scholarly journals Simultaneous detection of scale drop disease virus and Flavobacterium columnare from diseased freshwater-reared barramundi Lates calcarifer

2020 ◽  
Vol 140 ◽  
pp. 119-128
Author(s):  
P Kerddee ◽  
HT Dong ◽  
P Chokmangmeepisarn ◽  
C Rodkhum ◽  
P Srisapoome ◽  
...  
Keyword(s):  
Disease Virus ◽  
Dna Sequences ◽  
Simultaneous Detection ◽  
Flavobacterium Columnare ◽  
Lates Calcarifer ◽  
Specific Pcr ◽  
Liver And Kidney ◽  
Gill Filaments ◽  
Extensive Necrosis ◽  
Basophilic Inclusion

Freshwater farming of barramundi Lates calcarifer in Thailand is a growing sector in aquaculture, but mortalities due to infectious diseases are still a major threat to this industry. In 2018, an episode of severe mortality in juvenile barramundi was noted in a freshwater earth pond site. Fish presented with severe gill necrosis, as well as severe cutaneous hemorrhages, scale loss, and discoloration at the base of dorsal fin (saddleback lesions). Histopathology revealed extensive necrosis of skeletal muscle and gill filaments, as well as basophilic inclusion bodies and megalocytosis in muscle, gill, liver, and kidney. Scale drop disease virus (SDDV) infection was subsequently confirmed by virus-specific semi-nested PCR. Further, DNA sequences of the viral major capsid protein (MCP) and ATPase genes had a respective homology of 99.85 and 99.92% with sequences of SDDV infecting barramundi in saltwater culture. Gill necrosis and saddleback lesions are not typical lesions associated with scale drop syndrome. Their presence was explained by Flavobacterium columnare isolation from the gill, followed by positive F. columnare-specific PCR. To our knowledge, this is the first report of SDDV-associated mortality in freshwater-farmed barramundi. Furthermore, this mortality presented as a concurrent infection with SDDV and F. columnare, with typical lesions of both infections.

A PCR Method Based on 16S rRNA Sequence for Simultaneous Detection of the Genus Listeria and the Species Listeria monocytogenes in Food Products

2003 ◽  
Vol 66 (9) ◽  
pp. 1658-1665 ◽  
Author(s):  
LILACH SOMER ◽  
YECHEZKEL KASHI
Keyword(s):  
Listeria Monocytogenes ◽  
Dna Sequences ◽  
Simultaneous Detection ◽  
Food Products ◽  
Positive Control ◽  
Sensitive Detection ◽  
Rrna Gene ◽  
16S Rrna Sequence ◽  
Selective Enrichment ◽  
Specific Pcr

The genus Listeria comprises six closely related species, of which only Listeria monocytogenes is a human pathogen. The rapid and sensitive detection of L. monocytogenes is important in the food industry as well as in medical diagnosis. In this study, a PCR-based method for the rapid, specific, and sensitive detection of L. monocytogenes in food products was developed. The PCR is based on DNA sequences and primer pairs that are found within the 16S subunit of the rRNA gene and are specific to the Listeria genus and to L. monocytogenes within the Listeria genus. The primers for the Listeria genus and for L. monocytogenes were used in the same reaction mix for their simultaneous detection. In addition, a pair of bacterial primers universal to any bacterial DNA at the 16S subunit of the rRNA gene were developed as a positive control. For the detection of Listeria and L. monocytogenes in food products, the method includes selective enrichment for Listeria followed by DNA extraction and a specific PCR reaction. The method detects 1 to 5 CFU in a 25-g sample in ≤24 h. It can be easily incorporated into the routine screening of diverse food products and readily adapted for clinical use.

A validated semi-nested PCR for rapid detection of scale drop disease virus (SDDV) in Asian sea bass (Lates calcarifer)

2019 ◽  
Vol 268 ◽  
pp. 37-41 ◽  
Author(s):  
Onanong Charoenwai ◽  
Watcharachai Meemetta ◽  
Molrudee Sonthi ◽  
Ha Thanh Dong ◽  
Saengchan Senapin
Keyword(s):  
Disease Virus ◽  
Nested Pcr ◽  
Rapid Detection ◽  
Sea Bass ◽  
Lates Calcarifer ◽  
Asian Sea Bass

scholarly journals New PCR Primer Pairs Specific for Cryptococcus neoformans Serotype A or B Prepared on the Basis of Random Amplified Polymorphic DNA Fingerprint Pattern Analyses

1999 ◽  
Vol 37 (2) ◽  
pp. 315-320 ◽  
Author(s):  
Francisco Hideo Aoki ◽  
Tamae Imai ◽  
Reiko Tanaka ◽  
Yuzuru Mikami ◽  
Hideaki Taguchi ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Dna Sequences ◽  
Random Amplified Polymorphic Dna ◽  
Pcr Primers ◽  
Dna Fingerprint ◽  
Serotype A ◽  
Specific Pcr ◽  
Fingerprint Pattern ◽  
Serotype B ◽  
Pcr Primer

Thirty-three strains of Cryptococcus neoformans were isolated from clinical specimens, including specimens from AIDS patients in Brazil, and were classified into two serotypes; we detected 31 and 2 strains of serotypes A and B, respectively. Random amplified polymorphic DNA (RAPD) fingerprint pattern analyses of these strains of serotypes A and B showed that the patterns were similar for strains of each serotype when three 10-mer primers were used as the RAPD primers. Comparative studies of the fingerprint patterns of the study isolates with those of the reference strains also showed that the RAPD patterns for strains of each serotype were related and that most of the fingerprint bands existed commonly for all strains of each serotype tested. The common RAPD bands (an approximately 700-bp band for serotype A and an approximately 450-bp band for serotype B) were extracted and the DNA sequences were determined. Using this information, we prepared two and one PCR primer pairs which were expected to be specific for C. neoformans serotypes A and B, respectively. Use of each PCR primer combination thus prepared for serotype A or B was 100% successful in identifying the respectiveC. neoformans serotypes, including the 33 clinical isolates tested in the present study. Among these combinations, one for serotype A was found to amplify DNA from C. neoformans serotype B as well as serotype A. Serotype B-specific PCR primer pairs amplified DNA from not only serotype B strains but also from serotype C strains. The usefulness of other serotype-specific PCR primers for clinical C. neoformans isolates is discussed.

scholarly journals Identification and Characterization of Benzimidazole Resistance in Monilinia fructicola from Stone Fruit Orchards in California

2003 ◽  
Vol 69 (12) ◽  
pp. 7145-7152 ◽  
Author(s):  
Zhonghua Ma ◽  
Michael A. Yoshimura ◽  
Themis J. Michailides
Keyword(s):  
Dna Sequences ◽  
Point Mutations ◽  
Brown Rot ◽  
Stone Fruit ◽  
Monilinia Fructicola ◽  
Tubulin Gene ◽  
Benzimidazole Fungicides ◽  
Specific Pcr ◽  
Allele Specific ◽  
Fruit Orchards

ABSTRACT Low and high levels of resistance to the benzimidazole fungicides benomyl and thiophanate-methyl were observed in field isolates of Monilinia fructicola, which is the causative agent of brown rot of stone fruit. Isolates that had low levels of resistance (hereafter referred to as LR isolates) and high levels of resistance (hereafter referred to as HR isolates) were also cold and heat sensitive, respectively. Results from microsatellite DNA fingerprints showed that genetic identities among the populations of sensitive (S), LR, and HR isolates were very high (>0.96). Analysis of DNA sequences of theβ -tubulin gene showed that the LR isolates had a point mutation at codon 6, causing a replacement of the amino acid histidine by tyrosine. Codon 198, which encodes a glutamic acid in S and LR isolates, was converted to a codon for alanine in HR isolates. Based on these point mutations in the β-tubulin gene, allele-specific PCR assays were developed for rapid detection of benzimidazole-resistant isolates of M. fructicola from stone fruit.

scholarly journals Detection of Canine Parvovirus in Naturally Infected Dogs with Enteritis and Myocarditis by in Situ Hybridization

1997 ◽  
Vol 9 (3) ◽  
pp. 255-260 ◽  
Author(s):  
Whan-Gook Nho ◽  
Jung-Hyang Sur ◽  
Alan R. Doster ◽  
Soon-Bok Kim
Keyword(s):  
In Situ Hybridization ◽  
Capsid Protein ◽  
Dna Sequences ◽  
Middle Part ◽  
Canine Parvovirus ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Liver And Kidney ◽  
Labeled Probe

An improved method for the diagnosis of canine parvovirus using in situ hybridization in standard formalin-fixed, paraffin-embedded tissue sections was developed. A digoxigenin-labeled probe complementary to DNA sequences that code for the entire sequence of the capsid protein VP-1 and the middle part of the sequence of the capsid protein VP-2 was designed. Specific histologic localization of canine parvovirus-infected cells was demonstrated in small intestine, tonsil, lymph node, thymus, spleen, heart, liver, and kidney from dogs diagnosed at necropsy with canine parvovirus infection. The in situ hybridization accurately pinpointed the specific sites of viral infection. The detection of canine parvovirus in liver, kidney, and heart tissues together in the same pups could represent an enhanced virulence of this strain of canine parvovirus and suggests a broadened tissue tropism not seen before in Korean strains of canine parvovirus.

scholarly journals Allele-Specific PCR for the Detection of Azoxystrobin Resistance in Didymella bryoniae

Plant Disease ◽  
2014 ◽  
Vol 98 (12) ◽  
pp. 1681-1684 ◽  
Author(s):  
Mavis J. Finger ◽  
Venkatesan Parkunan ◽  
Pingsheng Ji ◽  
Katherine L. Stevenson
Keyword(s):  
Dna Sequences ◽  
Cyt B ◽  
Didymella Bryoniae ◽  
Specific Pcr ◽  
Pcr Product ◽  
Allele Specific ◽  
Polymerase Chain ◽  
Sensitive Allele ◽  
Cyt B Gene ◽  
Allele Specific Pcr

Gummy stem blight (GSB), caused by the fungus Didymella bryoniae, is considered the most widespread and destructive disease of watermelon in the southeastern United States. The quinone outside-inhibiting (QoI) fungicide azoxystrobin (AZO), which inhibits mitochondrial respiration by binding to the outer, quinone-oxidizing pocket of the cytochrome bc1 (cyt b) enzyme complex, was initially very effective in controlling GSB. However, resistance to AZO has been observed in D. bryoniae in many watermelon-producing regions. In this study, the DNA sequences of partial cyt b genes of four AZO-resistant (AZO-R) and four AZO-sensitive (AZO-S) isolates of D. bryoniae confirmed the amino acid substitution of glycine by alanine at the 143 codon (G143A) in the AZO-R isolates tested. Allele-specific primers were designed to detect the resistant or sensitive allele at codon 143 of the cyt b gene, which amplified a 165-bp polymerase chain reaction (PCR) product from genomic DNA of nine AZO-R and nine AZO-S isolates of D. bryoniae, respectively. The primer pairs did not amplify DNA from other pathogens tested in the study. The results indicated that the PCR assays developed in the study were specific in differentiating AZO-R and AZO-S isolates and could facilitate AZO resistance detection in D. bryoniae.

scholarly journals Evaluation of Single-Nucleotide Primer Extension for Detection and Typing of Phylogenetic Markers Used for Investigation of Microbial Communities

2009 ◽  
Vol 75 (9) ◽  
pp. 2850-2860 ◽  
Author(s):  
Marcell Nikolausz ◽  
Antonis Chatzinotas ◽  
András Táncsics ◽  
Gwenaël Imfeld ◽  
Matthias Kästner
Keyword(s):  
16S Rrna ◽  
Dna Sequences ◽  
Simultaneous Detection ◽  
Primer Extension ◽  
Detection Sensitivity ◽  
High Signal ◽  
Rrna Gene ◽  
Single Nucleotide ◽  
Specificity And Sensitivity ◽  
Single Nucleotide Primer Extension

ABSTRACT Single-nucleotide primer extension (SNuPE) is an emerging tool for parallel detection of DNA sequences of different target microorganisms. The specificity and sensitivity of the SNuPE method were assessed by performing single and multiplex reactions using defined template mixtures of 16S rRNA gene PCR products obtained from pure bacterial cultures. The mismatch discrimination potential of primer extension was investigated by introducing different single and multiple primer-target mismatches. The type and position of the mismatch had significant effects on the specificity of the assay. While a 3′-terminal mismatch has a considerable effect on the fidelity of the extension reaction, the internal mismatches influenced hybridization mostly by destabilizing the hybrid duplex. Thus, carefully choosing primer-mismatch positions should result in a high signal-to-noise ratio and prevent any nonspecific extension. Cyclic fluorescent labeling of the hybridized primers via extension also resulted in a significant increase in the detection sensitivity of the PCR. In multiplex reactions, the signal ratios detected after specific primer extension correlated with the original template ratios. In addition, reverse-transcribed 16S rRNA was successfully used as a nonamplified template to prove the applicability of SNuPE in a PCR-independent manner. In conclusion, this study demonstrates the great potential of SNuPE for simultaneous detection and typing of various nucleic acid sequences from both environmental and engineered samples.

Tests of conspecificity for closely related black fly (Diptera: Simuliidae) species of the Simulium striatum group in Thailand

Zootaxa ◽  
2017 ◽  
Vol 4231 (3) ◽  
pp. 421 ◽  
Author(s):  
SIRINAPA PANGJANDA ◽  
PAIROT PRAMUAL
Keyword(s):  
Dna Sequences ◽  
Developmental Stages ◽  
Species Group ◽  
Biological Species ◽  
Larval Habitat ◽  
Habitat Characteristics ◽  
Elongation Complex ◽  
Morphological Examination ◽  
Black Fly ◽  
Gill Filaments

Four black fly species of the Simulium striatum species group have been recorded in Thailand. These species are morphologically highly similar in all developmental stages except for the number and arrangement of the pupal gill filaments. In this study, we used multiple characters sources, including morphology, cytology, molecular biology and ecology, to test the hypothesis of conspecificity for S. quinquestriatum (Shiraki), S. nakhonense Takaoka & Suzuki and S. chiangmaiense Takaoka & Suzuki. A molecular study based on the cytochrome c oxidase subunit I (COI) was unable to separate these taxa. In contrast, the elongation complex protein 1 (ECP1) sequences clearly differentiate S. quinquestriatum from S. chiangmaiense and S. nakhonense. However, the latter two taxa could not be differentiated based on molecular DNA sequences. Simulium chiangmaiense and S. nakhonense are also similar in the larval habitat characteristics and have undifferentiated polytene chromosome banding patterns. Morphological examination of the number and arrangement of the pupal gill filaments found a number of intermediate forms. Therefore, S. chiangmaiense and S. nakhonense are apparently the same biological species that is polymorphic for the number and arrangement of gill filaments. Thus, we synonymized S. chiangmaiense with S. nakhonense. 

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