scholarly journals Laboratory Evidence of Norwalk Virus Contamination on the Hands of Infected Individuals

2013 ◽  
Vol 79 (24) ◽  
pp. 7875-7881 ◽  
Author(s):  
Pengbo Liu ◽  
Blanca Escudero ◽  
Lee-Ann Jaykus ◽  
Julia Montes ◽  
Rebecca M. Goulter ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Rna Extraction ◽  
High Pressure Processing ◽  
Norwalk Virus ◽  
Dot Blot ◽  
Definitive Evidence ◽  
Reverse Transcription Quantitative Pcr ◽  
Outbreak Investigations ◽  
Pcr Products ◽  
Polyethylene Glycol Precipitation

ABSTRACTHuman norovirus (NoV) outbreak investigations suggest that the hands of infected individuals play an important role in NoV transmission. However, there is no experimental evidence documenting the likelihood and degree of NoV contamination on hands. As part of a clinical trial designed to evaluate the efficacy of high-pressure processing for Norwalk virus (NV) inactivation in oysters, 159 hand rinse samples were collected from 6 infected and 6 uninfected subjects. NV was concentrated from the samples by polyethylene glycol precipitation, followed by RNA extraction using an automated guanidinium isothiocyanate-silica method. NV RNA was detected and quantified using multiple NV-specific reverse transcription-quantitative PCR (RT-qPCR) assays. A total of 25.4% (18/71) of the hand rinse samples collected from 6 infected volunteers were presumptively positive for NV, with an average of 3.86 log10genomic equivalent copies (GEC) per hand. Dot blot hybridization of PCR products obtained using a different primer set, and DNA sequencing of selected amplicons, provided further confirmation of the presence of NV in the hand rinses. NV contamination was also detected in two hand rinse samples obtained from one uninfected subject. These findings provide definitive evidence of NV contamination on the hands of infected subjects observed under controlled clinical research conditions. Such data support the need for better hand hygiene strategies to prevent NoV transmission.

HLA class II genotyping: two assay systems compared

1995 ◽  
Vol 41 (4) ◽  
pp. 553-556 ◽  
Author(s):  
J Thonnard ◽  
F Deldime ◽  
M Heusterspreute ◽  
B Delepaut ◽  
F Hanon ◽  
...  
Keyword(s):  
Large Scale ◽  
Clinical Laboratory ◽  
Blot Hybridization ◽  
Human Leukocyte ◽  
Hla Typing ◽  
High Rate ◽  
Polymorphism Analysis ◽  
Dot Blot ◽  
Pcr Products ◽  
Typing Methods

Abstract In the last few years, a variety of DNA-based human leukocyte antigen (HLA) typing methods have emerged, revealing the extreme polymorphism of HLA genes. This polymorphism makes it difficult for a clinical laboratory to establish the best HLA typing strategy. In this study we have compared two techniques for performing HLA-DRB typing: a commercial rapid assay based on the polymerase chain reaction (PCR) followed by reverse dot-blot hybridization of the PCR products (the Inno-LiPA assay), and a method based on PCR followed by restriction fragment length polymorphism analysis. We found that both methods provide reliable results with a high rate of concordance (97%) and that Inno-LiPA is convenient for large-scale routine typing. However, if a high-resolution allelic typing is required, each method lacks accuracy but using them in association improves the accuracy of the results.

scholarly journals First Report of Coleus blumei viroid 5 from Coleus blumei in India and Indonesia

Plant Disease ◽  
2013 ◽  
Vol 97 (4) ◽  
pp. 561-561 ◽  
Author(s):  
D. M. Jiang ◽  
S. F. Li ◽  
F. H. Fu ◽  
Z. J. Wu ◽  
L. H. Xie
Keyword(s):  
Blot Hybridization ◽  
Rna Extraction ◽  
Reference Sequence ◽  
Rt Pcr ◽  
Dot Blot ◽  
First Report ◽  
Coleus Blumei ◽  
Infectious Clones ◽  
Three Samples ◽  
Dark Green

Coleus blumei, which was found originally in Indonesia, is an ornamental plant grown worldwide. It can be infected by several viroids of the genus Coleviroid, family Pospiviroidae. Six main viroids that infect coleus have been reported: Coleus blumei viroid 1 through 6 (CbVd-1 ~ CbVd-6). Although CbVd-1 was first reported in a commercial coleus in Brazil in 1989 (1), and then in Germany, Japan, Canada, Korea, China, and India, CbVd-5 was reported only in China in 2009 (2). Symptoms caused by CbVd-5 varied depending on different cultivars, and in case of an unknown cultivar of “Red with dark green edge,” are very clear albino symptoms. From 2010 to 2011, 60 and 3 leaf samples of coleus were collected from Hyderabad, India, and Java, Indonesia, respectively, and subjected to low molecular weight RNA extraction according to Li et al. (3). The results of dot-blot hybridization using CbVd-5 cRNA probes and RT-PCR using CbVd-5 specific primers (CbVd-5-PF: 5′-TGACTAGAACAGTAGTAAAG-3′ / CbVd-5-PR: 5′-AATTGAGGTCAAACCTCTTT-3′) demonstrated that 28 out of the 60 samples from India and all three samples from Indonesia were positive for CbVd-5. The resulting RT-PCR fragments from one sample selected randomly from each country were cloned into the pMD18-T vector (Takara) and transformed into E. coli DH5α competent cells. Five positive clones of each sample were sequenced. The result of sequence analysis revealed that the similarities of CbVd-5 between the sequences we obtained and the reference sequence (GenBank Accession No. NC003683) were 97.8 to 100%. Bioassay using nine viroid-free coleus plants from three cultivars (three from each cultivar), inoculated with CbVd-5 infectious clones by stem slashing, demonstrated that CbVd-5 could induce albino symptom on the leaves of the unknown cultivar “Red with dark green edge” 2 months after inoculation. To our knowledge, this is the first report of CbVd-5 from India and Indonesia, and the second report of CbVd-5 in the world. Considering the effect of CbVd-5 on the appearance of coleus and its recombination ability, a certification program may be needed to control the spread of this viroid. References: (1) M. E. N. Fonseca et al. Fitopatol. Bras. 14:94, 1989. (2) W. Y. Hou et al. Arch. Virol. 154:315, 2009. (3) S. F. Li et al. Ann. Phytopathol. Soc. Jpn. 61:381, 1995.

scholarly journals Cucurbit leaf crumple virus Identified in Common Bean in Florida

Plant Disease ◽  
2009 ◽  
Vol 93 (3) ◽  
pp. 320-320 ◽  
Author(s):  
S. Adkins ◽  
J. E. Polston ◽  
W. W. Turechek
Keyword(s):  
Common Bean ◽  
Blot Hybridization ◽  
Cdna Probe ◽  
Hypervariable Region ◽  
Rt Pcr ◽  
Dot Blot ◽  
Fresh Market ◽  
Degenerate Primers ◽  
Pcr Products ◽  
Cucurbit Leaf Crumple Virus

Virus-like symptoms of leaf deformation and rugosity, especially of younger leaves, and a mild mosaic were observed on fresh market common (green) bean (Phaseolus vulgaris L.) plants in Hendry County in southwest Florida in December of 2007 and again in February of 2008. All bean fields were adjacent to watermelon fields in which Cucurbit leaf crumple virus (CuLCrV), Squash vein yellowing virus (SqVYV), and Papaya ringspot virus type W (PRSV-W) infections had previously been confirmed (fall of 2007) by PCR, reverse transcription (RT)-PCR, and/or ELISA. Whiteflies, Bemisia tabaci, were observed on both bean and watermelon plants in December and February. Fifteen samples (eleven with symptoms) were collected in December and two (both with symptoms) in February. Initial ELISA assays using commercially available antisera for potyviruses or Cucumber mosaic virus (Agdia, Elkhart, IN) were negative. Total nucleic acids were extracted and used for PCR testing. All samples tested negative by RT-PCR using specific primers for SqVYV, PRSV-W, and Cucurbit yellow stunting disorder virus, and degenerate primers for potyviruses. Ten of fifteen December samples (ten of eleven symptomatic samples) and both February samples yielded PCR products of the expected size with the degenerate begomovirus primers, PAR1c496/PAL1v1978, which amplify a portion of the begomovirus A component (3). PCR products from three December and both February samples were cloned and sequenced. The 1,159-nt PCR products shared 99% identity with each other and 96% identity with the corresponding region of A component sequences of Arizona and California CuLCrV isolates (GenBank Accession Nos. AF256200 and AF224760, respectively). Additional degenerate begomovirus primers PBL1v2040/PCRc154, which amplify a 381-nt portion of the hypervariable region of the begomovirus B component (3), and AC1048/AV494, which amplify a 533-nt portion of a conserved region of the coat protein gene (4), were used to confirm the identity of CuLCrV in the three December samples. The PBL1v2040/PCRc154 PCR products shared 98 to 99% identity with each other and 94 to 95% identity with the corresponding region of B component sequences of Arizona and California CuLCrV isolates (GenBank Accession Nos. AF327559 and AF224761, respectively), whereas the AC1048/AV494 PCR products shared 99% identity with each other and 97% identity with the corresponding region of A component sequences of Arizona and California CuLCrV isolates. Nucleic acid dot-blot hybridization assays of sap from homogenized leaves of the three December samples (from which the PCR product clones were obtained) with a digoxigenin-labeled CuLCrV cDNA probe also confirmed the presence of CuLCrV. Although CuLCrV has been reported to experimentally infect common bean and tobacco (2), to our knowledge, this is the first report of CuLCrV infecting any noncucurbit host in Florida. This finding suggests that CuLCrV may be more widely distributed than previously known in Florida (1) and that common bean (and potentially other legumes) are potential reservoirs for CuLCrV. References: (1) F. Akad et al. Plant Dis. 92:648, 2008. (2) J. K. Brown et al. Phytopathology 92:734, 2002. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

Genetic homogeneity within Leishmania (L.) infantum isolated from human and dogs: the relationship with the sandfly fauna distribution in endemic areas of Nueva Esparta State, Venezuela

Parasitology ◽  
2005 ◽  
Vol 130 (6) ◽  
pp. 611-619 ◽  
Author(s):  
N. M. RODRIGUEZ ◽  
Z. DE GUGLIELMO ◽  
M. A. BARRIOS ◽  
R. M. BARRIOS ◽  
O. ZERPA ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Restriction Analysis ◽  
Blot Hybridization ◽  
Lutzomyia Longipalpis ◽  
Dot Blot ◽  
Dot Blot Hybridization ◽  
Pcr Products ◽  
Species Specific ◽  
The Relationship

Leishmania infantum has been described as a highly polymorphic group of parasites, responsible for visceral leishmaniasis and cutaneous leishmaniasis. In this paper we report the life-cycle of L. (L.) infantum in an endemic area of visceral leishmaniasis in Venezuela, by using molecular diagnosis and characterization of parasites isolated from dogs, humans with visceral leishmaniasis and sand flies. The molecular characterization was carried out by use of kDNA restriction analysis, dot-blot hybridization with species-specific probes and RFLP of the PCR products. The results demonstrated that L. (L.) infantum is the parasite responsible for VL in the island. The parasites were revealed to be genetically homogeneous with no intra-specific differences between isolates from different individuals. The highest homology of the isolates was with L. (L.) infantum from the Old World rather than with L. (L.) chagasi from the New World. Additionally, we report the geographical distribution of Lutzomyia longipalpis, and the relationship with the transmission of L. (L.) infantum in the studied area.

scholarly journals Pepper huasteco virus and Pepper golden mosaic virus are Geminiviruses Affecting Tomatillo (Physalis ixocarpa) Crops in Mexico

Plant Disease ◽  
2001 ◽  
Vol 85 (12) ◽  
pp. 1291-1291 ◽  
Author(s):  
J. Méndez-Lozano ◽  
R. F. Rivera-Bustamante ◽  
C. M. Fauquet ◽  
R. De la Torre-Almaraz
Keyword(s):  
Mosaic Virus ◽  
Blot Hybridization ◽  
Amino Acid Level ◽  
Dot Blot ◽  
Stem Loop ◽  
Horticultural Crops ◽  
Hybridization Procedure ◽  
Pcr Products ◽  
Main Production ◽  
Production Areas

Whitefly-transmitted geminivirus diseases cause important losses in several horticultural crops in all areas in Mexico (1). Tomatillo is important in the Mexican diet since it is widely used to prepare many types of salsas and other dishes. As a result, tomatillo, also known as tomate verde (green tomato), is cultivated in 29 of 32 states in Mexico, with the main production areas located in the states of Morelos, Puebla, and Michoacán. Leaf samples of 105 tomatillo plants exhibiting yellowing, yellowing mosaic, leaf curl, bunchy top, and stunting were collected from the states of Puebla, Morelos, Estado de México, and Sinaloa. Symptomatic plants were associated with the presence of whiteflies in many fields and suggested a viral etiology. Total DNA extracted from symptomatic tomatillo plants was used as a template in a polymerase chain reaction (PCR)-based geminivirus detection procedure. MP16 and MP82 primers (2) were used to direct the amplification of a segment from the stem-loop structure in the intergenic region (IR) to a conserved region in the coat protein (CP) of begomoviruses (2). Sixty-nine percent (72/105) of the samples produced the expected PCR fragment (400 to 450 bp). Similar results were obtained with a dot-blot hybridization procedure using as a probe the component A of Pepper huasteco virus (PHV) under low stringency conditions. More than 50 PCR products were cloned and sequenced. Sequence analysis (nucleotide level for the IR; amino acid level for the CP) revealed that the tomatillo-infecting geminiviruses clustered into two main groups. The first group showed a high percent identity (average of 95.3% at the CP N terminus) to PHV, whereas the second showed a similarly high percent (average 93.8%) identity to Pepper golden mosaic virus (PepGMV, previously called Texas pepper geminivirus. Both PepGMV and PHV were found in all sampled areas. Although mixed infections (differentiated by the respective IR probes) of PHV and PepGMV were common (61%), single infections were also detected (PHV 27%; PepGMV 10%). The presence of begomoviruses in tomatillo crops has been previously reported (1); however, their identity as PHV and PepGMV was not confirmed. References: (1) I. Torres-Pacheco et al. Phytopathology 86:1186, 1996. (2) P. Umaharan et al. Phytophatology 88:1262, 1998.

scholarly journals Characterization of a Gene Identified in Pathotype 5 of the Clubroot Pathogen Plasmodiophora brassicae

2015 ◽  
Vol 105 (6) ◽  
pp. 764-770 ◽  
Author(s):  
H. Zhang ◽  
J. Feng ◽  
V. P. Manolii ◽  
S. E. Strelkov ◽  
S.-F. Hwang
Keyword(s):  
Plasmodiophora Brassicae ◽  
Blot Hybridization ◽  
Field Isolate ◽  
Pcr Analysis ◽  
In Planta ◽  
Dot Blot ◽  
Reverse Transcription Quantitative Pcr ◽  
Polymerase Chain ◽  
Differential Hosts

Clubroot caused by Plasmodiophora brassicae is an important disease of crucifers worldwide. Isolates of the pathogen can be classified into pathotypes according to their pathogenicity on differential hosts. In this study, the presence or absence of all database-available nonhousekeeping P. brassicae genes (118 in total) were assessed by polymerase chain reaction (PCR) analysis in isolates belonging to five P. brassicae pathotypes (2, 3, 5, 6, and 8 according to Williams’ differential set). One gene, designated Cr811, was present exclusively in the isolate of pathotype 5. This was further confirmed by dot blot hybridization and by PCR using alternative DNA preparations and primers. Reverse transcription quantitative PCR analysis indicated that in planta expression of Cr811 was up-regulated during canola infection, especially in the stage of secondary plasmodia. Primers specific to Cr811 could distinguish a field isolate of P. brassicae belonging to pathotype 5 from two other field isolates representing pathotypes 3 and 8. These findings suggest that Cr811 is a gene that is potentially involved in clubroot pathogenesis and that it also might serve as a molecular marker for differentiation of pathotype 5 from other pathotypes.

scholarly journals First Report of a Geminivirus Inducing Yellow Mottle in Okra (Abelmoschus esculentus) in Mexico

Plant Disease ◽  
2003 ◽  
Vol 87 (2) ◽  
pp. 202-202
Author(s):  
R. De La Torre-Almaráz ◽  
A. C. Monsalvo-Reyes ◽  
R. F. Rivera-Bustamante ◽  
J. Méndez-Lozano
Keyword(s):  
Blot Hybridization ◽  
Fruit Production ◽  
Dot Blot ◽  
Sequence Comparisons ◽  
First Report ◽  
Squash Leaf Curl Virus ◽  
Pcr Products ◽  
Yellow Mottle ◽  
Total Dna ◽  
Leaf Curl Virus

Okra is an annual vegetable species native to Africa. In Mexico, the states of Tamaulipas, Guerrero, and Morelos contain the most important okra-producing areas. Viral-like diseases have recently affected the fruit production. In the field, the most common symptoms encountered include yellow streak, distortion of fruits, and foliar yellow mottle. Total DNA extracts from symptomatic okra plants were used as a template for polymerase chain reaction (PCR)-based detection using begomovirus-specific primers. RepMot and CPMot primers (1) were used for the amplification of DNA fragments that included the Rep and coat protein (CP) genes of begomoviruses. PCR results suggested the presence of a begomovirus in symptomatic plants. Southern and dot blot hybridization analysis were performed using a DNA fragment containing the CP gene of Pepper huasteco virus as a probe. Hybridization conducted under low stringency conditions confirmed the presence of a geminivirus. Additionally, transmission by grafting and biolistic (total DNA extracts from symptomatic plants) inoculations induced consistently severe streak fruits and yellow mottle symptoms in okra plants. Cloning of the PCR products (approximately 632-bp fragment) was performed in the PCRTopo vector (Invitrogen, San Diego, CA). Cloned viral inserts were sequenced (Genbank Accession No. AF349113). Nucleotide sequence comparisons were performed using the Clustal Method (MegAlign, DNAStar software, Madison, WI) with the GenBank database. Analysis of the PCR products confirmed the begomovirus nature of the sequence. The first 64 amino acids of the CP had 89% identity with Squash leaf curl virus while the intergenic region had 85% identity with Sida golden mosaic virus. To our knowledge, this is the first report of a begomovirus infecting okra in Mexico. Reference: 1) J. T. Ascencio et al. Plant Dis. 86:692, 2002.

Construction and characterization of chromosome 1B specific DNA library of wheat

2005 ◽  
Vol 85 (2) ◽  
pp. 309-316
Author(s):  
Fa-Yun Zhang ◽  
Wei-Bo Yin ◽  
Rui Shi ◽  
Ying-Kao Hu ◽  
Yue-Ming Yan ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Wheat Chromosome ◽  
Single Copy ◽  
Triticum Aestivum L ◽  
Dot Blot ◽  
Insert Size ◽  
Pcr Products ◽  
Chromosome Specific Library

Chromosome 1B was microdissected and collected from chromosome spreads of wheat (Triticum aestivum L. ‘Jing 411’), using a glass needle. DNA of the isolated chromosome was amplified in vitro by Sau3A linker adaptor-mediated polymerase chain reaction (LA-PCR). The second-round PCR products were verified by Southern hybridization using DIG-labeled genomic DNA of wheat. The results initially showed the DNA was from wheat genome. A pair of SSR primers specific to chromosome 1B was used to verify the origin of the PCR products from the isolated chromosome. The results confirmed that the PCR products originated from chromosome 1B. The second round of PCR products from chromosome 1B were cloned into plasmid pUCm-T vectors to create a chromosome-specific library, which included approximately 248 000 recombinant clones. Characterization of 100 randomly selected clones of the library showed that the insert size ranged between 0.5 and 2.0 kb, with an average of 1 kb. Randomly selected 288 clones were characterized with dot blot hybridization, of which 57.2% were medium/high copy clones and 42.8% low/single copy clones. The application of this technique to establish high-density molecular maps for chromosome 1B is discussed. Key words: Wheat, chromosome microdissection, chromosome-specific library

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