Molecular differentiation of Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis by pyrosequencing

2013 ◽  
Vol 89 (1) ◽  
pp. 118-123 ◽  
Author(s):  
L. Sadaow ◽  
C. Tantrawatpan ◽  
P.M. Intapan ◽  
V. Lulitanond ◽  
T. Boonmars ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Trichinella Spiralis ◽  
Large Subunit ◽  
Pcr Amplification ◽  
Epidemiological Studies ◽  
Chain Reaction ◽  
Target Region ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Zoonotic Parasites

AbstractNematodes of the genus Trichinella which infect wildlife and domestic animals show a cosmopolitan distribution. These zoonotic parasites are the aetiological agents of a severe human disease, trichinellosis. Twelve taxa are recognized in the Trichinella genus, but they cannot be identified by morphology since they are sibling species/genotypes. For epidemiological studies, it is extremely important to identify each taxon since they have different distribution areas and host ranges. In the present study, polymerase chain reaction (PCR) amplification of the mitochondrial large subunit ribosomal RNA (lsu-RNA) gene coupled with a pyrosequencing technique was developed to distinguish among four Trichinella species: Trichinella spiralis, T. pseudospiralis, T. papuae and T. zimbabwensis. A PCR method was used to amplify the lsu-RNA of Trichinella sp. larvae in mouse muscles and single larvae collected from infected muscles by digestion. The results show that the four Trichinella species can be distinguished by using 26 nucleotides in the target region and the method is sensitive enough to identify individual larvae. The pyrosequencing provides a simple, rapid and high-throughput tool for the differentiation of Trichinella species.

scholarly journals Anaplasma marginale infection in cattle from south-western Amazonia

2010 ◽  
Vol 30 (3) ◽  
pp. 249-254 ◽  
Author(s):  
Luciana Gatto Brito ◽  
Márcia Cristina de Sena Oliveira ◽  
Rodrigo Barros Rocha ◽  
Francelino Goulart da Silva Netto ◽  
Adriana Denise Marim ◽  
...  
Keyword(s):  
High Frequency ◽  
Simple Procedure ◽  
Anaplasma Marginale ◽  
Epidemiological Data ◽  
Epidemiological Studies ◽  
Chain Reaction ◽  
Blood Clots ◽  
Pcr Method ◽  
The Mean ◽  
Polymerase Chain

The present study provides the first epidemiological data regarding infection by Anaplasma marginale in cattle reared in south-western Brazilian Amazonia. One simple procedure was adapted for the extraction of DNA from blood clots collected in seven microregions of Rondônia State and two mesoregions of Acre State. PCR method was used to asses the frequency of A. marginale infections in 4 to12-month-old cattle. The cattle infection was investigated by polymerase chain reaction (PCR) using the specific primer "msp5" for A. marginale. The DNA amplifications revealed that the mean frequency of A. marginale infection was 98.6% (1,627/1,650) in samples from Rondonia, and 92.87% (208/225) in samples from Acre. The high frequency of A. marginale infections in 4 to 12-month-old cattle indicate a situation of enzootic stability in the studied areas and are comparable to those detected by immunodiagnosis in different endemic regions in Brazil. The DNA extraction of clotted blood method described here can be used for epidemiological studies on anaplasmosis and other bovine hemoparasites.

Characterization of trypanosome infections by polymerase chain reaction (PCR) amplification in wild tsetse flies in Cameroon

Parasitology ◽  
1998 ◽  
Vol 116 (6) ◽  
pp. 547-554 ◽  
Author(s):  
I. MORLAIS ◽  
P. GREBAUT ◽  
J. M. BODO ◽  
S. DJOHA ◽  
G. CUNY
Keyword(s):  
Polymerase Chain Reaction ◽  
Infection Rate ◽  
Forest Type ◽  
Pcr Amplification ◽  
Tsetse Flies ◽  
Microscopical Examination ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Primer Sets

The polymerase chain reaction (PCR) method was used to characterize trypanosome infections in tsetse flies from 3 sleeping sickness foci in Cameroon. The predominant tsetse species found was Glossina palpalis palpalis. An average infection rate of 12·1% was revealed by microscopical examination of 888 non-teneral tsetse flies. PCR amplification analyses for trypanosome identification were carried out on 467 flies, with primer sets specific for Trypanosoma (Trypanozoon) brucei s.l., T. (Duttonella) vivax, T. (Nannomonas) simiae and forest type T. (Nannomonas) congolense. Of 467 flies 93 were positive by microscopical analysis while PCR succeeded in identifying 89 positive flies. Of the PCR-positive flies 34 (38·2%) were negative by microscopical examination. PCR amplification, when compared to the parasitological technique, gave a higher estimate of infection rate of trypanosomes in natural tsetse populations. The PCR technique did, however, fail to identify 40·9% (38/93) of the parasitologically positive flies. The reasons for this failure are discussed. The overall prevalence of mixed infections, assessed by PCR, was 37·1%; the majority (72·7%) involved T. brucei and forest type T. congolense.

scholarly journals Identification and Prevalence of Botrytis spp. from Blackberry and Strawberry Fields of the Carolinas

Plant Disease ◽  
2012 ◽  
Vol 96 (11) ◽  
pp. 1634-1637 ◽  
Author(s):  
Xingpeng Li ◽  
Dolores Fernández-Ortuño ◽  
Wenxuan Chai ◽  
Fei Wang ◽  
Guido Schnabel
Keyword(s):  
North Carolina ◽  
South Carolina ◽  
Pcr Amplification ◽  
Gray Mold ◽  
Chain Reaction ◽  
Reverse Primer ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Prevalent Species ◽  
Reference Strains

Gray mold disease of blackberry and strawberry is caused by Botrytis cinerea and B. caroliniana in the southeastern United States. In this study, methods to distinguish both species were established and their prevalence was determined in commercial blackberry and strawberry fields. Using DNA from B. cinerea and B. caroliniana reference strains, a species-differentiating polymerase chain reaction (PCR) amplification was developed that amplified G3PDH gene fragments of two different sizes depending on the species. The PCR is performed with three primers (two species-differentiating forward primers and one universal reverse primer) and amplified a 238-bp product from B. cinerea and a 536-bp fragment from B. caroliniana reference isolates. A total of 400 Botrytis isolates were collected from 6 commercial blackberry and 11 strawberry fields of the Carolinas and identified to the species level by the new PCR method. Both Botrytis spp. were identified in blackberry and strawberry fields, but B. caroliniana was less common than B. cinerea. Only 33 of 202 isolates from blackberry fields were identified as B. caroliniana, and the majority of these isolates came from two fields in South Carolina. Only 1 of 198 isolates from strawberries was identified as B. caroliniana, and this isolate was found in central North Carolina. B. cinerea but not B. caroliniana isolates sporulated on potato dextrose agar and Kings medium B. Our results show that B. cinerea and B. caroliniana coexist in at least some commercial blackberry and strawberry fields of the Carolinas, with B. cinerea being the more prevalent species.

What Molecular Biology has to Tell us at the Species Level in Lichenized Fungi

1998 ◽  
Vol 30 (4-5) ◽  
pp. 307-320 ◽  
Author(s):  
Paul D. Bridge ◽  
David L. Hawksworth
Keyword(s):  
Ribosomal Rna ◽  
Pcr Amplification ◽  
Fungal Genome ◽  
Species Boundaries ◽  
Chain Reaction ◽  
Variable Regions ◽  
Fungal Partner ◽  
Large Numbers ◽  
Polymerase Chain ◽  
Pcr Techniques

AbstractMolecular biological techniques now provide many opportunities for determining relationships between organisms. In lichen-forming fungi, polymerase chain reaction (PCR) amplification can be undertaken with primers that have enhanced specificity for fungi, allowing the recovery of precise regions of the genome of the fungal partner. Such PCR techniques are suitable for the analysis of both fresh and dried specimens of great age, and as the techniques require only minimal amounts of starting material, they are suitable for the analysis of large numbers of specimens. PCR can be used to examine both conserved and variable regions of the fungal genome that may be taxonomically significant, and variability in these sequences can be used to describe species boundaries. The ease of analysis of large numbers of specimens without the need to purify or culture the fungal partner makes analysis and comparison of infra- and inter-specific relationships possible, and provides valuable information into their genetic background. Most molecular analyses undertaken with lichen-forming fungi have considered variation within ribosomal RNA clusters, and in this paper we consider the application and interpretation of such PCR-based analysis in the circumscription of species and in determining concepts for individuality and infra-specific groupings.

scholarly journals First report of PCR-based detection of Helicobacter species DNA in Camelus dromedarius in Egypt

2020 ◽  
Vol 13 (9) ◽  
pp. 1898-1901
Author(s):  
Ahmed Youssef ◽  
Ahmed Afifi ◽  
Ayman Hamed ◽  
Mohamed Enany
Keyword(s):  
Ribosomal Rna ◽  
Conventional Polymerase Chain Reaction ◽  
Epidemiological Studies ◽  
Camelus Dromedarius ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Fecal Samples ◽  
First Report ◽  
16S Ribosomal Rna Gene ◽  
Polymerase Chain

Background and Aim: Helicobacter species infections have epidemiological and zoonotic impacts, and different species of Helicobacter have been implicated in infecting humans and animals. The aim of this study was to investigate Helicobacter species infections in Camelus dromedarius. Materials and Methods: Fecal samples were collected from 32 camels from 9 camel farms located at Ismailia Governorate, Egypt. The collected samples were investigated by bacteriological isolation and conventional polymerase chain reaction (PCR) assays targeting the 16S ribosomal RNA gene. Results: Although Helicobacter species could not be isolated from all the examined samples, Helicobacter DNA was detected in 2 (22.22%) of the 9 camel farms. Of the 32 camel fecal samples examined, 4 (12.5%) were positive for Helicobacter species as analyzed by the PCR assay. Conclusion: To the best of our knowledge, this is the first report of PCR-based detection of Helicobacter species infections in C. dromedarius. Further epidemiological studies are required to clarify Helicobacter species infections in camels.

Identification of Bacteroides forsythus in Subgingival Dental Plaque with the Aid of a Rapid PCR Method

1997 ◽  
Vol 76 (7) ◽  
pp. 1376-1380 ◽  
Author(s):  
J.H. Meurman ◽  
J. Wahlfors ◽  
A. Korhonen ◽  
P. Alakuijala ◽  
P. Väisänen ◽  
...  
Keyword(s):  
Dental Plaque ◽  
Subgingival Plaque ◽  
Chain Reaction ◽  
Rapid Pcr ◽  
Microbiological Methods ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Pre Treatment ◽  
Culture Positive ◽  
Positive Results

Bacteroides forsythus has been shown to be prevalent among patients with periodontitis. Conventional microbiological methods used to identify this bacterium, however, are laborious and time-consuming and are therefore not well-suited for screening purposes. We have developed a polymerase chain-reaction (PCR) method which is rapid, specific, and simple to perform and does not require other sample pre-treatment except a brief centrifugation. This method was applied to the detection of B. forsythus in subgingival plaque of 58 periodontitis patients. When compared with the results of conventional culturing, the PCR method always confirmed the culture-positive results, while none of the PCR negative samples was shown to be culture-positive. The PCR method appeared to give more than double the number of samples positive for B. forsythus than culturing (89.7% vs. 37.9%). The analysis requires less than 4 hrs to perform, and is specific only to B. forsythus and sensitive enough to detect fewer than 5 bacteria.

scholarly journals Amplification of human beta-actin gene by the reverse transcriptase-polymerase chain reaction: implications for assessment of RNA from formalin-fixed, paraffin-embedded material.

1996 ◽  
Vol 44 (10) ◽  
pp. 1205-1207 ◽  
Author(s):  
A Dakhama ◽  
V Macek ◽  
J C Hogg ◽  
R G Hegele
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Amplification ◽  
Housekeeping Gene ◽  
Chain Reaction ◽  
Viral Genomes ◽  
Negative Results ◽  
Beta Actin ◽  
Target Nucleic Acid ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain

The polymerase chain reaction (PCR) is a powerful method that allows enzymatic amplification of rate target nucleic acid sequences. It has been applied to the amplification of viral genomes from paraffin-embedded pathology specimens. However, interpretation of negative results requires amplification of a housekeeping gene such as beta-actin. In the present study we used specific oligonucleotide primers previously designed to amplify both the genomic DNA and the mRNA transcript from paraffin-embedded tissue. These products have predicted sizes of 250 BP and 154 BP, respectively, but our results showed that PCR amplification only (without reverse transcription) unexpectedly generated the 154-BP product. Further investigation of the nature of this product demonstrated that it originated from the amplification of DNA, not RNA. We conclude that the 154-BP product generated by these primers cannot be exclusively considered as beta-actin RNA product and should not be used to assess successful extraction of RNA, to ascertain its integrity, or to normalize for the total amount of RNA assayed by RT-PCR from paraffin-embedded tissue.

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