scholarly journals Molecular identification of a phytoplasma naturally infecting Populus nigra L. cv. Italica trees in Croatia

2002 ◽  
Vol 38 (SI 1 - 6th Conf EFPP 2002) ◽  
pp. S28-S30
Author(s):  
M. Šeruga ◽  
D. Škorić ◽  
S. Botti ◽  
S. Paltrinieri ◽  
N. Juretić ◽  
...  
Keyword(s):  
16S Rdna ◽  
Nested Pcr ◽  
Late Summer ◽  
Populus Nigra ◽  
Aster Yellows ◽  
Leaf Yellowing ◽  
Pcr Assays ◽  
Nested Pcr Assays ◽  
Zagreb Area ◽  
Lombardy Poplar

Leaf and branch samples of 10 Populus nigra L. cv. Italica trees were collected from the urban Zagreb area in late summer/autumn 2001. One of the trees exhibited leaf yellowing, overall sparse foliage, stunting and decline. Phytoplasma 16S rDNA was amplified in direct and nested PCR assays using universal and specific phytoplasma primer pairs, from nucleic acids extracted by two different procedures. Strong amplification signals were observed in samples from symptomatic Lombardy poplar as well as in samples from 4 of the asymptomatic trees. RFLP analyses of amplicons showed patterns characteristic of the phytoplasmas belonging to the Aster yellows group (16SrI). This is the first report of a phytoplasma naturally infecting poplar in Croatia.

scholarly journals First Report of an Aster Yellows Phytoplasma Associated with Cabbage in Southern Texas

Plant Disease ◽  
2001 ◽  
Vol 85 (4) ◽  
pp. 447-447 ◽  
Author(s):  
I.-M. Lee ◽  
R. A. Dane ◽  
M. C. Black ◽  
Noel Troxclair
Keyword(s):  
16S Rdna ◽  
Nested Pcr ◽  
Restriction Enzymes ◽  
Diagnostic Procedures ◽  
Acid Extraction ◽  
Insect Vector ◽  
Aster Yellows ◽  
First Report ◽  
Pcr Products ◽  
Aster Yellows Phytoplasma

In early spring 2000 carrot crops in southwestern Texas were severely infected by an outbreak of phyllody associated with aster yellows phytoplasma. Cabbage crops that had been planted adjacent to these carrot fields began to display previously unobserved symptoms characteristic of phytoplasma infection. Symptoms included purple discoloration in leaf veins and at the outer edges of leaves on cabbage heads. Proliferation of sprouts also occurred at the base of the stem and between leaf layers of some plants, and sprouts sometimes continued to proliferate on extended stems. About 5% of cabbage plants in the field exhibited these symptoms. Two symptomless and four symptomatic cabbage heads were collected in early April from one cabbage field. Veinal tissues were stripped from each sample and used for total nucleic acid extraction. To obtain specific and sufficient amount of PCR products for analysis, nested PCR was performed by using primer pairs (first with P1/P7 followed by R16F2n/R16R2) (1,2) universal for phytoplasma detection. A specific 16S rDNA fragment (about 1.2 kb) was strongly amplified from the four symptomatic but not from the two asymptomatic samples. The nested PCR products obtained from the four symptomatic samples were then analyzed by restriction fragment length polymorphism (RFLP) using the restriction enzymes MseI, HhaI, and HpaII, and the RFLP patterns were compared to the published patterns of known phytoplasmas (1). The resulting RFLP patterns were identical to those of a phytoplasma belonging to subgroup B of the aster yellows phytoplasma group (16SrI). These RFLP patterns were also evident in putative restriction sites observed in a 1.5 kbp nucleotide sequence of the 16S rDNA. This is the first report of aster yellows phytoplasma associated disease symptoms in cabbage in Texas. The occurrence of cabbage proliferation coincided with the presence of high populations of the insect vector, aster leafhopper. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) B. Schneider et al. 1995. Molecular and Diagnostic Procedures in Mycoplasmology, Vol. I. Academic Press, San Diego, CA.

scholarly journals Detection of Bovine and Porcine Adenoviruses for Tracing the Source of Fecal Contamination

2004 ◽  
Vol 70 (3) ◽  
pp. 1448-1454 ◽  
Author(s):  
Carlos Maluquer de Motes ◽  
Pilar Clemente-Casares ◽  
Ayalkibet Hundesa ◽  
Margarita Mart�n ◽  
Rosina Girones
Keyword(s):  
Nested Pcr ◽  
Fecal Contamination ◽  
Human Adenovirus ◽  
The Other ◽  
Animal Origin ◽  
Viral Contamination ◽  
Urban Sewage ◽  
Negative Results ◽  
Pcr Assays ◽  
Nested Pcr Assays

ABSTRACT In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera Mastadenovirus and Atadenovirus and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies.

Nested PCR assays with novel primers yield greater sensitivity to Tanzanian HIV-1 samples than a commercial PCR detection kit

1996 ◽  
Vol 62 (2) ◽  
pp. 131-141 ◽  
Author(s):  
Olov Grankvist ◽  
Lilian Walther ◽  
Ulla Bredberg-Rådén ◽  
Eligius Lyamuya ◽  
Fred Mhalu ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Pcr Detection ◽  
Pcr Assays ◽  
Nested Pcr Assays ◽  
Hiv 1

scholarly journals Malaria Parasites in Macaques in Thailand: Stump-Tailed Macaques (Macaca Arctoides) are New Natural Hosts for Plasmodium Knowlesi, P. Inui, P. Coatneyi and P. Fieldi.

2020 ◽  
Author(s):  
Wirasak Fungfuang ◽  
Chanya Udom ◽  
Daraka Tongthainan ◽  
Khamisah Abdul Kadir ◽  
Balbir Singh
Keyword(s):  
Nested Pcr ◽  
Plasmodium Knowlesi ◽  
Sampling Site ◽  
Macaca Arctoides ◽  
Malaria Parasites ◽  
Zoonotic Malaria ◽  
Natural Hosts ◽  
New Locations ◽  
Pcr Assays ◽  
Nested Pcr Assays

Abstract Background:Certain species of macaques are natural hosts ofPlasmodium knowlesi and P. cynomolgi, which can both cause malaria in humans, and P. inui, which can be experimentally transmitted to humans. A significant number of zoonotic malaria cases have been reported in humans throughout Southeast Asia, including Thailand. There have been only two studies undertaken in Thailand to identify malaria parasites in non-human primates in 6 provinces. The objective of this study was to determine the prevalence of P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldiin non-human primates from 4 new locations in Thailand. Methods:A total of 93 blood samples from Macaca fascicularis, M. leonina and M. arctoides were collected from four locations in Thailand: 32 were captive M. fascicularisfrom Chachoengsao Province (CHA), 4 were wild M. fascicularis from Ranong Province (RAN), 32 were wildM. arctoidesfromPrachuap Kiri Khan Province (PRA), and 25 were wild M. leoninafrom Nakornratchasima Province (NAK). DNA was extracted from these samples and analysed by nested PCR assays to detect Plasmodium, and subsequently to detectP. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi.Results:Twenty-seven of the 93 (29%) samples were Plasmodium-positive by nested PCR assays. Among wild macaques, all 4 M. fascicularis at RAN were infected with malaria parasites followed by 50% of 32 M. arctoides at PRA and 20% of 25 M. leonina at NAK. Only 2 (6.3%) of the 32 captive M. fascicularisat CHA were malaria-positive. All 5 species of Plasmodium were detected and 16 (59.3%) of the 27 macaques had single infections, 9had double and 2 had triple infections.The composition of Plasmodium species in macaques at each sampling site was different. Macaca arctoides from PRA were infected with P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi. Conclusions:The prevalence and species of Plasmodiumvaried among the wild and captive macaques, and betweenmacaques at 4 sampling sites in Thailand. Macaca arctoidesis a new natural host for P. knowlesi, P. inui,P. coatneyi and P. fieldi.

scholarly journals First Report of Aster Yellows Phytoplasma in Alfalfa

Plant Disease ◽  
1999 ◽  
Vol 83 (5) ◽  
pp. 488-488 ◽  
Author(s):  
R. D. Peters ◽  
M. E. Lee ◽  
C. R. Grau ◽  
S. J. Driscoll ◽  
R. M. Winberg ◽  
...  
Keyword(s):  
16S Rdna ◽  
Nested Pcr ◽  
Sequence Data ◽  
Sequence Similarity ◽  
Similarity Function ◽  
Sterile Water ◽  
Aster Yellows ◽  
First Report ◽  
Pcr Product ◽  
Aster Yellows Phytoplasma

Samples of alfalfa (Medicago sativa L.) leaves and stems showing symptoms of inter-veinal chlorosis and purpling, commonly associated with insect feeding, were collected from 8 sites in central and southern Wisconsin in autumn of 1998. Samples were frozen within 24 h of collection. Approximately 0.3 g of plant tissue from each sample was used for total DNA extraction according to the protocol of Zhang et al. (4), with minor modifications in grinding procedures and reagent volumes to optimize results. Nested polymerase chain reaction (PCR) was carried out by amplification of 16S rDNA with the universal primer pairs R16mF2/R16mR1 followed by R16F2n/R16R2 as described by Gunder-sen and Lee (1). Undiluted total sample DNA was used for the first amplification; PCR products were diluted (1:30) in sterile water prior to final amplification. Alfalfa DNA and sterile water were used as negative controls; DNA from phytoplasma causing X-disease in peach (CX) served as a positive control. Fragments of 16S rDNA from putative phytoplasmas amplified by PCR with the primer pair R16F2n/R16R2 were characterized by restriction endonuclease digestion (3). The resulting restriction fragment length polymorphism (RFLP) patterns were compared with patterns for known phytoplasmas described by Lee et al. (3). Products of nested PCR were also purified and sequenced with primers R16F2n/R16R2 and an automated DNA sequencer (ABI 377XL; C. Nicolet, Biotechnology Center, University of Wisconsin-Madison). Of 51 samples of alfalfa assessed, one sample from Evansville, WI, yielded a nested PCR product of the appropriate size (1.2 kb), indicating the presence of phytoplasma. Digestion of this product with various restriction enzymes produced RFLP patterns that were identical to those for phytoplasmas in the aster yellows phytoplasma subgroup 16SrI-A (3). Alignment of the DNA sequence of the nested PCR product from the positive sample with sequences found in the GenBank sequence data base (National Center for Biotechnology Information, Bethesda, MD) with the BLAST sequence similarity function confirmed this result. Although other phytoplasma strains (particularly those causing witches'-broom) have been reported to infect alfalfa (2), this is the first report of the presence of the aster yellows phytoplasma in the alfalfa crop. Vectors involved in transmission and the potential agronomic impacts of aster yellows phytoplasma in alfalfa are topics of current investigation. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) A.-H. Khadhair et al. Microbiol. Res. 152:269, 1997. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) Y.-P. Zhang et al. J. Virol. Methods 71:45, 1998.

Detection and identification of Toscana and other phleboviruses by RT-nested-PCR assays with degenerated primers

2003 ◽  
Vol 71 (1) ◽  
pp. 140-149 ◽  
Author(s):  
María-Paz Sánchez-Seco ◽  
José-Manuel Echevarría ◽  
Lourdes Hernández ◽  
Domingo Estévez ◽  
José-María Navarro-Marí ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Detection And Identification ◽  
Pcr Assays ◽  
Nested Pcr Assays

Development of conventional and nested PCR assays for the detection ofOphiocordyceps sinensis

2012 ◽  
Vol 53 (4) ◽  
pp. 340-347 ◽  
Author(s):  
Guo-Sheng Jin ◽  
Xiao-Liang Wang ◽  
Yi Li ◽  
Wen-Jing Wang ◽  
Rui-Heng Yang ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Pcr Assays ◽  
Nested Pcr Assays

scholarly journals Evaluation of Two Nested PCR Assays for Detection of Histoplasma capsulatum DNA in Human Tissue

2002 ◽  
Vol 40 (5) ◽  
pp. 1644-1647 ◽  
Author(s):  
R. Bialek ◽  
A. Feucht ◽  
C. Aepinus ◽  
G. Just-Nubling ◽  
V. J. Robertson ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Human Tissue ◽  
Histoplasma Capsulatum ◽  
Pcr Assays ◽  
Nested Pcr Assays
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