A critical assessment of two real-time PCR assays targeting the (SSU) rRNA and gdh genes for the molecular identification of Giardia intestinalis in a clinical laboratory

IntroductionGiardiasis is an intestinal diarrhoeal illness caused by the flagellate protozoan parasite Giardia intestinalis. Molecular techniques for the identification of G. intestinalis have generally been shown to offer a better detection rate of the parasite than the traditional faecal concentration and microscopy techniques.AimThe aim of this study was to critically assess the performance of a commercial and a published real-time PCR assay for their potential use as frontline tests for the diagnosis of giardiasis.MethodsA composite reference standard of enzyme immunoassay and rapid membrane test was used in a diagnostic accuracy study to assess the performance of Primerdesign's, and Verweij et alG. intestinalis real-time PCR assays, comparing them with the traditional ova, cysts and parasite microscopy test (OCP-M).ResultsThe Verweij real-time PCR used primers for the (SSU) rRNA gene, and produced a diagnostic sensitivity of 93.4% (95% CI 88.30% to 98.50%) and an efficiency of 100%. Primerdesign's real-time PCR used primers for the glutamate dehydrogenase gene and produced a diagnostic sensitivity of 61.5% (95% CI 51.50% to 71.50%) and an efficiency of 203%. The OCP-M sensitivity was 83.5% (95% CI 75.87% to 91.13%).ConclusionsThe Verweij real-time PCR was robust and the most sensitive assay suited for use as a first-line diagnostic test for giardiasis.

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Comparison of Three Real-Time PCR Assays Targeting the SSU rRNA Gene, the COWP Gene and the DnaJ-Like Protein Gene for the Diagnosis of Cryptosporidium spp. in Stool Samples

Pathogens ◽

10.3390/pathogens10091131 ◽

2021 ◽

Vol 10 (9) ◽

pp. 1131

Author(s):

Felix Weinreich ◽

Andreas Hahn ◽

Kirsten Alexandra Eberhardt ◽

Torsten Feldt ◽

Fred Stephen Sarfo ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Protein Gene ◽

Molecular Diagnostic ◽

Rrna Gene ◽

Ssu Rrna ◽

Ssu Rrna Gene ◽

Cryptosporidium Spp ◽

Stool Samples ◽

Pcr Assays

As qualified microscopy of enteric parasitoses as defined by high diagnostic accuracy is difficult to maintain in non-endemic areas due to scarce opportunities for practicing with positive sample materials, molecular diagnostic options provide less investigator-dependent alternatives. Here, we compared three molecular targets for the real-time PCR-based detection of Cryptosporidium spp. From a population of 1000 individuals comprising both Ghanaian HIV (human immunodeficiency virus) patients and military returnees after deployment in the tropics, stool samples were assessed for Cryptosporidium spp. by real-time PCR targeting the small subunit ribosomal RNA (SSU rRNA) gene, the Cryptosporidium oocyst wall (COWP) gene, and the DnaJ-like protein gene (DnaJ), respectively. In declining order, sensitivity of 100% for the SSU rRNA gene PCR, 90.0% for the COWP PCR and 88.8% for the DnaJ PCR, respectively, as well as specificity of 99.6% for the COWP PCR and 96.9% for both the SSU rRNA gene PCR and the DnaJ PCR, respectively, were recorded. Substantial agreement (kappa value 0.663) between the three assays was observed. Further, an accuracy-adjusted Cryptosporidium spp. prevalence of 6.0% was calculated for the study population. In conclusion, none of the assessed real-time PCR assays were associated with perfect test accuracy. However, a combination of highly sensitive SSU rRNA gene PCR for screening purposes and more specific COWP PCR for confirmatory testing should allow reliable diagnosis of Cryptosporidium spp. in stool samples even in low prevalence settings.

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Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR

Virology Journal ◽

10.1186/s12985-021-01541-z ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Ellen Kathrin Link ◽

Matthias Eddicks ◽

Liangliang Nan ◽

Mathias Ritzmann ◽

Gerd Sutter ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Diagnostic Sensitivity ◽

Locked Nucleic Acid ◽

Porcine Circovirus ◽

Amplification Efficiency ◽

Pcr Assays

Abstract Background The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR. Methods Based on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes. Results All eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g. Conclusion Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers.

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Detection of Four Plasmodium Species in Blood from Humans by 18S rRNA Gene Subunit-Based and Species-Specific Real-Time PCR Assays

Journal of Clinical Microbiology ◽

10.1128/jcm.42.12.5636-5643.2004 ◽

2004 ◽

Vol 42 (12) ◽

pp. 5636-5643 ◽

Cited By ~ 215

Author(s):

M. Rougemont ◽

M. Van Saanen ◽

R. Sahli ◽

H. P. Hinrikson ◽

J. Bille ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

18S Rrna ◽

Plasmodium Species ◽

18S Rrna Gene ◽

Rrna Gene ◽

Pcr Assays ◽

Species Specific

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Detection of Giardia intestinalis in water samples collected from natural water reservoirs and wells in northern and north-eastern Poland using LAMP, real-time PCR and nested PCR

Journal of Water and Health ◽

10.2166/wh.2017.039 ◽

2017 ◽

Vol 15 (5) ◽

pp. 775-787 ◽

Cited By ~ 8

Author(s):

Anna Lass ◽

Beata Szostakowska ◽

Krzysztof Korzeniewski ◽

Panagiotis Karanis

Keyword(s):

Surface Water ◽

Real Time ◽

Water Samples ◽

Real Time Pcr ◽

Nested Pcr ◽

Lamp Assay ◽

Giardia Intestinalis ◽

Detection Methods ◽

Rrna Gene ◽

Water Reservoirs

Giardia intestinalis is a protozoan parasite, transmitted to humans and animals by the faecal–oral route, mainly through contaminated water and food. Knowledge about the distribution of this parasite in surface water in Poland is fragmentary and incomplete. Accordingly, 36 environmental water samples taken from surface water reservoirs and wells were collected in Pomerania and Warmia-Masuria provinces, Poland. The 50 L samples were filtered and subsequently analysed with three molecular detection methods: loop-mediated isothermal amplification (LAMP), real-time polymerase chain reaction (real-time PCR) and nested PCR. Of the samples examined, Giardia DNA was found in 15 (42%) samples with the use of LAMP; in 12 (33%) of these samples, Giardia DNA from this parasite was also detected using real-time PCR; and in 9 (25%) using nested PCR. Sequencing of selected positive samples confirmed that the PCR products were fragments of the Giardia intestinalis small subunit rRNA gene. Genotyping using multiplex real-time PCR indicated the presence of assemblages A and B, with the latter predominating. The results indicate that surface water in Poland, as well as water taken from surface wells, may be a source of Giardia strains which are potentially pathogenic for humans. It was also demonstrated that LAMP assay is more sensitive than the other two molecular assays.

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Design and testing of real-time PCR primers for the quantification ofMethanoculleus,Methanosarcina,Methanothermobacter, and a group of uncultured methanogens

Canadian Journal of Microbiology ◽

10.1139/w08-157 ◽

2009 ◽

Vol 55 (5) ◽

pp. 611-616 ◽

Cited By ~ 32

Author(s):

Ingrid H. Franke-Whittle ◽

Marta Goberna ◽

Heribert Insam

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Biogas Production ◽

Pcr Primers ◽

Sybr Green ◽

Rrna Gene ◽

Pcr Assays ◽

Design And Testing ◽

Uncultured Archaea ◽

Detection And Quantification

In this study, 16S rRNA gene primers were designed to complement the suite of already available PCR primers for the detection of different methanogens involved in biogas production through anaerobic digestion by SYBR Green real-time PCR. Primers designed for use in TaqMan real-time PCR for the organisms Methanosaeta , Methanosarcina , and Methanoculleus have been described previously; however, we found that (i) the Methanoculleus primers were not specific to members of the genus and that (ii) the Methanosarcina primers did not work specifically with SYBR Green real-time PCR. Thus, we designed new primers for these and other methanogens, and we optimized SYBR Green real-time PCR assays. Primers were tested by end-point and real-time PCR, and they were found to work specifically and sensitively. Application of these primers will allow the detection and quantification of Methanoculleus, Methanosarcina, Methanothermobacter , and a group of yet uncultured archaea from anaerobic habitats.

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Validation of two commercial real-time PCR assays for rapid screening of the HLA-B*57:01 allele in the HIV clinical laboratory

Journal of Virological Methods ◽

10.1016/j.jviromet.2016.08.013 ◽

2016 ◽

Vol 237 ◽

pp. 18-24 ◽

Cited By ~ 1

Author(s):

Boaz Avidor ◽

Shirley Girshengorn ◽

Liran Giladi ◽

Shoshana Israel ◽

Rina Katz ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Clinical Laboratory ◽

Rapid Screening ◽

Pcr Assays

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Considerations for incorporating real-time PCR assays into routine marine biosecurity surveillance programmes: a case study targeting the Mediterranean fanworm (Sabella spallanzanii) and club tunicate (Styela clava)

Genome ◽

10.1139/gen-2018-0021 ◽

2019 ◽

Vol 62 (3) ◽

pp. 137-146 ◽

Cited By ~ 11

Author(s):

Susanna A. Wood ◽

Xavier Pochon ◽

Witold Ming ◽

Ulla von Ammon ◽

Chris Woods ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Molecular Techniques ◽

Extracellular Dna ◽

Occupancy Modelling ◽

Styela Clava ◽

Dna And Rna ◽

Marine Invasive Species ◽

Pcr Assays ◽

Species Specific

Molecular techniques may provide effective tools to enhance marine biosecurity surveillance. Prior to routine implementation, evidence-based consideration of their benefits and limitations is needed. In this study, we assessed the efficiency and practicality of visual diver surveys and real-time PCR assays (targeting DNA and RNA) for detecting two marine invasive species whose infestation levels varied between species and location: Sabella spallanzanii and Styela clava. Filtered water samples (n = 171) were collected in parallel with dive surveys at two locations as part of the New Zealand Marine High Risk Site Surveillance programme: Nelson Harbour (27 sites) and Waitemata Harbour (30 sites). Diver surveys resulted in a greater number of detections compared to real-time PCR: S. clava – 21 versus 5 sites in Nelson, 6 versus 1 in Auckland; S. spallanzanii – 18 versus 10 in Auckland, no detections in Nelson. Occupancy modelling derived detection probabilities for the real-time PCR for S. clava were low (14%), compared to S. spallanzanii (66%). This could be related to abundances, or species-specific differences in DNA shedding. Only one RNA sample was positive, suggesting that most detections were from extracellular DNA or non-viable fragments. While molecular methods cannot yet replace visual observations, this study shows they provide useful complementary information.

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A paradigm for the molecular identification of Mycobacterium species in a routine diagnostic laboratory

Journal of Medical Microbiology ◽

10.1099/jmm.0.46855-0 ◽

2007 ◽

Vol 56 (5) ◽

pp. 598-602 ◽

Cited By ~ 20

Author(s):

K. J. Williams ◽

C. L. Ling ◽

C. Jenkins ◽

S. H. Gillespie ◽

T. D. McHugh

Keyword(s):

Real Time ◽

Real Time Pcr ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Reference Laboratory ◽

Mycobacterium Fortuitum ◽

Diagnostic Laboratory ◽

Mycobacterium Chelonae ◽

Mycobacterium Xenopi ◽

Pcr Assays

The aim of this study was to improve the identification of Mycobacterium species in the context of a UK teaching hospital. Real-time PCR assays were established to enable the rapid differentiation between Mycobacterium tuberculosis (MTB) complex and Mycobacterium species other than tuberculosis (MOTT), followed by 16S rRNA gene sequencing for the speciation of MOTT. Real-time PCR assays gave comparable results to those from the reference laboratory. The implementation of these PCR assays using an improved bead extraction method has enhanced the mycobacterial diagnostic service at the Royal Free Hospital by providing a rapid means of differentiating between MTB complex and MOTT, and would be simple to implement in similar laboratories. Sequence analysis successfully identified a range of Mycobacterium spp. representative of those encountered in the clinical setting of the authors, including Mycobacterium avium complex, Mycobacterium fortuitum group, Mycobacterium chelonae–Mycobacterium abscessus group, Mycobacterium xenopi and Mycobacterium gordonae. It provides a useful tool for the identification of MOTT when clinically indicated.

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PCR and culture for diagnosis of Acanthamoeba keratitis

British Journal of Ophthalmology ◽

10.1136/bjophthalmol-2020-316730 ◽

2020 ◽

pp. bjophthalmol-2020-316730

Author(s):

Helene Yera ◽

Vichita Ok ◽

Fiona Lee Koy Kuet ◽

Naima Dahane ◽

Frédéric Ariey ◽

...

Keyword(s):

Real Time ◽

Internal Control ◽

Real Time Pcr ◽

Latent Class ◽

Rrna Gene ◽

Target Sequence ◽

Small Subunit Rrna ◽

Pcr Assay ◽

Corneal Scraping ◽

Pcr Assays

Background/AimsAcanthamoeba keratitis (AK) is a rare but sight-threatening infection. Molecular diagnosis of corneal scraping has improved the diagnosis of AK. Different molecular targets and conditions have been used in diagnosis thus far. In this study, we prospectively compared the performance of five PCR assays on corneal samples for the diagnosis of AK.Methods1217 corneal scraping samples were obtained from patients, for whom an AK was suspected. Sample processing involved both molecular diagnostics and culture. Acanthamoeba PCR assays detected different regions of the Acanthamoeba nuclear small-subunit rRNA gene: three final point PCR assays using Nelson, ACARNA and JDP1–JDP2 pairs of primers, and two real-time PCR assays using Acant primer-probe. Human DNA and internal control were co-amplified in the real-time PCR assay to ensure scraping quality and the absence of inhibitors. In the absence of a gold standard, the performance of each test was evaluated using latent class analysis. Genotypes of Acanthamoeba isolates were also characterised.ResultsEstimated prevalence of AK was 1.32%. The sensitivity of Acanthamoeba diagnostic PCRs (73.3% to 86.7%) did not differ significantly from that of culture (66.7%), or according to the target sequence or the technology. Sensitivity could be increased to 93.8% or 100% by combining two or three assays, respectively. PCR specificity (99.3% to 100%) differed between the assays. T4 was the predominant Acanthamoeba genotype (84.6%).ConclusionsCulture and a single PCR assay could lead to misdiagnosing AK. A combination of different PCR assays and improved sample quality could increase diagnosis sensitivity.

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Real-Time PCR Technology for Cancer Diagnostics

Clinical Chemistry ◽

10.1093/clinchem/48.8.1178 ◽

2002 ◽

Vol 48 (8) ◽

pp. 1178-1185 ◽

Cited By ~ 162

Author(s):

Philip S Bernard ◽

Carl T Wittwer

Keyword(s):

Clinical Practice ◽

Real Time ◽

Real Time Pcr ◽

Clinical Laboratory ◽

Melting Curve ◽

Cancer Diagnostics ◽

Molecular Techniques ◽

Melting Curve Analysis ◽

Oncology Research ◽

Tumor Profiling

Abstract Background: Advances in the biological sciences and technology are providing molecular targets for diagnosing and treating cancer. Current classifications in surgical pathology for staging malignancies are based primarily on anatomic features (e.g., tumor-node-metastasis) and histopathology (e.g., grade). Microarrays together with clustering algorithms are revealing a molecular diversity among cancers that promises to form a new taxonomy with prognostic and, more importantly, therapeutic significance. The challenge for pathology will be the development and implementation of these molecular classifications for routine clinical practice. Approach: This article discusses the benefits, challenges, and possibilities for solid-tumor profiling in the clinical laboratory with an emphasis on DNA-based PCR techniques. Content: Molecular markers can be used to provide accurate prognosis and to predict response, resistance, or toxicity to therapy. The diversity of genomic alterations involved in malignancy necessitates a variety of assays for complete tumor profiling. Some new molecular classifications of tumors are based on gene expression, requiring a paradigm shift in specimen processing to preserve the integrity of RNA for analysis. More stable markers (i.e., DNA and protein) are readily handled in the clinical laboratory. Quantitative real-time PCR can determine gene duplications or deletions. Furthermore, melting curve analysis immediately after PCR can identify small mutations, down to single base changes. These techniques are becoming easier and faster and can be multiplexed. Real-time PCR methods are a favorable option for the analysis of cancer markers. Summary: There is a need to translate recent discoveries in oncology research into clinical practice. This requires objective, robust, and cost-effective molecular techniques for clinical trials and, eventually, routine use. Real-time PCR has attractive features for tumor profiling in the clinical laboratory.

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