scholarly journals The Evaluation of Gastrointestinal Nematodes in Faecal Samples Using Multiplex Real-Time PCR Assays

2021 ◽  
Author(s):  
Nikol Reslová ◽  
Lucie Skorpikova ◽  
Iveta Angela Kyrianova ◽  
Jaroslav Vadlejch ◽  
Johan Höglund ◽  
...  
Keyword(s):  
Real Time ◽  
Species Identification ◽  
Real Time Pcr ◽  
Quantitative Estimation ◽  
False Negative ◽  
Infection Intensity ◽  
Multiplex Assays ◽  
Ashworthius Sidemi ◽  
Faecal Samples ◽  
Pcr Assays

Abstract Background: The diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods and followed by larval culture techniques. Such an approach is laborious, time-consuming, requires a skilled expert and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identification of nematode species and, therefore, may be valuable in sustainable parasite control strategies.Methods: Two multiplex real-time PCR assays for specific detection of six main GIN species, including an internal amplification control to avoid false negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi‑quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms and results were compared to those from faecal egg counts (FEC) using the Concentration McMaster technique, and larval cultures (LC).Results: Multiplex real-time PCR assays showed great specificity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing 3 false negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp.Conclusions: Our multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identification from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic efficacy, where it is important to identify species surviving after treatment.

scholarly journals The identification and semi-quantitative assessment of gastrointestinal nematodes in faecal samples using multiplex real-time PCR assays

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Nikol Reslova ◽  
Lucie Skorpikova ◽  
Iveta Angela Kyrianova ◽  
Jaroslav Vadlejch ◽  
Johan Höglund ◽  
...  
Keyword(s):  
Real Time ◽  
Species Identification ◽  
Real Time Pcr ◽  
Quantitative Estimation ◽  
False Negative ◽  
Infection Intensity ◽  
Multiplex Assays ◽  
Ashworthius Sidemi ◽  
Faecal Samples ◽  
Pcr Assays

Abstract Background The diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods, followed by larval culture (LC) techniques. Such an approach is laborious, time-consuming, requires a skilled expert, and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identification of nematode species and, therefore, may be valuable in sustainable parasite control strategies. Methods Two multiplex real-time polymerase chain reaction (PCR) assays for specific detection of five main and one invasive GIN species, including an internal amplification control to avoid false-negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi-quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms, and results were compared to those from faecal egg counts (FEC) using the concentration McMaster technique and LC. Results Multiplex real-time PCR assays showed great specificity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing three false-negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp. Conclusions Our multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identification from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic efficacy, where it is important to identify species surviving after treatment. Graphical Abstract

scholarly journals Simple Technique for Internal Control of Real-Time Amplification Assays

2004 ◽  
Vol 50 (5) ◽  
pp. 819-825 ◽  
Author(s):  
Siegfried Burggraf ◽  
Bernhard Olgemöller
Keyword(s):  
Melting Point ◽  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
False Negative ◽  
Hybridization Probe ◽  
Melting Point Analysis ◽  
Point Analysis ◽  
Pcr Assays ◽  
Time Amplification

Abstract Background: In real-time PCR assays, the most accurate way to identify false-negative results, e.g., those caused by PCR inhibitors, is to add to samples an internal control that will be coamplified with the target (e.g., pathogen) DNA. Current internal control procedures, however, which usually involve the introduction of a DNA fragment, are complex, time-consuming, and expensive. Methods: Single-stranded oligonucleotides, which contain little more than primer and probe binding sites, were used as internal controls in real-time PCR assays. Mismatches were included in the probe-binding region of the internal control oligonucleotide (ICO) to prevent probe–control hybridization during the fluorescence acquisition step of the PCR. Amplified ICOs were detected by melting point analysis. ICOs could be added directly to the sample material before DNA extraction. Results: To demonstrate the feasibility of the new approach, we designed ICOs for the LightCycler hybridization probe assays for Mycobacterium tuberculosis complex, hepatitis B virus, herpes simplex virus, and varicella zoster virus. In each case, the controls did not interfere with detection of the pathogen, but were clearly detectable during a subsequent melting point analysis. Conclusions: A single-stranded oligonucleotide that mimics the target region of the pathogen but is clearly distinguishable from the target during melting point analysis can serve as a simple, cost-effective internal control for real-time amplification assays. Such control oligonucleotides are easy to design and inexpensive. A costly second probe system is not necessary. Moreover, the internally controlled assay uses only one fluorescence detection channel of the instrument, leaving the second channel free for multiplex applications.

Real-time PCR assays for strain-specific quantification of probiotic strains in human faecal samples

2012 ◽  
Vol 27 (1-2) ◽  
pp. 58-64 ◽  
Author(s):  
Heli Karjalainen ◽  
Terhi Ahlroos ◽  
Eveliina Myllyluoma ◽  
Soile Tynkkynen
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Faecal Samples ◽  
Probiotic Strains ◽  
Pcr Assays

scholarly journals Development and application of a canine endogenous internal positive control for use in real-time PCR assays

2018 ◽  
Vol 30 (5) ◽  
pp. 789-792 ◽  
Author(s):  
Joseph J. Modarelli ◽  
Pamela J. Ferro ◽  
Maria D. Esteve-Gasent
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Positive Control ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Negative Results ◽  
False Negative Results ◽  
Internal Positive Control ◽  
Pcr Assays

Real-time PCR (rtPCR) tests have become a method of choice in many diagnostic settings, both animal and human. A concern remains, however, regarding rtPCR assay inhibition during nucleic acid extraction and/or rtPCR reaction process that may result in false-negative results. The use of an internal positive control, either endogenous or exogenous, to mitigate this issue has become more commonplace. We identified and standardized an endogenous internal positive control that can be utilized in rtPCR assays targeting canine-specific pathogens in either a singleplex or multiplex format. The target chosen for the endogenous internal positive control (EIPC-K9) was a highly conserved region in canine mitochondrial DNA. Samples from 240 dogs and 11 other species were screened with EIPC-K9; all canine samples were detected, and no cross-amplification with other species tested was observed. Additionally, no inhibition was noted when comparing singleplex to multiplex rtPCR formats.

scholarly journals Pentaplexed Quantitative Real-Time PCR Assay for the Simultaneous Detection and Quantification of Botulinum Neurotoxin-Producing Clostridia in Food and Clinical Samples

2010 ◽  
Vol 76 (13) ◽  
pp. 4387-4395 ◽  
Author(s):  
Sebastian Kirchner ◽  
K. Melanie Krämer ◽  
Martin Schulze ◽  
Diana Pauly ◽  
Daniela Jacob ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Target Genes ◽  
False Negative ◽  
Simultaneous Detection ◽  
Locked Nucleic Acid ◽  
Clinical Samples ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Pcr Assays

ABSTRACT Botulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 103 and 105 CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F.

DEVELOPMENT OF MULTIPLEX REAL-TIME PCR ASSAY FOR SIX PATHOGENS: RAPID TOOL FOR DIAGNOSIS OF BACTERIAL MENINGITIS

2019 ◽  
pp. 60-66
Author(s):  
Viet Quynh Tram Ngo ◽  
Thi Ti Na Nguyen ◽  
Hoang Bach Nguyen ◽  
Thi Tuyet Ngoc Tran ◽  
Thi Nam Lien Nguyen ◽  
...  
Keyword(s):  
Bacterial Meningitis ◽  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Methods ◽  
Type B ◽  
E Coli ◽  
Pcr Assays ◽  
Set Up ◽  
Etiological Agents ◽  
Suis Serotype

Introduction: Bacterial meningitis is an acute central nervous infection with high mortality or permanent neurological sequelae if remained undiagnosed. However, traditional diagnostic methods for bacterial meningitis pose challenge in prompt and precise identification of causative agents. Aims: The present study will therefore aim to set up in-house PCR assays for diagnosis of six pathogens causing the disease including H. influenzae type b, S. pneumoniae, N. meningitidis, S. suis serotype 2, E. coli and S. aureus. Methods: inhouse PCR assays for detecting six above-mentioned bacteria were optimized after specific pairs of primers and probes collected from the reliable literature resources and then were performed for cerebrospinal fluid (CSF) samples from patients with suspected meningitis in Hue Hospitals. Results: The set of four PCR assays was developed including a multiplex real-time PCR for S. suis serotype 2, H. influenzae type b and N. meningitides; three monoplex real-time PCRs for E. coli, S. aureus and S. pneumoniae. Application of the in-house PCRs for 116 CSF samples, the results indicated that 48 (39.7%) cases were positive with S. suis serotype 2; one case was positive with H. influenzae type b; 4 cases were positive with E. coli; pneumococcal meningitis were 19 (16.4%) cases, meningitis with S. aureus and N. meningitidis were not observed in any CSF samples in this study. Conclusion: our in-house real-time PCR assays are rapid, sensitive and specific tools for routine diagnosis to detect six mentioned above meningitis etiological agents. Key words: Bacterial meningitis, etiological agents, multiplex real-time PCR

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