Isolation and characterization of Listeria monocytogenes from environmental and clinical sources by culture and PCR-RFLP methods

Background and Objectives: Due to the widespread distribution of Listeria monocytogenes in environmental and animal sources and serious clinical complications in human, this study was aimed to isolate L. monocytogenes from water and clin- ical specimens by culture and PCR methods and to investigate the presence of hlyA and inlA virulence genes. Materials and Methods: Water and clinical samples of vaginal and fecal were screened for the presence of L. monocyto- genes by phenotypic and standard biochemical tests. PCR amplification was performed on extracted DNA using primers based on the hlyA and inlA genes. A 733-bp fragment of inlA gene was used for investigation of polymorphism using RFLP analysis. Results: In total, 45 phenotypically and molecularly confirmed L. monocytogenes strains were isolated from different sourc- es including 30 (16.7%) from water, 9 (11.3%) from vaginal swabs and 6 (7.5%) from fecal samples. RFLP analysis of PCR products using AluI and Tsp509I restriction enzymes, generated two profiles with 8 to 10 bands ranging in size from 15 to 210 bp. The majority of water and clinical isolates were classified in profile 2. Conclusion: We demonstrated 45 L. monocytogenes isolates from tested water and clinical samples by phenotypic and mo- lecular tests. The majority of the isolates were classified in the same RFLP profile, showing the water as a potential source of clinical complications in patients in the region of study.

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PCR amplification and sequence analyses of reverse transcriptase-like genes in Crinipellis perniciosa isolates

Fitopatologia Brasileira ◽

10.1590/s0100-41582007000500001 ◽

2007 ◽

Vol 32 (5) ◽

pp. 373-380 ◽

Cited By ~ 3

Author(s):

Jorge F. Pereira ◽

Mariana D.C. Ignacchiti ◽

Elza F. Araújo ◽

Sérgio H. Brommonschenkel ◽

Júlio C.M. Cascardo ◽

...

Keyword(s):

Reverse Transcriptase ◽

Pathogenic Fungus ◽

Pcr Amplification ◽

Restriction Enzymes ◽

Sequence Comparisons ◽

Copy Numbers ◽

A Genome ◽

Close Relationship ◽

Pcr Products ◽

Broom Disease

Reverse transcriptase (RT) sequence analysis is an important technique used to detect the presence of transposable elements in a genome. Putative RT sequences were analyzed in the genome of the pathogenic fungus C. perniciosa, the causal agent of witches' broom disease of cocoa. A 394 bp fragment was amplified from genomic DNA of different isolates of C. perniciosa belonging to C-, L-, and S-biotypes and collected from various geographical areas. The cleavage of PCR products with restriction enzymes and the sequencing of various RT fragments indicated the presence of several sequences showing transition events (G:C to A:T). Southern blot analysis revealed high copy numbers of RT signals, forming different patterns among C-, S-, and L-biotype isolates. Sequence comparisons of the predicted RT peptide indicate a close relationship with the RT protein from thegypsy family of LTR-retrotransposons. The possible role of these retrotransposons in generating genetic variability in the homothallic C. perniciosa is discussed.

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RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb

AJP Renal Physiology ◽

10.1152/ajprenal.1995.268.6.f1224 ◽

1995 ◽

Vol 268 (6) ◽

pp. F1224-F1228 ◽

Cited By ~ 10

Author(s):

P. Borensztein ◽

M. Froissart ◽

K. Laghmani ◽

M. Bichara ◽

M. Paillard

Keyword(s):

Rat Kidney ◽

Pcr Amplification ◽

Restriction Enzymes ◽

Pcr Analysis ◽

Thick Ascending Limb ◽

Rt Pcr ◽

Medullary Thick Ascending Limb ◽

Pcr Products ◽

Polymerase Chain ◽

Nhe Isoforms

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.

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Taxa of Leafhoppers Carrying Phytoplasmas at Sites of Ash Yellows Occurrence in New York State

Plant Disease ◽

10.1094/pdis.2000.84.2.134 ◽

2000 ◽

Vol 84 (2) ◽

pp. 134-138 ◽

Cited By ~ 21

Author(s):

G. T. Hill ◽

W. A. Sinclair

Keyword(s):

New York ◽

New York State ◽

Pcr Amplification ◽

Restriction Enzymes ◽

Disease Group ◽

York State ◽

Aster Yellows ◽

Pcr Products ◽

Polymerase Chain ◽

Abundant Genus

Leafhopper (Homoptera: Cicadellidae) populations were sampled and leafhopper carriers of ash yellows (AshY) phytoplasmas were identified as first steps toward vector identification. Nearly 5,000 leafhoppers were collected in malaise traps at two sites of high AshY incidence in New York state in 1996 and 1997. These insects comprised 33 taxa, including representatives of 13 genera known to contain phytoplasma vectors. The most abundant genus was Scaphoideus, with numbers about six times greater than any other genus. A total of 1,632 insects were assayed individually for phytoplasmas by polymerase chain reaction (PCR) amplification of phytoplasmal 16S rDNA and restriction fragment length polymorphism analyses of PCR products using restriction enzymes TaqI and RsaI separately. Phytoplasmas were detected in 35 insects, all but one in the subfamily Deltocephalinae. AshY phytoplasmas were detected in 19 of 812 individuals of Scaphoideus spp. and 1 of 87 of Colladonus clitellarius. Phytoplasmas of the Prunus X-disease group were detected in 1 Scaphoideus sp., 4 individuals of C. clitellarius, and 4 of 83 Scaphytopius acutus individuals. Phytoplasmas of the aster yellows group were detected in 1 of 68 individuals of Gyponana spp. and 5 of S. acutus. AshY phytoplasma carriers merit testing for possible vector ability.

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Detection and characterization of Leptospira spp. isolated from aborted bovine clinical samples

Acta Veterinaria Brno ◽

10.2754/avb201281010021 ◽

2011 ◽

Vol 81 (1) ◽

pp. 21-25 ◽

Cited By ~ 1

Author(s):

Hassan Momtaz ◽

Saadat Moshkelani

Keyword(s):

Public Health Problem ◽

Rflp Analysis ◽

Restriction Enzymes ◽

Clinical Samples ◽

Dairy Herds ◽

Important Public Health Problem ◽

Pcr Rflp ◽

Beef Herds ◽

Bovine Abortion

Leptospira is recognized as an important public health problem worldwide, especially in tropical countries, and is a common cause of abortion in dairy and beef herds. The aim of the present study was to detect and characterize Leptospira as the causative agent of abortion in cattle using a PCR-RFLP in Chaharmahal va Bakhtiari and Isfahan provinces, Iran. A total of 220 bovine aborted foetuses and 120 vaginal discharges from an aborted calf were collected from 64 commercial dairy herds. After isolation of 60 Leptospira spp. from samples, RFLP analysis was carried out with HindIII and HaeIII restriction enzymes in reference strains and isolated for characterization. In a total of 340 specimens, 46 (20.9%) and 14 (11.66%) were identified positive for Leptospira spp. from aborted bovine foetuses and vaginal discharges, respectively. The present results also suggest that L. interrogans serovar hardjo has the highest prevalence in the region under study and L. hardjo is a major pathogen causing bovine abortion in Chaharmahal va Bakhtiari and Isfahan provinces of Iran.

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A Sida sp. Is a New Host for “Candidatus Phytoplasma brasiliense” in Brazil

Plant Disease ◽

10.1094/pdis-09-10-0647 ◽

2011 ◽

Vol 95 (3) ◽

pp. 363-363 ◽

Cited By ~ 3

Author(s):

B. Eckstein ◽

J. C. Barbosa ◽

J. A. M. Rezende ◽

I. P. Bedendo

Keyword(s):

16S Rdna ◽

Sequence Similarity ◽

Rflp Analysis ◽

Restriction Enzymes ◽

Sao Paulo ◽

São Paulo ◽

New Host ◽

Nucleotide Sequence Alignment ◽

Pcr Products

Sida is a genus of flowering herbs in the family Malvaceae, which includes several species that are weeds in Brazil. Plants of a Sida sp. exhibiting symptoms characterized by stunting, chlorosis, small leaves, and witches'-broom, indicative of infection by phytoplasmas, were found in a field previously cultivated with tomato, located in the region of Campinas, State of São Paulo, in December 2008. To demonstrate the presence of phytoplasmas in diseased tissues, DNA was extracted from shoots and leaves from three symptomatic and eight asymptomatic plants. Nested PCR was performed using primers P1/Tint followed by primer pair R16F2n/R16R2 (1). DNA fragments of 1.2 kb, corresponding to 16S rDNA, were amplified only for DNA from two symptomatic samples. Phytoplasma identification was initially carried out by restriction fragment length polymorphism (RFLP) analysis through digesting the PCR products with the restriction enzymes AluI, HhaI, HaeIII, HpaII, MseI, and RsaI. The two phytoplasma isolates found to be infecting a Sida sp. showed identical RFLP patterns, which were indistinguishable from the phytoplasma previously reported in association with hibiscus (Hibiscus rosa-sinensis) witches'-broom in Brazil (2). Nucleotide sequence alignment revealed that 16S rDNA of both phytoplasma isolates found in a Sida sp. (GenBank Accession No. HQ230579) shared 99.9% sequence similarity with 16S rDNA from hibiscus witches'-broom phytoplasma (HibWB) (GenBank Accession No. AF147708). HibWB is the representative of the 16SrXV group and it was proposed as a putative species nominated “Candidatus Phytoplasma brasiliense” (2). The disease is frequently observed in hibiscus plants used as ornamentals in the states of São Paulo (4) and Rio de Janeiro (2). “Ca. Phytoplasma brasiliense” has only been reported in Brazil to be infecting hibiscus (2,4) and periwinkle (Catharanthus roseus) (3). The presence of a phytoplasma belonging to group 16SrXV in a Sida sp. expands its natural host range. The role of this weed as a potential source of inoculum for crops should be investigated. References: (1) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) H. G. Montano et al. Int. J. Syst. Evol. Microbiol. 51:1109, 2001. (3) H. G. Montano et al. Plant Dis. 85:1209, 2001. (4) E. G. Silva et al. Summa Phytopathol. 35:234, 2009.

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Identification of phytoplasma strains associated with witches’ broom and yellowing in Ziziphus jujube nurseries in Iran

Phytopathologia Mediterranea ◽

10.36253/phyto-10857 ◽

2020 ◽

Vol 59 (1) ◽

pp. 55-61

Author(s):

Ghobad BABAEI ◽

Seyyed Alireza ESMAEILZADEH-HOSSEINI ◽

Mahbobeh ZANDIAN ◽

Vahid NIKBAKHT

Keyword(s):

Restriction Analysis ◽

Rflp Analysis ◽

Restriction Enzymes ◽

Reference Pattern ◽

Consensus Sequences ◽

Rdna Sequences ◽

Rflp Profile ◽

Ziziphus Jujube ◽

East Of Iran ◽

Virtual Rflp

Phytoplasma symptoms, including proliferation, witches’ broom, leaf rolling and yellowing, were observed in jujube (Ziziphus jujube) nurseries in the East of Iran. Total nucleic acid was extracted from symptomatic and symptomless plants, and was tested for phytoplasma presence using nested PCR. Amplicons of about 1.8 kb (primer pair P1/P7) and 1.25 kb (R16F2n/R16R2) were obtained from all symptomatic plants but not from symptomless plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R2 amplicons using KpnI, HaeIII, RsaI, AluI, HpaII, HhaI, TaqI, MseI, BfaI and ThaI restriction enzymes showed two RFLP patterns referable to 16SrI and 16SrVI phytoplasma groups. The consensus sequences of Z. jujube yellowing and witches’ broom of six samples correspond to ‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma trifolii’-related strains. Two R16F2n/R16R2 16S rDNA sequences representative of each RFLP profile, one each from witches’ broom (accession number MK379605) and yellowing (MK379604) host symptoms, were submitted to the GenBank. Phylogenetic analysis confirmed that the phytoplasma strains associated with jujube yellowing clustered within the 16SrI phytoplasma clade, and those associated with witches’ broom clustered within the 16SrVI clade. Restriction analysis confirmed that virtual RFLP patterns of the jujube yellowing and witches’ broom phytoplasma strains were identical to the reference pattern of 16SrI-B and 16SrVI-A. This is the first report of these phytoplasma strains associations with witches’ broom and yellowing in jujube plants.

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Direct Identification in Food Samples of Listeria spp. and Listeria monocytogenes by Molecular Methods

Applied and Environmental Microbiology ◽

10.1128/aem.68.12.6273-6282.2002 ◽

2002 ◽

Vol 68 (12) ◽

pp. 6273-6282 ◽

Cited By ~ 77

Author(s):

Luca Cocolin ◽

Kalliopi Rantsiou ◽

Lucilla Iacumin ◽

Carlo Cantoni ◽

Giuseppe Comi

Keyword(s):

Listeria Monocytogenes ◽

Denaturing Gradient Gel Electrophoresis ◽

Direct Detection ◽

Pcr Amplification ◽

Food Samples ◽

Detection And Identification ◽

Optimized Protocol ◽

Pcr Products ◽

Gradient Gel Electrophoresis ◽

Species Specific

ABSTRACT A new molecular approach for the detection and identification of Listeria spp. and Listeria monocytogenes in food is presented here. The method is based on the PCR amplification of a fragment of the iap gene from the five species belonging to the genus and on the analysis of the PCR products obtained by denaturing gradient gel electrophoresis (DGGE). The protocol was first optimized by using strains from international collections. Based on the differences present in the sequences amplified, it was possible to obtain species-specific DGGE migration that allowed fast and easy identification of L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii. Moreover, for L. monocytogenes serotypes, partial differentiation was possible. The optimized protocol was used for identification of Listeria strains traditionally isolated from food and for direct detection and identification of Listeria members in food after an overnight enrichment. Identification of 48 food isolates and direct detection of Listeria spp. in 73 food samples show the potential of the method that can be used as a fast screening test to investigate the presence of Listeria spp. and L. monocytogenes in food.

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Responses of Six Eurasian Ulmus Cultivars to a North American Elm Yellows Phytoplasma

Plant Disease ◽

10.1094/pdis.2000.84.12.1266 ◽

2000 ◽

Vol 84 (12) ◽

pp. 1266-1270 ◽

Cited By ~ 13

Author(s):

W. A. Sinclair ◽

A. M. Townsend ◽

H. M. Griffiths ◽

T. H. Whitlow

Keyword(s):

Pcr Amplification ◽

Rflp Analysis ◽

Restriction Enzymes ◽

Phloem Necrosis ◽

Fluorescence Test ◽

Polymerase Chain ◽

Elm Yellows ◽

Control Tree ◽

Eurasian Species ◽

Dapi Fluorescence

Elms (genus Ulmus) of six clonal cultivars representing Eurasian species and hybrids were grafted when 2 to 3 years old with bark patches from U. rubra infected with an elm yellows phytoplasma or were left untreated as controls. The cultivars were U. glabra × minor ‘Pioneer’, U. minor × parvifolia ‘Frontier’, U. parvifolia ‘Pathfinder’, U. wilsoniana ‘Prospector’, and the complex hybrids ‘Homestead’ and ‘Patriot’. Trees were evaluated for infection and symptoms 1 or 2 years after inoculation. Infection was detected via the 4′,6-diamidino-2-phenylindol e·2HCl (DAPI) fluorescence test in 26 of 86 grafted trees representing five cultivars. Infection of selected trees was confirmed by polymerase chain reaction (PCR) amplification of a fragment of phytoplasmal rDNA, and the phytoplasma was identified by restriction fragment length polymorphism (RFLP) analysis of the amplified DNA using restriction enzymes AluI, RsaI, and TaqI. Elm yellows phytoplasma was also identified by nested PCR and RFLP analysis in two of seven inoculated, healthy-appearing, DAPI-negative trees and one noninoculated control tree. All RFLP profiles were identical to that of reference strain EY1. Phytoplasma-associated symptoms, observed in five cultivars, included suppressed growth, progressive size reduction of apical shoots and leaves, chlorosis, foliar reddening, witches'-brooms, and dieback. Phyto-plasma was not detected in cv. Homestead. Possible resistance of this cultivar to elm yellows phytoplasma was indicated by localized phloem necrosis in stems below inoculum patches.

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Genotyping of Trichomonas vaginalis isolates from women in Shahrekord city (Southwestern Iran)

Genetika ◽

10.2298/gensr1703059k ◽

2017 ◽

Vol 49 (3) ◽

pp. 1059-1070 ◽

Cited By ~ 1

Author(s):

Bahman Khalili ◽

Payam Ghasemi-Dehkordi ◽

Gholamreza Pourshahbazi ◽

Hossein Yousofi-Darani ◽

Morteza Hashemzadeh-Chaleshtori ◽

...

Keyword(s):

Trichomonas Vaginalis ◽

Accurate Determination ◽

Geographical Area ◽

Rflp Analysis ◽

Restriction Enzymes ◽

Nested Polymerase Chain Reaction ◽

Pcr Products ◽

Southwestern Iran ◽

Different Strains ◽

High Level

Trichomonas vaginalis is a causative agent of vaginitis in female and urethritis in men. It is primarily transmitted by sexually route. It is known that each geographical area has its own set of Trichomonas vaginalis strain. Parasite strains in each region have its specific characterizations and different strains of the parasite are able to cause various diseases with the acuity and severity. The aim of this study was to determine the genotyping of Trichomonas vaginalis strains in the Shahrekord city (Chaharmahal Va Bakhtiari province, southwest Iran). A total of 1725 vaginal samples were taken from clinically suspected women for Trichomonas vaginalis infection and 21 specimens were diagnosed as positive by direct smear wet mount and culture repeated passage of the parasite in the modified TYI-S-33 medium. The genomic DNA was extracted from each sample and the nested polymerase chain reaction was applied using specific oligonucleotide primers for actin gene amplification. Finally, the restriction fragment length polymorphism using RsaI, MseI, and HindII restriction enzymes were done on PCR products for genotyping. PCR-RFLP analysis of 21 positive cases (1.22%) was showing the most frequent genotype was H (8 cases), followed by G (4 cases), E (3 cases), and P (2 cases). N and I genotypes were detected in each 1 case. Also, there was 2 cases mix (E and H) genotype. The findings of the present work were showed 7 different genetic strains in isolated Trichomonas vaginalis from symptomatic and asymptomatic women in Shahrekord city. In this study high level of H genotype in referred women in Shahrekord city was observed and H, G, E, and I genotypes were may be related to burning and itching as well as H, P, and mix genotypes were associated with malodorous discharge with pelvic pain in this region of Iran. For a suggestion, it would be better in further studies the accurate determination of genetic diversity of this parasite done in Chaharmahal Va Bakhtiari province and other parts of the country.

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First Report of an Aster Yellows Subgroup 16SrI-B Phytoplasma Infecting Chayote in Costa Rica

Plant Disease ◽

10.1094/pdis.2002.86.3.330c ◽

2002 ◽

Vol 86 (3) ◽

pp. 330-330 ◽

Cited By ~ 3

Author(s):

W. Villalobos ◽

L. Moreira ◽

C. Rivera ◽

K. D. Bottner ◽

I.-M. Lee

Keyword(s):

Costa Rica ◽

Rflp Analysis ◽

Restriction Enzymes ◽

Potential Threat ◽

Nested Polymerase Chain Reaction ◽

Aster Yellows ◽

First Report ◽

Rdna Sequences ◽

Pcr Products ◽

Production Area

An outbreak of a witches' broom disease affected approximately 20% of plants in several chayote (Sechium edule (Jacq.) Schwartz) fields in the commercial production area of the Ujarrás Valley, Cartago Province, Costa Rica during 2000 and 2001. Affected chayote plants exhibited symptoms, including basal proliferation with severe foliage reduction, aborted flowers, and deformed fruits, suggestive of phytoplasmal infection. Two other symptomatic cucurbit species growing near the chayote fields were also identified. These species were tacaco plants (S. tacaco (Pitt.) C. Jeffrey), an edible cucurbit for domestic marketing in Costa Rica, showing severe size reduction of leaves and fruits, and Rytidostylis carthaginensis (Jacq.) Kuntze, a weed in chayote and tacaco fields, exhibiting abnormal tendril proliferation. Plants were analyzed for phytoplasma infection by a nested polymerase chain reaction (PCR) assay, using the universal rRNA primer pair P1/P7 followed by R16F2n/R16R2 (2). Phytoplasmas were detected in all symptomatic samples (18 chayote, 6 tacaco, and 3 weed) tested but were undetectable in all asymptomatic samples (10 chayote, 6 tacaco, and 2 weed). Restriction fragment length polymorphism (RFLP) analysis of PCR products (16S rDNA sequences) by separate digestion with eight restriction enzymes (RsaI, HhaI, KpnI, BfaI, HaeIII, HpaII, AluI, MseI) revealed that a phytoplasma belonging to subgroup 16SrI-B in the aster yellows phytoplasma group (16SrI) was associated with chayote witches' broom (CWB). The same or very similar phytoplasmas were found in both symptomatic tacaco and R. carthaginensis plants. Phylogenetic analysis of 16SrDNA sequences also confirmed the CWB phytoplasma to be most similar to members of subgroup 16SrI-B. Similar diseases in chayote and other cucurbits have been reported in Brazil (3), Taiwan (1), and Mexico (4). The CWB phytoplasma differs from the phytoplasma (16SrIII-J subgroup) associated with chayote in Brazil. The identities of phytoplasmas associated with cucurbits in Taiwan and Mexico are unknown. The occurrence of an aster yellows group phytoplasma in chayote may pose a potential threat to continued production and exportation of this cash crop. To our knowledge, this is the first report of 16SrI-B subgroup phytoplasmas in naturally infected cucurbits in Costa Rica. References: (1) T. G. Chou et al. Plant Dis. Rep. 60:378, 1976. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) H. G. Montano et al. Plant Dis. 84:429, 2000. (4) E. Olivas. Rev. Fitopatol. (Lima) 13:14, 1978.

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