Bleeding canker of horse chestnut (Aesculus hippocastanum) in Ireland: incidence, severity and characterization using DNA sequences and real-time PCR

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species–derived material in concentrate feed mixtures and fish meals.

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PO 8441 EXPERIMENTAL COMPARISON OF SENSITIVITY OF LAMP AND REAL-TIME PCR

BMJ Global Health ◽

10.1136/bmjgh-2019-edc.98 ◽

2019 ◽

Vol 4 (Suppl 3) ◽

pp. A37.3-A38

Author(s):

Jacques Kaboré ◽

Hamidou Ilboudo ◽

Charlie FA Compaoré ◽

Oumou Camara ◽

Mohamed Bamba ◽

...

Keyword(s):

Real Time ◽

Dna Sequences ◽

Filter Paper ◽

Real Time Pcr ◽

Sleeping Sickness ◽

Taqman Probe ◽

Experimental Comparison ◽

Analytical Sensitivity ◽

Molecular Tools ◽

Detection Techniques

BackgroundHuman African trypanosomiasis, or sleeping sickness, remains a serious problem in tropical Africa. Timely diagnosis of this disease requires systematic population screening, particularly for Trypanosoma brucei gambiense, which has a long asymptomatic period.The lack of sensitivity and specificity of conventional diagnostic tests has led in recent years to the use of molecular tools. Amplification of parasite-specific DNA sequences significantly improved diagnosis of infection. However, these molecular tools still have some limitations especially in the case of low parasitaemia. Furthermore, research is still needed to make molecular detection a real control tool for the fight against sleeping sickness. The purpose of this study is to determine the threshold of sensitivity of real-time PCR using the 18S and TgsGp primers and of the LAMP technique, applied in the DiTECT-HAT project as molecular reference tests.MethodsWe used serial dilutions containing 0, 1, 10, 100, 103, 104, 105, 106 parasites per ml of blood. Samples were extracted, and DNA was amplified.ResultsThe analytical sensitivity of the 18S real-time PCR with the Taqman probe of the filter paper samples is 100 parasites/ml and that of the TgsGp real-time PCR with the Taqman probe of filter paper samples is 104 parasites/ml. For Lamp technique, the analytical sensitivity is 103 parasites/ml.ConclusionThis study shows that a ‘negative PCR’ would not mean ‘no parasite’. It suggests that DNA detection techniques should still be improved.

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Application of Quantitative Real-Time PCR in the Detection of Prion-Protein Gene Species-Specific DNA Sequences in Animal Meals and Feedstuffs

Journal of Food Protection ◽

10.4315/0362-028x-69.4.891 ◽

2006 ◽

Vol 69 (4) ◽

pp. 891-896 ◽

Cited By ~ 14

Author(s):

FEDERICA BELLAGAMBA ◽

SERGIO COMINCINI ◽

LUCA FERRETTI ◽

FRANCO VALFRÈ ◽

VITTORIO M. MORETTI

Keyword(s):

Real Time ◽

Prion Protein ◽

Dna Sequences ◽

Real Time Pcr ◽

Animal Feed ◽

Quantitative Estimation ◽

Taqman Assay ◽

Quantitative Real Time Pcr ◽

The Real ◽

Species Specific

This study describes a method for quantitative and species-specific detection of animal DNA from different species (cattle, sheep, goat, swine, and chicken) in animal feed and feed ingredients, including fish meals. A quantitative real-time PCR approach was carried out to characterize species-specific sequences based on the amplification of prion-protein sequence. Prion-protein species-specific primers and TaqMan probes were designed, and amplification protocols were optimized in order to discriminate the different species with short PCR amplicons. The real-time quantitative PCR approach was also compared to conventional species-specific PCR assays. The real-time quantitative assay allowed the detection of 10 pg of ruminant, swine, and poultry DNA extracted from meat samples processed at 130°C for 40 min, 200 kPa. The origin of analyzed animal meals was characterized by the quantitative estimation of ruminant, swine, and poultry DNA. The TaqMan assay was used to quantify ruminant DNA in feedstuffs with 0.1% of meat and bone meal. In conclusion, the proposed molecular approach allowed the detection of species-specific DNA in animal meals and feedstuffs.

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Real-time PCR assays targeting unique DNA sequences of fish-pathogenic Francisella noatunensis subspecies noatunensis and orientalis

Diseases of Aquatic Organisms ◽

10.3354/dao02514 ◽

2012 ◽

Vol 101 (3) ◽

pp. 225-234 ◽

Cited By ~ 15

Author(s):

S Duodu ◽

P Larsson ◽

A Sjödin ◽

E Soto ◽

M Forsman ◽

...

Keyword(s):

Real Time ◽

Dna Sequences ◽

Real Time Pcr ◽

Pcr Assays

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Conventional PCR Detection and Real-Time PCR Quantification of Reniform Nematodes

Plant Disease ◽

10.1094/pdis-12-11-1033-re ◽

2012 ◽

Vol 96 (12) ◽

pp. 1757-1762 ◽

Cited By ~ 8

Author(s):

Ronald J. Sayler ◽

Courtney Walker ◽

Fiona Goggin ◽

Paula Agudelo ◽

Terrence Kirkpatrick

Keyword(s):

Real Time ◽

Dna Sequences ◽

Real Time Pcr ◽

Its Region ◽

Taqman Probe ◽

Reniform Nematode ◽

Rotylenchulus Reniformis ◽

Conventional Pcr ◽

Diagnostic Assays ◽

Reniform Nematodes

Reniform nematode (Rotylenchulus reniformis) is a relatively recent introduction into the continental United States that can cause major yield losses on a variety of important crops including cotton and soybeans. DNA sequences from the internal transcribed spacer (ITS) region of this nematode were used to design primers for conventional and real-time PCR, as well as a TaqMan probe. These primers amplified DNA of reniform nematode isolates from a wide geographic range but did not detect genetically related species or other pathogenic nematodes found in production fields including Meloidogyne incognita and Heterodera glycines. Both SYBR green and TaqMan assays reliably quantified as little as 100 fg of reniform nematode DNA, and could be used to quantify as few as five reniform nematodes. An inexpensive and rapid DNA extraction protocol for high throughput diagnostic assays is described.

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Tentacle Probes™: Differentiation of Difficult Single-Nucleotide Polymorphisms and Deletions by Presence or Absence of a Signal in Real-Time PCR

Clinical Chemistry ◽

10.1373/clinchem.2007.091488 ◽

2007 ◽

Vol 53 (12) ◽

pp. 2042-2050 ◽

Cited By ~ 9

Author(s):

Brent C Satterfield ◽

David A Kulesh ◽

David A Norwood ◽

Leonard P Wasieloski ◽

Michael R Caplan ◽

...

Keyword(s):

Real Time ◽

Dna Sequences ◽

Real Time Pcr ◽

False Positive ◽

Yersinia Pseudotuberculosis ◽

Melting Curve ◽

Melting Curve Analysis ◽

Specific Cell ◽

Single Nucleotide ◽

Positive Results

AbstractBackground: False-positive results are a common problem in real-time PCR identification of DNA sequences that differ from near neighbors by a single-nucleotide polymorphism (SNP) or deletion. Because of a lack of sufficient probe specificity, post-PCR analysis, such as a melting curve, is often required for mutation differentiation.Methods: Tentacle Probes™, cooperative reagents with both a capture and a detection probe based on specific cell-targeting principles, were developed as a replacement for 2 chromosomal TaqMan–minor groove binder (MGB) assays previously developed for Yersinia pestis and Bacillus anthracis detection. We compared TaqMan-MGB probes to Tentacle Probes for SNP and deletion detection based on the presence or absence of a growth curve.Results: With the TaqMan-MGB Y. pestis yp48 assays, false-positive results for Yersinia pseudotuberculosis occurred at every concentration tested, and with the TaqMan-MGB B. anthracis gyrA assays, false-positive results occurred in 21 of 29 boil preps of environmental samples of near neighbors. With Tentacle Probes no false-positive results occurred.Conclusions: The high specificity exhibited by Tentacle Probes may eliminate melting curve analysis for SNP and deletion mutation detection, allowing the diagnostic use of previously difficult targets.

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Detection of Nontuberculous Mycobacterium by Real Time PCR from Variety of Clinical Specimens

Pulse ◽

10.3329/pulse.v9i1.31874 ◽

2017 ◽

Vol 9 (1) ◽

pp. 15-21

Author(s):

MM Rahman ◽

R Rahim ◽

F Nasrin ◽

AH Rasel ◽

A Khaled ◽

...

Keyword(s):

Real Time ◽

Dna Sequences ◽

Real Time Pcr ◽

Respiratory Infections ◽

Multidrug Resistant ◽

Rapid Identification ◽

Nontuberculous Mycobacterium ◽

Specific Primers ◽

Clinical Specimens ◽

Mdr Tb

Aim: Nontuberculous mycobacterium (NTM) causes many types of infections including respiratory and non-respiratory infections such as skin and soft tissue infections, lymphadenitis, meningitis, gastro-intestinal infections, disseminated infections and even intravenous catheter-related infections. Increasing incidence of NTM is reported worldwide in last decade. However, incidence of NTM in Bangladesh is not known as detection of NTM is not undergoing in Bangladesh which is necessary to know as these NTM species are resistant to first-line anti-TB drugs and, when mistaken for M. tuberculosis, give rise to erroneous identification of multidrug-resistant TB (MDR-TB). We wanted to know the existence of NTM from various clinical specimens including tissues from tuberculosis suspected patients visited in Apollo Hospitals Dhaka in 2013 to 2015.Material and Method: Sample processing, DNA extraction and real time PCR (polymerase chain reaction) were done according to the commercial LyteStar TB/NTM PCR kit developed by Altona Diagnostics, Germany. The target DNA sequences are amplified with IS61 10-specific primers for MTB complex and ITS-specific primers for NTM. Probes specific for MTB complex and NTM DNA are labeled with fluorophore dye FAM and HEX, respectively. We have analyzed 579 clinical specimens from tuberculosis suspected patient.Result: Among 579 specimen different types of tissues were 201 and histopathology data were available for 166 cases. In tissues NTM was detected by PCR in 3 1(19%) cases, 8 of which were compatible with histopathology findings and rest 23 cases showed no evidence of granulomatous lesion. We analyzed 378 different varieties of clinical specimens such as sputum, bronchial lavages, body fluids, pus and swabs. Among 378 samples 215 samples were requested for AFB staining. NTM was detected by PCR in 19(8.8%) samples and out of 19 NTM positive specimens only one was AFB positive.Conclusion: This is the first report in the country about detection of NTM in variety of clinical specimens and warrants further elaborate investigation. Our results showed that PCR is an effective tool for the rapid identification of NTM from tissues and AFB negative clinical specimens having suspicion for mycobacterial infection.Pulse Vol.9 January-December 2016 p.15-21

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Bovine ticks harbour a diverse array of microorganisms in Pakistan

Parasites & Vectors ◽

10.1186/s13071-019-3862-4 ◽

2020 ◽

Vol 13 (1) ◽

Cited By ~ 12

Author(s):

Abdul Ghafar ◽

Alejandro Cabezas-Cruz ◽

Clemence Galon ◽

Dasiel Obregon ◽

Robin B. Gasser ◽

...

Keyword(s):

Real Time ◽

High Throughput ◽

Dna Sequences ◽

Real Time Pcr ◽

Distribution Patterns ◽

Diagnostic Methods ◽

Mixed Infections ◽

Ecological Zones ◽

Wide Range ◽

Relationship Of

Abstract Background Ticks and tick-borne pathogens (TTBP) are a major constraint to livestock production in Pakistan; despite a high prevalence of TTBPs, knowledge on the capacity of Pakistani ticks to carry pathogens and endosymbionts is limited. Furthermore, mixed infections with multiple microorganisms further complicate and limit the detection potential of traditional diagnostic methods. The present study investigated the tick-borne microorganisms in bovine ticks in Pakistan, employing a high-throughput microfluidic real-time PCR based technique. Methods Ticks were collected from clinically healthy cattle (n = 116) and water buffaloes (n = 88) from 30 villages across six districts located in five agro-ecological zones (AEZs) of Pakistan from September to November 2017. The microfluidic real-time PCR was used to test the genomic DNA of individual ticks for the presence of 27 bacterial and eight parasitic microorganisms. Phylogenetic methods were used to assess the genetic relationship of DNA sequences determined herein. Results PCR detected DNA of at least one microorganism in each of 221 ticks tested (94.4%, 221/234). DNA-based detection inferred that single pathogens/endosymbionts were the most common (43.4%, 96/221) followed by double (38.9%, 86/221), triple (14.5%, 32/221), quadruple (2.3%, 5/221) and quintuple (0.9%, 2/221) mixed infections. Piroplasms (Babesia/Theileria spp.) were the most prevalent (31.6%, 74/234), followed by Ehrlichia spp. (20%, 47/234) and Anaplasma marginale (7.7%, 18/234). Anaplasma phagocytophilum, A. ovis, A. centrale, Babesia ovis, Borrelia spp., Rickettsia spp., R. massiliae, Bartonella spp. and Hepatozoon spp. were also detected. Endosymbionts such as Francisella-like (91.5%, 214/234) and Coxiella-like (1.3%, 3/234) organisms were also detected in ticks. The highest diversity of microorganisms was detected in Hyalomma anatolicum ticks (test-positive for 14/14 microorganisms), followed by Rhipicephalus microplus (4/14), Hy. hussaini (3/14) and Rh. annulatus (2/14). Ticks collected from cattle carried significantly more frequently piroplasms (41.2%, 54/131; P < 0.05) than those from buffaloes (19.4%, 20/103). However, the overall prevalence of microorganisms did not vary significantly among ticks from the two host species as well as across different AEZs. Conclusions To our knowledge, this is the first study to investigate a wide range of tick-borne microorganisms in bovine ticks using a high-throughput diagnostic method from different AEZs in Pakistan. These findings will aid in establishing the distribution patterns and the control of tick-borne pathogens of bovines in Pakistan.

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The utility and application of real-time PCR and FISH in the detection of single-copy gene targets inEscherichia coliO157:H7 andSalmonellaTyphimurium

Canadian Journal of Microbiology ◽

10.1139/w09-126 ◽

2010 ◽

Vol 56 (3) ◽

pp. 254-262 ◽

Cited By ~ 10

Author(s):

Merriam Haffar ◽

Kimberley A. Gilbride

Keyword(s):

Real Time ◽

Dna Sequences ◽

Real Time Pcr ◽

Receptor Gene ◽

Detection Limits ◽

Single Copy ◽

Target Cells ◽

Single Copy Gene ◽

Gene Copies ◽

Copy Gene

The ultimate specificity in molecular-based assays for pathogen detection relies on the design of the primers and probes. Their ability to hybridize to DNA sequences found only in pathogens can be realized by designing primers and probes that are complementary to pathogen-specific virulence genes. This study evaluates the detection and enumeration strengths of real-time PCR (qPCR) and fluorescent in situ hybridization (FISH) for selected waterborne pathogens and their ultimate applicability within a monitoring framework. Detection limits calculated in the qPCR assay were 150 tir (intimin protein receptor) gene copies for Escherichia coli O157:H7 and 2 × 103invA (inner membrane invasive protein) gene copies for Salmonella enterica serovar Typhimurium. Detection limits were, however, at least 100-fold less sensitive in wastewater extracts, partly because of the inhibitory effect of the wastewater itself. Fluorescent signals from hybridized whole target cells were below the detection limit of the FISH assay. While this research demonstrates the potential detection strength of qPCR, it highlights the need for strong dependable primer and probe sets among PCR and FISH methodologies as well as the need for further signal amplification with DNA-targeted FISH for single-copy gene targets within environmental samples.

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