rDNA-RFLP identification ofCandidaspecies in immunocompromised and seriously diseased patients

PCR was used to amplify a targeted region of the ribosomal DNA of 76 Candida spp. isolates from immunocompromised and seriously diseased patients. Thirty-seven strains isolated from different anatomical sites of 11 patients infected with HIV (Vitória, ES, Brazil), 26 isolates from patients under treatment at Odilon Behrens Hospital and 13 isolates from skin and urine samples from São Marcos Clinical Analysis Laboratory (Belo Horizonte, Brazil) were scored. Fragments of rDNA were amplified using primer pairs ITS1-ITS4, for the amplification of ITS1 and ITS2 regions, including the gene for the 5.8s subunit. Amplification resulted in fragments ranging in size from 350 to 950 bp. Amplicons were digested with eight restriction enzymes. A pattern of species-specificity among the different medically important Candida species could be identified following restriction digestion of the PCR products. Candida albicans was the species most frequently observed, except for the group of newborns under treatment at the Odilon Behrens Hospital and for the isolates from the clinical analysis laboratory. C. parapsilosis was the species most frequently observed in these two groups.Key words: polymerase chain reaction, restriction fragment length polymorphism, candidosis, Candida spp.

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Identification of Medically Important Candida Species by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Analysis of the rDNA ITS1 and ITS2 Regions

Indian Journal of Medical Microbiology ◽

10.4103/ijmm.ijmm_17_102 ◽

2017 ◽

Vol 35 (4) ◽

pp. 535-542 ◽

Cited By ~ 2

Author(s):

Suphi Bayraktar ◽

Nizami Duran ◽

Gülay Gülbol Duran ◽

Naciye Eryilmaz ◽

Hayat Aslan ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Restriction Fragment Length Polymorphism ◽

Length Polymorphism ◽

Fragment Length ◽

Fragment Length Polymorphism ◽

Polymorphism Analysis ◽

Chain Reaction ◽

Polymerase Chain ◽

Its1 And Its2 ◽

Restriction Fragment Length

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Discrimination of Diploid Crucifer Species Using PCR-RFLP of Chloroplast DNA

HortScience ◽

10.21273/hortsci.39.7.1575 ◽

2004 ◽

Vol 39 (7) ◽

pp. 1575-1577 ◽

Cited By ~ 6

Author(s):

Claudia Cunha ◽

Muhammet Tonguç ◽

Phillip D. Griffiths

Keyword(s):

Chloroplast Dna ◽

Dna Sequences ◽

Raphanus Sativus ◽

Fragment Length Polymorphism ◽

Diploid Species ◽

Restriction Enzymes ◽

Chain Reaction ◽

Species Identity ◽

Pcr Rflp ◽

Polymerase Chain

Chloroplast DNA (cpDNA) was used to identify polymorphisms between crucifer species using the polymerase chain reaction-random fragment-length polymorphism (PCR-RFLP) technique. Ten primer pairs based on cpDNA gene sequences were used to amplify cpDNA fragments in Brassica oleracea L., B. rapa L., B. nigra (L.) Koch, B. napus L., B. carinata Braun, B. juncea (L.) Czern, and Raphanus sativus L. accessions. Amplified DNA sequences were then digested using 11 restriction enzymes to identify polymorphisms between the 7 species. Of the 110 combinations, 38 generated polymorphisms that discriminated one or more of the species. Genotyping of these polymorphisms in 10 accessions of each of the diploid species (B. oleracea, B. nigra, B. rapa and R. sativus) did not reveal segregating polymorphisms among accessions within species, indicating that they can be used to help determine species identity. Ten accessions of each of the amphidiploids B. napus, B. carinata and B. juncea were genotyped to infer their maternal ancestry. The diploid source of cpDNA in B. carinata was B. nigra in all accessions tested and B. rapa for nine of ten B. juncea accessions tested. Two B. napus accessions amplified polymorphisms shared with B. rapa, and eight accessions produced unique polymorphisms from neither B. rapa, B. oleracea or B. nigra. The polymorphisms identified in this study can be used to help confirm identity of the diploid crucifer species for taxonomic and conservation studies.

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Molecular Species Identification of Candida from Blood Samples of Intensive Care Unit Patients by Polymerase Chain Reaction – Restricted Fragment Length Polymorphism

Journal of Laboratory Physicians ◽

10.4103/0974-2727.98661 ◽

2012 ◽

Vol 4 (01) ◽

pp. 001-004 ◽

Cited By ~ 4

Author(s):

Ramraj Vijayakumar ◽

Sidhartha Giri ◽

Anupma Jyoti Kindo

Keyword(s):

Intensive Care Unit ◽

Polymerase Chain Reaction ◽

Intensive Care ◽

Length Polymorphism ◽

Fragment Length Polymorphism ◽

Chain Reaction ◽

Candida Spp ◽

Blood Samples ◽

Pcr Rflp ◽

Polymerase Chain

ABSTRACT Introduction: Candida spp is an emerging cause of blood stream infections worldwide. Delay in speciation of Candida isolates by conventional methods and resistance to antifungal drugs (especially fluconazole, amphotericin B, etc.) in various Candida species are some of the factors responsible for the increase in morbidity and mortality due to candidemia. So, the rapid detection and identification of Candida isolates from blood is very important for the proper management of patients having candidemia. Materials and Methods: In this study, we have used polymerase chain reaction (PCR) - restriction fragment length polymorphism (RFLP) as a method for the speciation of Candida isolates from blood samples of intensive care unit (ICU) patients. PCR was used to amplify the ITS-1 and ITS-2 regions of Candida spp using universal primers ITS-1 and ITS-4. The amplified product was digested using Msp I restriction enzyme by RFLP. Results and Discussion: The method PCR-RFLP helped in identifying five medically important Candida spp (C. tropicalis, C. albicans, C. parapsilosis, C. krusei and C. glabrata) from blood. This method is rapid, reliable, easy and cost-effective and can be used in routine laboratory diagnostics for the rapid identification of Candida isolates from blood. Conclusion: PCR-RFLP is an easy, rapid and highly valuable tool which can be used in routine diagnostic laboratories to speciate Candida isolates obtained from blood. This rapid method of speciation will help clinicians to decide on empirical therapy in candidemia cases before antifungal susceptibility results are available.

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Profile of follicle-stimulating hormone and polymorphism of follicle-stimulating hormone receptor in Madrasin cattle with ovarian hypofunction

Veterinary World ◽

10.14202/vetworld.2020.879-883 ◽

2020 ◽

Vol 13 (5) ◽

pp. 879-883

Author(s):

Budi Utomo ◽

Emmanuel Djoko Putranto ◽

Amaq Fadholly

Keyword(s):

Fragment Length Polymorphism ◽

Follicle Stimulating Hormone ◽

Restriction Enzymes ◽

Chain Reaction ◽

Blood Samples ◽

Fshr Gene ◽

Polymerase Chain ◽

Genetic Makeup ◽

Restriction Fragment Length ◽

Stimulating Hormone

Background and Aim: The follicle-stimulating hormone (FSH) gene is an essential regulator of fertility in livestock. This study aims to provide information on the genetic makeup of Madrasin cattle experiencing hypofunction by the FSH profile and FSH receptors (FSHR) polymorphism. Materials and Methods: Blood samples were collected from the Bangkalan regency in Indonesia. DNA was isolated and purified following the extraction protocol of polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism. Results: Our results showed that the FSH gene had a band length of 310 bp and produce two alleles (A and B) with restriction enzymes at 250 bp, 230 bp, and 145 bp. Furthermore, the FSHR gene had a band length of 303 bp and produced two homozygous genotypes: GG at bp 239 and CC at bp 188. Conclusion: Based on these differences, there was no change in allele frequency and genotype between Madura and Madrasin cattle due to crossbreeding with Limousin cattle. Thus, further detailed investigations of Madrasin cattle are required to elucidate the profile of the LH and LHR genes.

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Prevalence of Candida bloodstream isolates from patients in two hospitals in Vietnam

Iranian Journal of Microbiology ◽

10.18502/ijm.v11i2.1071 ◽

2019 ◽

Author(s):

Nguyen Duy Bac1 ◽

Le Tran Anh2 ◽

Le Bach Quang ◽

Nguyen Khac Luc ◽

Tran Thi ◽

...

Keyword(s):

Fragment Length Polymorphism ◽

Blood Stream ◽

Blood Cultures ◽

Ho Chi Minh City ◽

Military Hospital ◽

Chain Reaction ◽

Candida Spp ◽

Pcr Rflp ◽

Polymerase Chain ◽

Epidemiological Profile

Background and Objectives: Identification of yeasts provides helpful information for appropriate administration of an- ti-fungal treatments; however, few reports from the Vietnam have been published. This study has been performed to find the prevalence of Candida blood stream isolates from patients in two hospitals in Vietnam. Materials and Methods: Candida spp. were isolated from blood cultures in two hospitals, Vietnam between May 2013 and May 2015. Participating hospitals were 103 Military Hospital, Ha Noi city (550 beds) and Cho Ray Hospital, Ho Chi Minh city (1800 beds). All the bloodstream isolates were identified to species level by the germ tube test and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). In addition, unknown isolates were subjected to PCR sequencing. Results: A total of 93 Candida isolates were isolated from blood cultures during the study period. The results of this study showed that C. tropicalis (n = 47, 50.54%) was the most common agent, followed by Candida albicans/dubliniensis (n = 18, 19.35%), C. parapsilosis (n = 16, 17.20%), C. glabrata (n = 6, 6.45%), C. mesorugosa (n = 5, 5.38%) and C. krusei (n = 1, 1.08%), respectively. Conclusion: The frequency of the non-albicans Candida species in blood is increasing, especially C. tropicalis. Addi- tional investigations should be made to clarify the epidemiological profile of invasive Candida bloodstream in Vietnam.

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Rhizoctonia spp. Recovered from Strawberry Roots in Central Coastal California

Phytopathology ◽

10.1094/phyto.2000.90.4.345 ◽

2000 ◽

Vol 90 (4) ◽

pp. 345-353 ◽

Cited By ~ 33

Author(s):

Frank N. Martin

Keyword(s):

Polymerase Chain Reaction ◽

Fragment Length Polymorphism ◽

Coastal Region ◽

Rflp Analysis ◽

Restriction Enzymes ◽

Molecular Techniques ◽

Chain Reaction ◽

Polymerase Chain ◽

Collection Sites ◽

Restriction Fragment Length

Rhizoctonia spp. were commonly recovered from the roots of strawberry plants growing in nonfumigated soil in the central coastal region of California. With the exception of one multinucleate isolate of R. solani (frequency of recovery of 0.8%), all other isolates were binucleate and were in anastomosis groups (AG) A, G, or I. AGs-A and -I were recovered from all five collection sites, whereas AG-G was recovered from only two sites. AG-A was the most commonly isolated AG, followed by AGs-I and -G. Similar levels of virulence were observed among the different AGs, but differences in virulence were observed among isolates in the same AG. Evaluating anastomosis grouping by pairing isolates recovered from strawberry with known tester isolates did not always yield a positive anastomosis reaction, even though both isolates anastomosed with other members of the same AG. Subsequent investigations with multiple isolates in the same AG from the same collection location confirmed that there was a lack of anastomosis or weak anastomosis reactions for some combinations of pairings, highlighting the need for to use multiple tester isolates or molecular techniques for AG determination. Restriction fragment length polymorphism (RFLP) analysis of a polymerase chain reaction-amplified region of the rDNA was effective for differentiating AGs. Sixteen RFLP groups were observed after cluster analysis with data for the size of the amplified products and fragment sizes after digestion with four restriction enzymes. Although each AG had isolates in multiple RFLP groups, any one individual RFLP group contained isolates of only a single AG. There was no consistent correlation between RFLP group and location of isolate collection.

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Detection of New Ethylene-Producing Bacteria, Pseudomonas syringae pvs. cannabina and sesami, by PCR Amplification of Genes for the Ethylene-Forming Enzyme

Phytopathology ◽

10.1094/phyto.1997.87.12.1192 ◽

1997 ◽

Vol 87 (12) ◽

pp. 1192-1196 ◽

Cited By ~ 24

Author(s):

M. Sato ◽

K. Watanabe ◽

M. Yazawa ◽

Y. Takikawa ◽

K. Nishiyama

Keyword(s):

Pseudomonas Syringae ◽

Fragment Length Polymorphism ◽

Pcr Amplification ◽

Chain Reaction ◽

Pcr Assay ◽

Enzyme Gene ◽

Indigenous Plasmid ◽

Pcr Products ◽

Polymerase Chain ◽

Primer Sets

Strains of Pseudomonas syringae (78 strains and 43 pathovars) and other strains (79) of plant and insect origin were examined for the presence of the ethylene-forming enzyme gene (efe) by polymerase chain reaction (PCR) assay. The sequence of the efe gene of P. syringae pv. phaseolicola PK2 was used to design two primer sets for amplification of the gene. In addition to P.syringae pv. phaseolicola (the “kudzu strain”) and P.syringae pv. glycinea, which were efficient ethylene producers, several strains of P.syringae pvs. sesami and cannabina generated PCR products of the predicted size. A DNA probe of the efe gene, isolated from strain PK2, hybridized to these PCR products, indicating homology to the P.syringae pv. phaseolicola efe gene. PCR restriction fragment length polymorphism analyses suggested that these four pathovars harbor a similar efe gene. Furthermore, the probe hybridized to an indigenous plasmid of P.syringae pv. cannabina, suggesting that the efe gene could be located on a plasmid in this pathovar, but did not hybridize to plas-mids of P.syringae pv. sesami strains. P.syringae pvs. sesami and cannabina strains produced ethylene in King's medium B at levels similar to those of P.syringae pvs. phaseolicola and glycinea. Thus, two new ethylene-producing bacteria were detected by the PCR assay.

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A simple PCR-based method for the rapid and accurate identification of spider mites (Tetranychidae) on cassava

Scientific Reports ◽

10.1038/s41598-020-75743-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Tatiana M. Ovalle ◽

Aymer Andrés Vásquez-Ordóñez ◽

Jenyfer Jimenez ◽

Soroush Parsa ◽

Wilmer J. Cuellar ◽

...

Keyword(s):

Fragment Length Polymorphism ◽

Crop Protection ◽

Restriction Enzymes ◽

Mite Species ◽

Morphological Identification ◽

Chain Reaction ◽

Accurate Identification ◽

Polymerase Chain ◽

Cassava Crop ◽

Mitochondrial Cytochrome Oxidase

Abstract The morphological identification of mites entails great challenges. Characteristics such as dorsal setae and aedeagus are widely used, but they show variations between populations, and the technique is time consuming and demands specialized taxonomic expertise that is difficult to access. A successful alternative has been to exploit a region of the mitochondrial cytochrome oxidase I (COI) gene to classify specimens to the species level. We analyzed the COI sequences of four mite species associated with cassava and classified them definitively by detailed morphological examinations. We then developed an identification kit based on the restriction fragment length polymorphism–polymerase chain reaction of subunit I of the COI gene focused on the three restriction enzymes AseI, MboII, and ApoI. This set of enzymes permitted the simple, accurate identification of Mononychellus caribbeanae, M. tanajoa, M. mcgregori, and Tetranychus urticae, rapidly and with few resources. This kit could be a vital tool for the surveillance and monitoring of mite pests in cassava crop protection programs in Africa, Asia, and Latin America.

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Survey of mutations in prolificacy genes in Santa Ines and Morada Nova sheep

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/1678-4162-9339 ◽

2017 ◽

Vol 69 (4) ◽

pp. 1047-1053

Author(s):

G.M.L. Holanda ◽

J.C. Oliveira ◽

D.M.F. Silva ◽

S.S.N. Rocha ◽

V. Pandolfi ◽

...

Keyword(s):

Restriction Site ◽

Fragment Length Polymorphism ◽

Nucleotide Sequencing ◽

Chain Reaction ◽

Specific Primers ◽

Pcr Rflp ◽

Pcr Products ◽

Polymerase Chain ◽

Santa Inês ◽

Restriction Fragment Length

ABSTRACT Polymorphisms in the BMP-15 gene related to Galway (FecXG) and Inverdale (FecXI) and in the BMPR-1B gene known as Booroola (FecB) mutations were investigated using the Polymerase Chain Reaction - Restriction Fragment Length Polymorphism (PCR-RFLP) method, on sheep from the breeds Santa Inês (n= 574) and Morada Nova (n=282). DNA was extracted and amplified through PCR with specific primers that introduced a restriction site in association with the mutation. The PCR products were submitted to endonucleases. The experiment found no FecXG and FecXI mutations. Six samples of animals with multiple offspring/birth history presented polymorphism for FecB similar to control samples, but this pattern was not confirmed by nucleotide sequencing. Although the absence of these mutations in the studied breeds, other factors related to prolificacy should be investigated to explain the inherent prolificity mechanisms.

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Identitas dan Keragaman Genetik Begomovirus yang Berasosiasi dengan Penyakit Keriting pada Tomat Berdasarkan Teknik Polymerase Chain Reaction (PCR)- Restriction Fragment Length Polymorphism (RFLP)

Jurnal AgroBiogen ◽

10.21082/jbio.v4n1.2008.p9-17 ◽

2016 ◽

Vol 4 (1) ◽

pp. 9 ◽

Cited By ~ 1

Author(s):

Tri Joko Santoso ◽

Sri H. Hidayat ◽

M. Herman ◽

H. Aswidinnoor ◽

Sudarsono Sudarsono

Keyword(s):

Polymerase Chain Reaction ◽

Restriction Fragment Length Polymorphism ◽

Length Polymorphism ◽

Fragment Length ◽

Fragment Length Polymorphism ◽

Restriction Enzymes ◽

Chain Reaction ◽

Polymerase Chain ◽

Leaf Curl Virus ◽

Restriction Fragment Length

Begomoviruses, members of the Geminivirus, are considered as emerging plant viruses. This was due to the increasing incidences and severities of the diseases in a number of economically important crops, including tomato. Genetic diversities of the Begomovirus isolates infecting tomato (Lycopersicon esculentum) of several areas in Indonesia were analyzed by using Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (PCR-RFLP) technique. A 1500 base pairs of PCR fragments amplified by using degenerate primers for Begomovirus was digested using four restriction enzymes, i.e., DraI, EcoRI, RsaI, and PstI. The pattern of RE digested fragments of 8 Begomovirus isolates and the predicted RFLP fragments of the Begomovirus isolates in the GeneBank database were used to determine the genetic identities and diversities among the isolates. Positive results of the PCR amplifications proved that diseased tomato plant samples collected from 8 locations in Java and Sumatra were infected with at least one Begomovirus isolate. The PCR amplification products, which were digested using the four restriction enzymes indicated the presence of polimorfisms among the DNA fragments of the Begomovirus isolates. Identifications of the Begomovirus indicated that the Brastagi, Bogor, Sragen, Ketep, and Boyolali isolates were Tomato Leaf Curl Virus (ToLCV); the isolates from Malang and Blitar isolates were Ageratum Yellow Vein Virus (AYVV), while one isolate from Kaliurang was Tomato Yellow Leaf Curl Virus (TYLCV). Results of the phylogenetic analysis of the 8 Begomovirus isolates based on Begomoviruses from the DNA database indicated that they belonged to three different groups.

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