scholarly journals RAPID PCR–BASED DETECTION OPTIMIZATION OF PORCINE DNA IN GELATIN CAPSULE SHELL

2018 ◽  
Vol 10 (6) ◽  
pp. 217
Author(s):  
Amalia Sitti Khayyira ◽  
Viktoria Mardhika Estepane ◽  
Amarila Malik
Keyword(s):  
Molecular Detection ◽  
Universal Primers ◽  
Gelatin Capsule ◽  
Duplex Pcr ◽  
Rapid Pcr ◽  
Capsule Shell ◽  
Pcr Rflp ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Bovine Species

Objective: This study was conducted to optimize the genomic deoxyribonucleic acid (DNA) based molecular detection of gelatin derived from porcine by performing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and duplex PCR method employing cyt B gene.Methods: Optimization was carried out for DNA extraction, PCR conditions, and the sensitivity of the PCR-RFLP method. Due to the very low DNA trace in gelatin after the various manufacturing process, the extraction was optimized to obtain sufficient DNA which was visible on the agarose gel. PCR-RFLP was carried out using universal primers and BsaJI restriction enzyme, and duplex PCR was carried out using two sets of porcine-specific primers. Porcine and bovine DNA were mixed in various concentration to confirm sensitivity of both methods, i.e. 100%, 50%, 10%, 1%, 0.5%, 0.1%, 0.05%, and 0.01%Results: Both methods, PCR-RFLP, and Duplex PCR, were able to detect as low as 0.01% porcine DNA, indicated by the presence of porcine DNA amplicon bands (131 bp and 228 bp for PCR-RFLP, 212 bp and 398 bp for duplex PCR). Although DNA bands presented in low intensity, identification of porcine and bovine species and estimation of DNA quantities were possible.Conclusion: Both conventional PCR methods, i.e. PCR-RFLP and Duplex PCR, were sensitive, specific, and suitable as a rapid initial detection method for molecular detection of porcine in gelatin capsule shells.

scholarly journals Identification of Eutypa lata by PCR-RFLP

Plant Disease ◽  
2004 ◽  
Vol 88 (9) ◽  
pp. 925-929 ◽  
Author(s):  
P. E. Rolshausen ◽  
F. Trouillas ◽  
W. D. Gubler
Keyword(s):  
Universal Primers ◽  
Phaeomoniella Chlamydospora ◽  
Phomopsis Viticola ◽  
Eutypa Lata ◽  
Incorrect Diagnosis ◽  
Pcr Rflp ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Rflp Assay

Eutypa lata is a vascular canker pathogen of woody plants commonly diagnosed by isolating the pathogen from infected tissue. Related fungi from the same family, the Diatrypaceae, also have been found in association with grapevine in Californian vineyards. An in situ polymerase chain reaction (PCR) method has been developed for detection of E. lata in infected wood tissue. However, our results indicate that this method also would amplify other Diatrypaceous fungi, which could potentially lead to an incorrect diagnosis. Therefore, we developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay. The internal transcribed spacer (ITS)1/5.8S/ITS2 ribosomal DNA region was amplified by PCR using universal primers, and RFLP patterns were determined after digestion with AluI. The restriction profiles obtained served to distinguish E. lata from wood trunk pathogens of grapevine (Phomopsis viticola, Botryodiplodia sp., Phaeoacremonium aleophilum, and Phaeomoniella chlamydospora), Diatrypaceous fungi (Diatrype sp., Diatrypella sp., Eutypella vitis, and Eutypa leptoplaca), and Cryptovalsa sp. found on dead wood of grapevine, and other Eutypa spp. (E.petrakii var. hederae, E. astroidea, E. crustata, and E. lejoplaca), with the exception of E. armeniacae, which we regard as a synonym for E. lata, and E. laevata, which has not been found on grapevine.

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.

Levels of Ancylostoma infections and phylogenetic analysis of cox 1 gene of A. ceylanicum in stray cat faecal samples from Guangzhou, China

2015 ◽  
Vol 90 (4) ◽  
pp. 392-397 ◽  
Author(s):  
W. Hu ◽  
X.G. Yu ◽  
S. Wu ◽  
L.P. Tan ◽  
M.R. Song ◽  
...  
Keyword(s):  
South China ◽  
Potential Risk ◽  
Stray Cats ◽  
Faecal Samples ◽  
High Prevalence ◽  
Pcr Rflp ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Stray Cat ◽  
A Minor

AbstractAncylostoma ceylanicum is a common zoonotic nematode. Cats act as natural reservoirs of the hookworm and are involved in transmitting infection to humans, thus posing a potential risk to public health. The prevalence of feline A. ceylanicum in Guangzhou (South China) was surveyed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). In total, 112 faecal samples were examined; 34.8% (39/112) and 43.8% (49/112) samples were positive with hookworms by microscopy and PCR method, respectively. Among them, 40.8% of samples harboured A. ceylanicum. Twelve positive A. ceylanicum samples were selected randomly and used for cox 1 sequence analysis. Sequencing results revealed that they had 97–99% similarity with A. ceylanicumcox 1 gene sequences deposited in GenBank. A phylogenetic tree showed that A. ceylanicum isolates were divided into two groups: one comprising four isolates from Guangzhou (South China), and the other comprising those from Malaysia, Cambodia and Guangzhou. In the latter group, all A. ceylanicum isolates from Guangzhou were clustered into a minor group again. The results indicate that the high prevalence of A. ceylanicum in stray cats in South China poses a potential risk of hookworm transmission from pet cats to humans, and that A. ceylanicum may be a species complex worldwide.

Rapid Molecular Typing of Methicillin-ResistantStaphylococcus aureusby PCR-RFLP

2001 ◽  
Vol 22 (5) ◽  
pp. 294-298 ◽  
Author(s):  
Thomas A. Wichelhaus ◽  
Klaus-Peter Hunfeld ◽  
Boris Böddinghaus ◽  
Peter Kraiczy ◽  
Volker Schàfer ◽  
...  
Keyword(s):  
Fragment Length Polymorphism ◽  
Restriction Analysis ◽  
Protein A ◽  
Hypervariable Region ◽  
Epidemiological Surveillance ◽  
Typing Method ◽  
Rapid Pcr ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Rflp Typing

AbstractObjective:To establish a new, rapid, and reliable genotypic fingerprinting technique for methicillin-resistantStaphylococcus aureus(MRSA) typing in routine epidemiological surveillance.Design:The method is based on polymerase chain reaction (PCR) restriction fragment-length polymorphism (RFLP) followingHaeII digestion of simultaneously amplified parts of the protein A gene, the coagulase gene, and the hypervariable region adjacent tomecA. A total of 46 MRSA initial isolates were analyzed, including 14 isolates from five countries; the six German epidemic strains; 16 isolates from the Frankfurt metropolitan area, which were known to be heterogeneous by pulsed-field gel electrophoresis (PFGE); and 10 isolates obtained during three epidemics, all of which displayed an identical genotype.Results:Restriction analysis by PCR-RFLP permitted discrimination of 10 of 14 international isolates, all six German epidemic strains, and 15 of 16 national isolates. It also confirmed the homogeneous character of the 10 outbreak isolates.Conclusions:This new and rapid PCR-RFLP typing method is an attractive tool in routine epidemiological surveillance. Its impressive characteristics are ease of performance and interpretation, while at the same time guaranteeing good discriminatory power, reproducibility, and typeability.

scholarly journals Molecular Detection of Plasmid-Mediated Quinolone Resistance in Ciprofloxacin-Resistant Escherichia coli from Urine of Patients attending Garki Hospital, Abuja, Nigeria

2020 ◽  
Vol 1 (4) ◽  
Author(s):  
Moses Oghenaigah Eghieye ◽  
Istifanus Haruna Nkene ◽  
Rejoice Helma Abimiku ◽  
Yakubu Boyi Ngwai ◽  
Ibrahim Yahaya ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Urinary Tract ◽  
Molecular Detection ◽  
Quinolone Resistance ◽  
Chain Reaction ◽  
E Coli ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Tract Infections

Urinary tract infections (UTIs) caused by Escherichia coli (E. coli) is common worldwide; and its successful treatment using antibiotics is limited by acquisition of resistance by the bacteria. This study investigated the occurrence of plasmid-mediated quinolone resistance (PMQR) genes in ciprofloxacin-resistant E. coli from urine of patients with suspected cases of UTIs attending Garki Hospital Abuja (GHA), Nigeria. A total of 8 confirmed ciprofloxacin-resistant E. coli was screened for carriage of PMQR genes using polymerase chain reaction (PCR) method. The occurrences of the PMQR genes detected were in the order: aac-(6′)-Ib-cr (87.5%) > qnrB (50.0%) > qnrS (37.5%) > oqxAB (12.5%) > qnrA(0.0%). qnrB and qnrS did not exist alone, but in combination with other genes; aac-(6′)-Ib-crexisted both alone and in combination with others; the most prevalent patterns of existence were aac-(6′)-Ib-cr alone and aac-(6′)-Ib-cr + qnrB + qnrS at 25.0% each. This study has shown that the ciprofloxacin-resistant E. coli harbored aac-(6′)-Ib-cr, qnrB, qnrS and oqxAB PMQR genes, with aac-(6′)-Ib-cr being the most prevalent. The genes were present either alone or in combination with one another. This has implication for the clinical application of fluoroquinolones to treat UTI in the study location and environs. 

scholarly journals Molecular Detection and PCR-RFLP Analysis of Mucoviscosity-Associated Gene A (magA) in Clinical Isolates of Multidrug-Resistant Klebsiella pneumoniae in Bangladesh

2020 ◽  
Vol 14 (1) ◽  
pp. 196-204
Author(s):  
Md. Hazrat Ali ◽  
Saeed Anwar ◽  
Nusrat Jahan Toma ◽  
Ikram Rafid ◽  
Md. Kamrul Hasan ◽  
...  
Keyword(s):  
Molecular Detection ◽  
Clinical Specimen ◽  
Rflp Analysis ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Specific Gene ◽  
Multicenter Cohort ◽  
Pcr Rflp ◽  
Invasive Infections ◽  
Polymerase Chain

Background and Objective: The mucoviscosity associated gene A (magA) in the hypermucoviscous variants of K. pneumoniae is reported to be associated with invasive infections and considered a virulence factor. We sought to analyze the magA genes in K. pneumoniae isolates in the clinical specimen collected from Bangladesh. Methods: We established a multicenter cohort of patients with Klebsiella infection hospitalized at 05 different hospitals between September 2016 and April 2017. We collected 313 K. pneumoniae isolates from patients who consented to participate in the study. The isolates were evaluated for harboring the magA genes using a single-tube multiplexed polymerase chain reaction. The magA genes were analyzed by PCR-RFLP technique using two enzymes, namely PciI and SmaI. Antibiogram assay using 12 commercially available antibiotic discs was performed on all the isolates. Results: The presence of K. pneumoniae specific gene (ureD) was confirmed in all the isolates. The percentage of isolates harboring the magA gene was 7.34%(23 isolates), the majority of which was collected from the patients admitted in intensive care units (16 isolates, 69.6%), and infectious diseases wards (5 isolates, 21.7%). PCR-RFLP analysis revealed that for 7 out of 23 isolates, where Sma1 could not cleave the magA gene. All the isolates showed resistance to ampicillin, carbenicillin cefradine, chloramphenicol, erythromycin, kanamycin, and sulphamethoxazole, though the extent was varying. However, imipenem showed 100% sensitivity to all the tested isolates. Conclusion: This study demonstrates the presence of the magA gene in multidrug-resistant clinical isolates of K. pneumoniae collected from Bangladesh.

scholarly journals Pathology of Fowl Paratyphoid and Molecular Detection of Its Pathogen in Layer Chickens

2021 ◽  
Vol 24 (2) ◽  
pp. 47-57
Author(s):  
J Alam ◽  
T Chakma ◽  
MS Islam ◽  
MT Islam ◽  
MAHNA ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Salmonella Enteritidis ◽  
Molecular Detection ◽  
Inflammatory Cells ◽  
Chain Reaction ◽  
Biochemical Tests ◽  
Salmonella Spp ◽  
Gross Lesions ◽  
Pcr Method ◽  
Polymerase Chain

The study was aimed to ascertain the pathology of fowl paratyphoid and molecular detection of its causal agent (Salmonella spp) in chickens. Pathological and swab samples were collected from layers in Gazipur district, Bangladesh. For observing the gross and microscopic lesions of different organs necropsy and histopathology were done, and to isolate and identify the Salmonella spp, different bacteriological tests and Polymerase Chain Reaction (PCR) were performed. Swabs from 150 chickens showed 66% of salmonellosis. Gram’s staining of isolated bacteria showed pink colored rod shaped bacilli. In biochemical tests, Salmonella fermented dextrose, maltose, xylose, arabinose, dulcitol, mannitol except lactose and sucrose. Investigation of gross lesions at necropsy revealed hemorrhage and congestion in intestine, liver, spleen and ovaries. Necrotic foci were found in liver and spleen, and button like ulceration in cecal tonsils as well. Microscopic lesions included hemorrhage and focal necrosis in liver and spleen. Congestion and infiltrations of inflammatory cells were observed in small intestine. Ovary was hemorrhagic and there was infiltration of heterophils. Biochemically positive and isolated Salmonella organisms were confirmed by PCR method using invA and IE1 primers. The final results showed that a total of 91.7% Salmonella suspected cultures were confirmed as Salmonella Enteritidis. Ann. Bangladesh Agric. (2020) 24(2) : 47-57

scholarly journals Comparison of Three Strategies for Sequencing Rabies Viral G Gene

2013 ◽  
Vol 2 (1) ◽  
Author(s):  
Shengli Meng ◽  
Wenli Lv ◽  
Jie Wu ◽  
Jilin Wang ◽  
Gelin Xu ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Low Cost ◽  
Cost Effective ◽  
Brain Homogenate ◽  
Universal Primers ◽  
Viral Gene ◽  
Gene Sequences ◽  
Rt Pcr ◽  
Pcr Method ◽  
Polymerase Chain

<p>It is essential to rapidly, low-cost and precisely determining gene sequences of rabies virus. Reverse transcription-polymerase chain reaction can be used to identify rabies viral G gene sequences. In this study, we evaluated three methods, conventional RT-PCR and direct sequencing, adapter RT-PCR and sequencing with universal primers, conventional RT-PCR and cloning sequencing with universal primers, to detect rabies in animal brain homogenate. Four rabies isolates recovered from Fuyang city of Anhui province were diagnosed as positive using the fluorescentantibody test, rapid rabies enzyme immunodiagnosis methods and the mouse inoculation test. The results indicated that the adapter RT-PCR method and sequencing with universal primers is extremely well suited for sequencing rabies viral gene, which is rapid, cost-effective and precise compared with other two methods.<strong></strong></p>

scholarly journals A New Member of the Clover Proliferation Phytoplasma Group (16SrVI) Associated with Elm Yellows in Illinois

Plant Disease ◽  
2003 ◽  
Vol 87 (3) ◽  
pp. 241-246 ◽  
Author(s):  
K. A. Jacobs ◽  
I.-M. Lee ◽  
H. M. Griffiths ◽  
F. D. Miller ◽  
K. D. Bottner
Keyword(s):  
Disease Severity ◽  
Universal Primers ◽  
Specific Primers ◽  
Detection Rates ◽  
Ulmus Americana ◽  
Disease Symptoms ◽  
Lower Trunk ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Elm Yellows

A disease with symptoms similar to elm yellows (EY) was noticed in the early 1990s in suburban Chicago, IL. More than 1,000 mature American elms (Ulmus americana) have since died. Infected trees varied in the incidence and severity of canopy yellowing, leaf epinasty, butterscotch discoloration, and wintergreen odor of the phloem, but all developed a sparse and clumpy crown, uniformly necrotic phloem, and died within 2 years of showing canopy symptoms. Because symptoms were expressed irregularly and phytoplasma detection results by a commercial diagnostic company were inconsistent, a study was initiated to determine if EY phytoplasma was the causal agent. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods using universal or EY phytoplasma specific primers were employed to detect putative phytoplasma(s) associated with 10 trees of varied disease severity within the outbreak region and 10 asymptomatic trees from an uninfected area (controls). Nested PCR using universal primers revealed that 90% of trees from the outbreak region were positive for phytoplasma while asymptomatic elms from another location (controls) tested negative. Phytoplasma-positive trees ranged in disease severity from 1 (asymptomatic) to 5 (near death). Inner bark samples chiseled from the lower trunk had higher phytoplasma detection rates than foliage or drill shavings. RFLP analyses and DNA sequencing of 16S rDNA indicated that the phytoplasma recovered from dying elms in Arlington Heights is not related to the reference EY phytoplasma (group16SrV). It is most closely related to clover proliferation (CP) phy-toplasma (group 16SrVI), and we have designated it Illinois Elm Yellows (ILEY) phytoplasma, and assigned it to a new taxonomic subgroup (16SrVI-C). EY phytoplasma was not detected in any samples, but two ILEY phytoplasma positive trees also were positive for aster yellows (AY) phytoplasma. ILEY phytoplasma was not detected in local leafhopper populations trapped in elm trees between May and September 2000. This is the first report of a phytoplasma related to CP phytoplasma causing elm yellows disease symptoms.

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