Consensus nested PCR amplification and sequencing of diverse reptilian, avian, and mammalian orthoreoviruses

2009 ◽  
Vol 133 (1-2) ◽  
pp. 34-42 ◽  
Author(s):  
James F.X. Wellehan ◽  
April L. Childress ◽  
Rachel E. Marschang ◽  
April J. Johnson ◽  
Elaine W. Lamirande ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Pcr Amplification

scholarly journals Detection of Pneumocystis in lungs of bats from Brazil by PCR amplification

2009 ◽  
Vol 29 (6) ◽  
pp. 469-473 ◽  
Author(s):  
Edna Maria Cavallini Sanches ◽  
Susi M. Pacheco ◽  
Alison S. Cericatto ◽  
Rosane M. Melo ◽  
Edson Molleta Colodel ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Urban Areas ◽  
Pcr Amplification ◽  
Small Subunit ◽  
Rrna Gene ◽  
Wild Animals ◽  
Tadarida Brasiliensis ◽  
Desmodus Rotundus ◽  
Mato Grosso ◽  
Wide Range

Pneumocystis has been isolated from a wide range of unrelated mammalian hosts, including humans, domestic and wild animals. It has been demonstrated that the genome of Pneumocystis of one host differs markedly from that of other hosts. Also, variation in the chromosome and DNA sequence of Pneumocystis within a single host species has been observed. Since information about the occurrence and nature of infections in wild animals is still limited, the objective of this work was to detect the presence of Pneumocystis sp. in lungs of bats from two states from Brazil by Nested-PCR amplification. The bats, captured in caves and in urban areas, were obtained from the Program of Rabies Control of two States in Brazil, Mato Grosso and Rio Grande do Sul, located in the Mid-Western and Southern regions of the country, respectively. DNAs were extracted from 102 lung tissues and screened for Pneumocystis by nested PCR at the mtLSU rRNA gene and small subunit of mitochondrial ribosomal RNA (mtSSU rRNA). Gene amplification was performed using the mtLSU rRNA, the primer set pAZ102H - pAZ102E and pAZ102X - pAZY, and the mtSSU rRNA primer set pAZ102 10FRI - pAZ102 10R-RI and pAZ102 13RI - pAZ102 14RI. The most frequent bats were Tadarida brasiliensis (25), Desmodus rotundus (20), and Nyctinomops laticaudatus (19). Pneumocystis was more prevalent in the species Nyctinomops laticaudatus (26.3% = 5/19), Tadarida brasiliensis (24% = 6/25), and Desmodus rotundus (20% = 4/20). Besides these species, Pneumocystis also was detected in lungs from Molossus molossus (1/11, 9.1%), Artibeus fimbriatus (1/1, 100%), Sturnira lilium (1/3, 33.3%), Myotis levis (2/3, 66.7%)and Diphylla ecaudata (1/2, 50%). PCR products which could indicate the presence of Pneumocystis (21.56%) were identified in DNA samples obtained from 8 out of 16 classified species from both states (5 bats were not identified). This is the first report of detection of Pneumocystis in bats from Brazil.

scholarly journals Whole blood Nested PCR and Real-time PCR amplification ofTalaromyces marneffeispecific DNA for diagnosis

2015 ◽  
Vol 54 (2) ◽  
pp. 162-168 ◽  
Author(s):  
Sha Lu ◽  
Xiqing Li ◽  
Richard Calderone ◽  
Jing Zhang ◽  
Jianchi Ma ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Whole Blood ◽  
Nested Pcr ◽  
Pcr Amplification

scholarly journals Methacarn Fixation for Genomic DNA Analysis in Microdissected, Paraffin-embedded Tissue Specimens

2002 ◽  
Vol 50 (9) ◽  
pp. 1237-1245 ◽  
Author(s):  
Chikako Uneyama ◽  
Makoto Shibutani ◽  
Naoya Masutomi ◽  
Hironori Takagi ◽  
Masao Hirose
Keyword(s):  
Nested Pcr ◽  
Genomic Dna ◽  
Hippocampal Neurons ◽  
Dna Analysis ◽  
Extraction Procedure ◽  
Pcr Amplification ◽  
Single Step ◽  
Superior Performance ◽  
Cresyl Violet ◽  
Rat Tissues

We recently found methacarn to be a versatile fixative for analysis of RNA and protein applicable for microdissected specimens from paraffin-embedded tissue (PET). In this study we investigated the performance of methacarn for genomic DNA analysis using microdissected rat tissues. We found that extensive portions of DNA up to 2.8 kb could be amplified by nested PCR using DNA templates extracted by a simple and rapid extraction procedure from a 1 × 1-mm area of cerebral cortex of a 10-μm-thick section. By nested PCR, a 522-bp fragment from a single cell could be amplified in 20% of cresyl violet-stained Purkinje cells, and the minimal number of cells required, as estimated using hippocampal neurons, was on the order of 10-20. Although tissue staining with hematoxylin and eosin affected the PCR, amplification of a 522-bp fragment was successful, with 150-270 cells by 35 cycles of single-step PCR. Immunostaining resulted in a substantial decrease of yield and degradation of extracted DNA. However, even after immunostaining, a 184-bp DNA fragment could be amplified with 150-270 cells by 35 cycles of PCR. The results thus demonstrate the superior performance of methacarn to that reported with formalin in genomic DNA analysis using microdissected PET specimens.

Rapid HLA-DRB1 genotyping by nested PCR amplification

1992 ◽  
Vol 39 (2) ◽  
pp. 68-73 ◽  
Author(s):  
G. Bein ◽  
R. Gläser ◽  
H. Kirchner
Keyword(s):  
Nested Pcr ◽  
Pcr Amplification

Detection of filamentous genus Gordonia in foam samples using genus-specific primers combined with PCR – denaturing gradient gel electrophoresis analysis

2007 ◽  
Vol 53 (6) ◽  
pp. 768-774 ◽  
Author(s):  
Fo-Ting Shen ◽  
Hsuan-Ru Huang ◽  
A.B. Arun ◽  
Hui-Ling Lu ◽  
Ta-Chen Lin ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Wastewater Treatment Plants ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Gradient Gel Electrophoresis

A nested-PCR amplification combined with denaturing gradient gel electrophoresis (PCR–DGGE) approach was used to detect and identify Gordonia populations from wastewater treatment plant foam samples. The PCR-amplified region (position 722–1119) by specifically designed primers G699F and G1096R covered the hypervariable region of the Gordonia 16S rRNA gene sequence. This approach successfully distinguished Gordonia species to the interspecies level. The differential ability of PCR–DGGE analysis was effectively used to separate 12 Gordonia species belonging to different 16S rRNA gene-based phylogenetic lineages into 8 groups. Based on this method, the minimum limit of Gordonia detection was 5 × 104CFU·g–1in the seeded soil samples. The PCR–DGGE bands obtained were excised and identified by sequence analysis. Gordonia polyisoprenivorans , Gordonia amicalis , DGGE type II Gordonia species, and an uncertain Gordonia species dominated the activated sludge foam samples. Results of this study indicate that the detection and analyses of genus Gordonia within a complex microbial community could be accomplished using the PCR–DGGE approach to a larger extent, with certain limitations. Detection of diverse Gordonia populations in foam samples from wastewater treatment plants revealed the significant role of Gordonia in biological foaming during wastewater treatment. The nested-PCR amplification and DGGE can be used as a diagnostic tool for the early detection of foaming incidents in wastewater treatment plants using Gordonia as indicator organism.

Abnormally Small BCR-ABL Transcripts in CML Patients before and during Imatinib Treatment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2153-2153 ◽  
Author(s):  
Jamshid S. Khorashad ◽  
Jeffrey H. Lipton ◽  
David Marin ◽  
Dragana Milojkovic ◽  
Nicholas C.P. Cross ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Genetic Instability ◽  
Fusion Gene ◽  
Direct Sequencing ◽  
Pcr Amplification ◽  
Kinase Domain ◽  
Current Evidence ◽  
Imatinib Treatment ◽  
Abl Kinase ◽  
Detection Frequency

Abstract Neoplastic cells bearing fusion genes that express an activated tyrosine kinase may set the scene for accumulation of genetic lesions by dysregulating DNA damage repair mechanisms and causing genetic instability. The observation that the BCR-ABL fusion gene alters pre-mRNA splicing in a variety of other genes including Ikaros and PYK2 supports this hypothesis. However, the only current evidence for acquired genetic change in the BCR-ABL gene itself is limited to finding mutations in the BCR-ABL kinase domain in patients treated with imatinib mesylate (IM). Here we report the observation that some patients with CML have abnormally small BCR-ABL transcripts both before and during treatment. Patients with sub-optimal response to IM are screened for mutations specifically within the BCR-ABL kinase domain by performing nested PCR, thereby excluding amplification of the non-translocated ABL allele. In the first round PCR amplification is performed across the fusion and the amplicons generated are subjected to a second round to yield an expected 863 bp (containing ABL exons 4 through 9 and thus the entire BCR-ABL kinase domain) PCR fragment. Smaller amplicons were observed in 49 (9.9%) of the 494 CML patients investigated. There was marked variation in the mRNA species when the abnormally small amplicons were subjected to direct sequencing; we found exon skipping, intra-exon splicing and insertion of intronic sequences. Similarly, in some cases the open reading frame was maintained whilst in others there were frame shifts leading to premature stop codons. The commonest finding, (22 of the 49 patients) was skipping of ABL exon 7 from codons 362 to 424, which includes the activation loop of the kinase domain. The smaller amplicons persisted even after the first round products had been diluted to 1:160. We also noted that the normal 863 bp fragment was present in some cases but was not detectable in others; its absence could reflect preferential amplification of the smaller transcripts. In a number of cases the same abnormally short amplicons were identified in the same patient studied serially on three or more separate occasions. We subsequently performed a second round of nested PCR with primers designed to amplify across the BCR-ABL junction such that the product included sequences from BCR 13 to ABL exon 9. With these new primers the detection frequency of abnormally small transcripts was increased. Furthermore, we observed the smaller transcripts in all of the 12 patients tested prior to beginning IM therapy. We then sought to determine if the normal ABL allele was involved; in order to avoid amplifying the BCR-ABL kinase domain, we performed a single round of PCR and restricted the analysis to patients in complete cytogenetic remission (CCyR). Only the expected 863 bp amplicon was observed in cDNA samples from 19 CML patients in CCyR whose BCR-ABL/ABL ratios ranged from 0.01 to 0.98. Furthermore, the smaller amplicons were not observed in cDNA samples from 20 normal individuals. We conclude that these abnormalities may result either from aberrant alternative splicing or from spontaneous deletions, or from a combination of both mechanisms. They may be a manifestation of the genetic instability believed to be an integral feature of CML.

Detection of Flexibacter maritimus in fish tissue using nested PCR amplification

2003 ◽  
Vol 26 (2) ◽  
pp. 65-70 ◽  
Author(s):  
C Cepeda ◽  
S Garcia-Marquez ◽  
Y Santos
Keyword(s):  
Nested Pcr ◽  
Pcr Amplification ◽  
Fish Tissue
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