be cloned. Commercially available kits exploit the fact that PCR-amplified products have an overhanging 3' deoxyadenosine residue at each end when certain DNA polymerases are used. This allows cloning of the product into a sequencing-ready vector containing a complementary deoxy-thymidine overhang, in many cases, without requiring the product to be purified or further modified. Alternatively, the PCR products can be cloned by "filling" overhanging residues followed by blunt-end ligation procedures.

Recent Advances in Marine Biotechnology, Vol. 8 ◽

10.1201/9781482279986-14 ◽

2003 ◽

pp. 221-221

Keyword(s):

16S Rrna ◽

Dna Polymerases ◽

Pcr Amplification ◽

16S Rrna Genes ◽

Rrna Genes ◽

Universal Primers ◽

Standard Methods ◽

Pcr Products ◽

Total Community ◽

Total Community Dna

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First Report of a Group 16SrII Phytoplasma Infecting Chickpea in Oman

Plant Disease ◽

10.1094/pd-90-0973c ◽

2006 ◽

Vol 90 (7) ◽

pp. 973-973 ◽

Cited By ~ 8

Author(s):

N. A. Al-Saady ◽

A. M. Al-Subhi ◽

A. Al-Nabhani ◽

A. J. Khan

Keyword(s):

Nested Pcr ◽

Animal Feed ◽

Restriction Enzymes ◽

Universal Primers ◽

Polymorphism Analysis ◽

Disease Symptoms ◽

First Report ◽

Pcr Products ◽

Polymerase Chain ◽

Group 2

Chickpea (Cicer arietinum), locally known as “Dungo”, is grown for legume and animal feed mainly in the interior region of Oman. During February 2006, survey samples of chickpea leaves from plants showing yellows disease symptoms that included phyllody and little leaf were collected from the Nizwa Region (175 km south of Muscat). Total nucleic acid was extracted from asymptomatic and symptomatic chickpea leaves using a cetyltrimethylammoniumbromide method with modifications (3). All leaf samples from eight symptomatic plants consistently tested positive using a polymerase chain reaction assay (PCR) with phytoplasma universal primers (P1/P7) that amplify a 1.8-kb phytoplasma rDNA product and followed by nested PCR with R16F2n/R16R2 primers yielding a product of 1.2 kb (2). No PCR products were evident when DNA extracted from healthy plants was used as template. Restriction fragment length polymorphism analysis of nested PCR products by separate digestion with Tru9I, HaeIII, HpaII, AluI, TaqI, HhaI, and RsaI restriction enzymes revealed that a phytoplasma belonging to group 16SrII peanut witches'-broom group (2) was associated with chickpea phyllody and little leaf disease in Oman. Restriction profiles of chickpea phytoplasma were identical with those of alfalfa witches'-broom phytoplasma, a known subgroup 16SrII-B strain (3). To our knowledge, this is the first report of phytoplasma infecting chickpea crops in Oman. References: (1) A. J. Khan et al. Phytopathology, 92:1038, 2002. (2). I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998 (3) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA. 81:8014, 1984.

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Phylogenetic Relationships of Local Durian Species based on Morphological Characteristics and PCR-RFLP Analysis of the Ribosomal Internal Transcribed Spacer (ITS) DNA

Biosaintifika Journal of Biology & Biology Education ◽

10.15294/biosaintifika.v8i3.6748 ◽

2016 ◽

Vol 8 (3) ◽

pp. 362

Author(s):

Fidia Fibriana ◽

Lutfia Nur Hadiyanti

Keyword(s):

Phylogenetic Relationships ◽

Internal Transcribed Spacer ◽

Morphological Characteristics ◽

Biology Education ◽

Rflp Analysis ◽

Restriction Pattern ◽

Internal Transcribed Spacers ◽

Central Java ◽

Pcr Rflp ◽

Pcr Products

In this study, twenty local durian accessions obtained from Central Java in situ collection were characterized using the morphological characteristics and the restriction patterns which generated from the region spanning the internal transcribed spacers ITS LEU and ITS 4. Morphological characteristics of durian leaf, stem, tree, and fruit showed variations for the different accessions, whereas polymerase chain reaction (PCR) products of ribosomal DNA region showed a low length of variation. The size of the PCR products and the restriction analyses with the restriction endonucleases Bsp1431yielded a restriction pattern for each accessions. The results of this study can be utilized by local durian farmers as a preliminary reference for durian propagation. The data obtained need to be supported by further research using the other molecular markers to obtain more accurate data. The clear identity of durian species can help the management of propagation systems by farmers to get superior local durian.How to CiteFibriana, F., & Hadiyanti, L. N. (2016). Phylogenetic Relationships of Local Durian Species based on Morphological Characteristics and PCR-RFLP Analysis of the Ribosomal Internal Transcribed Spacer (ITS) DNA. Biosaintifika: Journal of Biology & Biology Education, 8(3), 362-370.

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