A Novel PCR Method for Identifying Plankton in Cases of Death by Drowning

2003 ◽  
Vol 43 (1) ◽  
pp. 23-30 ◽  
Author(s):  
Sumiko Abe ◽  
Miwako Suto ◽  
Hidemasa Nakamura ◽  
Hirobumi Gunji ◽  
Kouichi Hiraiwa ◽  
...  
Keyword(s):  
Euglena Gracilis ◽  
Water Samples ◽  
Human Tissue ◽  
Skeletonema Costatum ◽  
Pcr Analysis ◽  
Blood Samples ◽  
Human Dna ◽  
Pcr Products ◽  
Pcr Method ◽  
Death By Drowning

We present a new PCR method for identifying plankton in cases of death by drowning. We designed four primer pairs for chlorophyll-related genes of Euglena gracilis (EG) and Skeletonema costatum (SK), which are commonly distributed in water. The primers were selected from sequences coding chloroplast/chlorophyll apoprotein of EG (EG1 and EG2) and fucoxanthin-chlorophyll a/c harvesting protein of SK (SK1 and SK2). With EG1 or EG2, up to 2 fg of EG-DNA was identified, and 0.2 pg of SK-DNA was detectable with SK1 or SK2. No PCR products were amplified from green vegetables (komatsuna, spinach, parsley) or human DNA with the four primer pairs. Regardless of the origin, seawater or fresh water, most diatoms were detectable with primer pairs of EG1 and EG2. With SK1, only Centrales diatoms were identified, and five diatom strains originating from seawater were detectable with SK2. EG1 and EG2 gave rise to PCR products from most water samples. By using Percoll®, plankton was easily isolated from human tissue or blood samples and good results of PCR analysis were obtained in cases of death by drowning.

scholarly journals Identification of Theileria Species in Sheep and Vector Ticks Using PCR Method in Zabol, Eastern Iran

2019 ◽  
Author(s):  
Fateme Zarei ◽  
Maryam Ganjali ◽  
Reza Nabavi
Keyword(s):  
Molecular Study ◽  
Epidemiological Studies ◽  
Pcr Analysis ◽  
Blood Samples ◽  
Control Programs ◽  
Theileria Lestoquardi ◽  
Eastern Iran ◽  
High Prevalence ◽  
Rhipicephalus Bursa ◽  
Pcr Method

Background: Theileria is a protozoal parasite that belongs to the phylum Apicomplexa. Theileriosis is an important tick-borne disease caused by various species of Theileria. Among these species, T. lestoquardi (T. hirci) is highly pathogenic, while other species such as T. ovis make Subclinical and mild infections in small ruminant. Therefore, the precise identification of the species and the vector ticks are very essential for epidemiological studies and the design of control programs. Methods: This research was conducted with the aim of molecular study to identify Theileria species and vectors in Zabol, eastern Iran in 2015. The presence of Theileria in 80 blood samples and vector ticks was evaluated using PCR method. Results: Of 80 blood samples, PCR analysis showed that 50 samples (62.5%) were infected with Theileria. The eval­uation of the first phase PCR with Nested PCR showed that infections with Theileria ovis and Theileria lestoquardi were 67.45% and 32.55% cases respectively. Overall, 110 ticks (78 males and 32 females) were collected and generally two genera and six spe­cies including Rhipicephalus bursa (9.1%), Rh. sanguineus (29.1%), Rh. turanicus (10.9%) Hyalomma asiati­cum asiati­cum (23.63%), Hy. excavatum (10.9%), Hy. anatolicum (16.37%) were detected. After evaluating ticks infection by PCR method, three species of Rh. turanicus, Rh. sanguineus and Hy. asiaticum asiaticum, were infected. Conclusion: Theileria ovis has a high prevalence among the sheep of zabol and Hy. asiaticum asiaticum, Rh. sanguineus and Rh. turanicus may be the main vectors of Theileria species in this area.

scholarly journals Genotyping and phylogenetic analysis of free-living amoeba (Acanthamoeba and Naegleria) in treated and untreated water in the northeastern provinces of Iran

2021 ◽  
Author(s):  
Yousef Sharifi ◽  
Omid Ahmadi ◽  
Bibi Razieh Hossini Farash ◽  
Nazgol Khosravinia ◽  
Reza Fotouhi-Ardakani ◽  
...  
Keyword(s):  
Water Quality ◽  
Water Samples ◽  
Taxonomic Status ◽  
Water Quality Monitoring ◽  
Pcr Analysis ◽  
Growth Media ◽  
Genotype Distribution ◽  
Free Living ◽  
Northern Iran ◽  
Pcr Products

Abstract Free-living amoebae (FLA) are widely distributed protozoa in natural or man-made aquatic environments without the need for a host organism for survival. Several strains of FLA are known to be pathogenic. As of date, there is inadequate data on the geographical distribution of FLA in northeastern and northern Iran. This study aimed to investigate the prevalence and genotype distribution of Acanthamoeba and Naegleria in drinking water and surface water samples in northern and northeastern Iran. A total of 60 water samples were collected and filtered from various sources for the presence of amoebae. DNA extraction was performed, and PCR confirmed the presence of FLA. PCR products were sequenced to identify the species/genotype. Phylogenetic relationships and taxonomic status constructed using MEGA X software. The findings on growth media showed 35% (21/60) and 26% (16/60) were positive for Acanthamoeba and Naegleria, respectively, while PCR analysis also obtained similar results. All isolates of Acanthamoeba were identified as T4 genotype. Poor water quality, as well as insufficient preservation and treatment, might indicate that chlorine disinfection is ineffective in removing contamination of amoebas in treated water samples. Therefore, regular water quality monitoring is essential to control amoeba's growth, reducing the risk of human infections with FLA.

PCR Analysis of Bacteria and Fungi in Early Phase of Hematopoietic Stem Cell Transplantation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1963-1963
Author(s):  
Atsushi Fujieda ◽  
Akiko Nakamura ◽  
Kohshi Ohishi ◽  
Fumihiko Monma ◽  
Masahiro Masuya ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Blood Culture ◽  
Febrile Neutropenia ◽  
Stenotrophomonas Maltophilia ◽  
Pcr Analysis ◽  
Hematopoietic Stem ◽  
Blood Samples ◽  
Pcr Products ◽  
Bacteria And Fungi

Abstract Abstract 1963 Purpose: Management of febrile neutropenia in patients receiving allogeneic hematopoietic stem cell transplantation (HCT) is crucial for successful treatment, but precise pathogen identification is difficult because the sensitivity of blood culture remains low, making the antimicrobial therapy empiric. This study aimed to detect and identify bacterial and fungal pathogens from peripheral blood samples during early phase of HCT, using highly sensitive PCR method which is able to detect and identify a broad range of bacteria and fungi (J Clin Microbiol. 48(6):2030-6, 2010) and to assess the clinical usefulness of PCR analysis in managing febrile neutropenia after HCT. Patients and Methods: Ten consecutive patients who underwent HCT in our institute between June 2007 and December 2009 were enrolled. Eight patients received TBI-based conventional and two received reduced-intensity preparative regimens. All patients received antibacterial prophylaxis with fluoroquinolone and antifungal prophylaxis by mold-active azole with temporary use of micafungin. We prospectively performed PCR analysis and blood culture of blood samples at least once a week for the first 30 days of HCT. For species identification, positive PCR products were sequenced. Results: In a total of 56 analyses, bacteria were detected in 23 cases with PCR but in only one case with blood culture. In the febrile patients, 18 cases out of 37 PCR analyses were positive for bacteria, while, in afebrile cases, 5 cases out of 19 PCR analyses were positive for bacteria. In 17 pathogen identified cases, 14 were gram positive cocci and 3 were gram negative rods. Most of the identified pathogens were oral and intestinal bacteria reflecting grade 3 severe mucositis. Fever was manageable in most cases with empiric use of antibiotics, but several series of antibiotic change was required in 2 cases, in which bacteria susceptible to specific antibiotics such as Stenotrophomonas maltophilia were detected. In several cases suffering from severe diarrhea, pathogen could not be identified because PCR products formed multiple peaks. Fungi were detected in only 4 cases, of which 2 cases were provable Aspergillus infection and another 2 cases possessed no other evidence of fungal infection. Conclusion: Bacterial pathogens mostly associated with oral and intestinal mucositis were detected with PCR in approximately half cases of febrile neutropenia, but fever was manageable in most cases with empiric antibiotic therapy by experienced physicians. PCR analysis was considered to be useful in febrile neutropenia due to bacteria susceptible to specific antibiotics such as Stenotrophomonas maltophilia. More rapid and simplified approach which targets clinically important pathogens such as Stenotrophomonas maltophilia that requires specific use of antibiotics may be useful and reasonable to provide support for the choice of antibiotics in clinical practice. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Simplified PCR analysis of a mutation in the NHEJ1 gene causing collie eye anomaly in some dog breeds

2010 ◽  
Vol 55 (No. 8) ◽  
pp. 346-350
Author(s):  
J. Dostál ◽  
P. Horák ◽  
A. Hrdlicová ◽  
A. Stratil
Keyword(s):  
Population Studies ◽  
Pcr Analysis ◽  
Time Saving ◽  
The Czech Republic ◽  
Blood Samples ◽  
Pcr Method ◽  
Clinical Variation ◽  
Thermal Cycler ◽  
Intron 4 ◽  
Phenotypic Development

Collie eye anomaly (CEA) is an inherited eye disease affecting development of the choroids and sclera segregating in several, mostly herding breeds of dog. Phenotypic development of the disease varies greatly in the affected animals. Genetic control of its clinical variation is unknown so far. Affected dogs share a 7.8 kb deletion in intron 4 of the NHEJ1 gene. We report here population studies of 379 dogs (Australian Shepherd, Border Collie, Rough Collie, Smooth Collie, Shetland Sheep Dog, and Nova Scotia Duck Tolling Retriever) from breeders in the Czech Republic. A simple PCR method using a Piko<sup>TM</sup> Thermal Cycler and unclotted blood samples was employed for the analysis of the NHEJ1 gene. No isolation of DNA from blood samples before PCR was needed. The method is time-saving and gives excellent results. Frequencies of the disease allele in each breed were calculated (0.045, 0.194, 0.797, 0.367, 0.429 and 0.244, respectively). An improvement of genetic health of the breeds on the basis of allele frequencies is discussed.

Acanthamoeba Species in Tap Water, Egypt

2017 ◽  
Vol 9 (1) ◽  
Author(s):  
Ahmad Z Al-Herrawy ◽  
Mohamed A Marouf ◽  
Mahmoud A. Gad
Keyword(s):  
Water Samples ◽  
Tap Water ◽  
Pcr Analysis ◽  
Pulmonary Infections ◽  
Clinical Syndromes ◽  
Acanthamoeba Species ◽  
Amebic Encephalitis ◽  
Acanthamoeba Culbertsoni ◽  
Autumn And Winter

Genus Acanthamoeba causes 3 clinical syndromes amebic keratitis, granulomatous amebic encephalitis and disseminated granulomatous amebic disease (eg, sinus, skin and pulmonary infections). A total of 144 tap water samples were collected from Giza governorate, Egypt. Samples were processed for detection of Acanthamoeba species using non-nutrient agar (NNA) and were incubated at 30oC. The isolates of Acanthamoeba were identified to species level based on the morphologic criteria. Molecular characterization of the Acanthamoeba isolates to genus level was performed by using PCR. The obtained results showed that the highest occurrence percentage of Acanthamoeba species in water samples was observed in summer season (38.9%), then it decreased to be 30.6% in spring and 25% in each of autumn and winter. PCR analysis showed that 100% of 43 Acanthamoeba morphologically positive samples were positive by genus specific primer. In the present study eight species of Acanthamoeba can be morphologically recognized namely Acanthamoeba triangularis, Acanthamoeba echinulata, Acanthamoeba astronyxis, Acanthamoeba comandoni, Acanthamoeba griffini, Acanthamoeba culbertsoni, Acanthamoeba quina and Acanthamoeba lenticulata. In conclusion, the most common Acanthamoeba species in tap water was Acanthamoeba comandoni

PCR Detection of Alternaria spp. in Processed Foods, Based on the Internal Transcribed Spacer Genetic Marker

2011 ◽  
Vol 74 (2) ◽  
pp. 240-247 ◽  
Author(s):  
MIGUELÁNGEL PAVÓN ◽  
ISABEL GONZÁLEZ ◽  
MARÍA ROJAS ◽  
NICOLETTE PEGELS ◽  
ROSARIO MARTÍN ◽  
...  
Keyword(s):  
Food Processing ◽  
Internal Transcribed Spacer ◽  
Rapid Identification ◽  
Food Samples ◽  
Pcr Analysis ◽  
Infant Food ◽  
Rrna Gene ◽  
Tomato Pulp ◽  
Pcr Method ◽  
Fungal Contaminants

The genus Alternaria is considered one of the most important fungal contaminants of vegetables, fruits, and cereals, producing several mycotoxins that can withstand food processing methods. Conventional methods for Alternaria identification and enumeration are laborious and time-consuming, and they might not detect toxigenic molds inactivated by food processing. In this study, a PCR method has been developed for the rapid identification of Alternaria spp. DNA in foodstuffs, based on oligonucleotide primers targeting the internal transcribed spacer (ITS) 1 and ITS2 regions of the rRNA gene. The specificity of the Alternaria-specific primer pair designed (Dir1ITSAlt–Inv1ITSAlt) was verified by PCR analysis of DNA from various Alternaria spp., and also from several fungal, bacterial, yeast, animal, and plant species. The detection limit of the method was 102 CFU/ml in viable culture, heated culture, or experimentally inoculated tomato pulp. The applicability of the method for detection of Alternaria spp. DNA in foodstuffs was assessed by testing several commercial samples. Alternaria DNA was detected in 100% of spoiled tomato samples, 8% of tomato products, and 36.4% of cereal-based infant food samples analyzed.

scholarly journals Novel Endogenous Retrovirus in Rabbits Previously Reported as Human Retrovirus 5

2002 ◽  
Vol 76 (14) ◽  
pp. 7094-7102 ◽  
Author(s):  
David J. Griffiths ◽  
Cécile Voisset ◽  
Patrick J. W. Venables ◽  
Robin A. Weiss
Keyword(s):  
Sequence Analysis ◽  
Dna Sequences ◽  
Inflammatory Diseases ◽  
Endogenous Retrovirus ◽  
European Rabbit ◽  
Pcr Analysis ◽  
Northern Hybridization ◽  
Human Dna ◽  
Integration Sites ◽  
Human Retrovirus

ABSTRACT Human retrovirus 5 (HRV-5) represented a fragment of a novel retrovirus sequence identified in human RNA and DNA preparations. In this study, the genome of HRV-5 was cloned and sequenced and integration sites were analyzed. Using PCR and Southern hybridization, we showed that HRV-5 is not integrated into human DNA. A survey of other species revealed that HRV-5 is present in the genomic DNA of the European rabbit (Oryctolagus cuniculus) and belongs to an endogenous retrovirus family found in rabbits. The presence of rabbit sequences flanking HRV-5 proviruses in human DNA extracts suggested that rabbit DNA was present in our human extracts, and this was confirmed by PCR analysis that revealed the presence of rabbit mitochondrial DNA sequences in four of five human DNA preparations tested. The origin of the rabbit DNA and HRV-5 in human DNA preparations remains unclear, but laboratory contamination cannot explain the preferential detection of HRV-5 in inflammatory diseases and lymphomas reported previously. This is the first description of a retrovirus genome in rabbits, and sequence analysis shows that it is related to but distinct from A-type retroelements of mice and other rodents. The species distribution of HRV-5 is restricted to rabbits; other species, including other members of the order Lagomorpha, do not contain this sequence. Analysis of HRV-5 expression by Northern hybridization and reverse transcriptase PCR indicates that the virus is transcribed at a low level in many rabbit tissues. In light of these findings we propose that the sequence previously designated HRV-5 should now be denoted RERV-H (for rabbit endogenous retrovirus H).

scholarly journals Simplified Fluorescent Multiplex PCR Method for Evaluation of the T-Cell Receptor Vβ-Chain Repertoire

2005 ◽  
Vol 12 (4) ◽  
pp. 477-483 ◽  
Author(s):  
Sanjit Fernandes ◽  
Surendra Chavan ◽  
Vivek Chitnis ◽  
Nina Kohn ◽  
Savita Pahwa
Keyword(s):  
T Cell ◽  
Multiplex Pcr ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
Clinical Samples ◽  
Cdr3 Length ◽  
Pcr Products ◽  
Pcr Method ◽  
T Cell Receptor Vβ

ABSTRACTRationale: evaluation of the T-cell receptor (TCR) Vβ-chain repertoire by PCR-based CDR3 length analysis allows fine resolution of the usage of the TCR Vβ repertoire and is a sensitive tool to monitor changes in the T-cell compartment. A multiplex PCR method employing 24 labeled upstream Vβ primers instead of the conventionally labeled downstream Cβ primer is described. Method: RNA was isolated from purified CD4 and CD8 T-cell subsets from umbilical cord blood and clinical samples using TRI reagent followed by reverse transcription using a Cβ primer and an Omniscript RT kit. The 24 Vβ primers were multiplexed based on compatibility and product sizes into seven reactions. cDNA was amplified using 24 Vβ primers (labeled with tetrachloro-6-cardoxyfluorescein, 6-carboxyfluorescein, and hexachloro-6-carboxyfluorescein), an unlabeled Cβ primer, and Taqgold polymerase. The fluorescent PCR products were resolved on an automated DNA sequencer and analyzed using the Genotyper 2.1 software. Results: Vβ spectratypes of excellent resolution were obtained with RNA amounts of 250 ng using the labeled Vβ primers. The resolution was superior to that obtained with the labeled Cβ primer assay. Also the numbers of PCRs were reduced to 7 from the 12 required in the Cβ labeling method, and the sample processing time was reduced by half. Conclusion: The method described for T-cell receptor Vβ-chain repertoire analysis eliminates tedious dilutions and results in superior resolution with small amounts of RNA. The fast throughput makes this method suitable for automation and offers the feasibility to perform TCR Vβ repertoire analyses in clinical trials.

scholarly journals Microscopic And Molecular Identification of Anaplasma Ovis in Small Ruminants in Duhok Province in Kurdistan Region of Iraq

2020 ◽  
Vol 23 (2) ◽  
pp. 228-235
Author(s):  
Adnan Ahmed ◽  
Jassim M Abdo
Keyword(s):  
Microscopic Examination ◽  
Prevalence Rate ◽  
Small Ruminants ◽  
Surface Protein ◽  
Positive Sample ◽  
Pcr Analysis ◽  
Anaplasma Ovis ◽  
Blood Samples ◽  
Major Surface Protein ◽  
Major Surface

In last ten years, there has been a developing enthusiasm for microscopic organisms from the genus Anaplasma, particularly the species A. ovis. It is associated with the pathogenic action of these microscopic organisms in livestock. Anaplasma ovis is a tick-borne obligate intracellular rickettsial bacterium that causes anaplasmosis in domestic and wild small ruminants. The samples of the present study were collected from small ruminants from inside seven distinct regions (Akre, Simele, Zummar, Feshchapoor, Deraboon, Bajed Kandal,Karoda)of Duhok province, 389 (goats 75 and sheep 314) during the period of April and May 2018, blood sample were taken and thin smear was formed, after Giemsa’s staining the slide is observed under microscope. In this study used Giemsa stain for microscopic examination out of 389 animals 250 were found positive for Anaplasma ovis infection with a prevalence rate of 64.26 % and 139 of them were negative with a prevalence rate of 35.73 %. According to the species of animals, the highest prevalence of A. ovis infection in animals by using microscopic examination was 67.83 %, 213 positive sample from total 314 blood samples from sheep and lowest prevalence was 49.33 %, 37 positive sample from total 75 blood samples from goats. PCR analysis of 100 blood samples obtained from total 250 positive blood samples after DNA extraction and measure of concentration and purity we used 2 primers that target major surface protein 4 (MSP4) in A. ovis genomic DNA. The results of PCR test with major surface protein 4 primer was 83 samples positive from total 100 samples, According to the species of animals, the highest prevalence of A. ovis was 83.7 %, 72 positive sample from total 86 blood samples from sheep and lowest prevalence was 78.5 %, 11 positive sample from total 14 blood samples from goats.

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