Specific Real-Time PCR for Simultaneous Detection and Identification ofLegionella pneumophilaSerogroup 1 in Water and Clinical Samples

ABSTRACTLegionella pneumophila, a bacterium that replicates within aquatic amoebae and persists in the environment as a free-living microbe, is the causative agent of Legionnaires' disease. Among the manyLegionellaspecies described,L. pneumophilais associated with 90% of human disease, and within the 15 serogroups (Sg),L. pneumophilaSg1 causes more than 84% of Legionnaires' disease worldwide. Thus, rapid and specific identification ofL. pneumophilaSg1 is of the utmost importance for evaluation of the contamination of collective water systems and the risk posed. Previously we had shown that about 20 kb of the 33-kb locus carrying the genes coding for the proteins involved in lipopolysaccharide biosynthesis (LPS gene cluster) byL. pneumophilawas highly specific for Sg1 strains and that three genes (lpp0831,wzm, andwzt) may serve as genetic markers. Here we report the sequencing and comparative analyses of this specific region of the LPS gene cluster inL. pneumophilaSg6, -10, -12, -13, and -14. Indeed, thewzmandwztgenes were present only in the Sg1 LPS gene cluster, which showed a very specific gene content with respect to the other five serogroups investigated. Based on this observation, we designed primers and developed a classical and a real-time PCR method for the detection and simultaneous identification ofL. pneumophilaSg1 in clinical and environmental isolates. Evaluation of the selected primers with 454Legionellaand 38 non-Legionellastrains demonstrated 100% specificity. Sensitivity, specificity, and predictive values were further evaluated with 209 DNA extracts from water samples of hospital water supply systems and with 96 respiratory specimens. The results showed that the newly developed quantitative Sg1-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-riskL. pneumophilaSg1 in water and clinical samples.

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Improvement of Legionnaires’ disease diagnosis using real-time PCR assay: a retrospective analysis, Italy, 2010 to 2015

Eurosurveillance ◽

10.2807/1560-7917.es.2018.23.50.1800032 ◽

2018 ◽

Vol 23 (50) ◽

Cited By ~ 2

Author(s):

Maria Luisa Ricci ◽

Antonella Grottola ◽

Giulia Fregni Serpini ◽

Antonino Bella ◽

Maria Cristina Rota ◽

...

Keyword(s):

Real Time ◽

Legionella Pneumophila ◽

Real Time Pcr ◽

Reference Method ◽

Disease Diagnosis ◽

Pcr Assay ◽

Predictive Values ◽

The Real ◽

Legionnaires Disease ◽

Antigen Tests

Aim To evaluate real-time PCR as a diagnostic method for Legionnaires’ disease (LD). Detection of Legionella DNA is among the laboratory criteria of a probable LD case, according to the European Centre for Disease Prevention and Control, although the utility and advantages, as compared to culture, are widely recognised. Methods Two independent laboratories, one using an in-house and the other a commercial real-time PCR assay, analysed 354 respiratory samples from 311 patients hospitalised with pneumonia between 2010–15. The real-time PCR reliability was compared with that of culture and urinary antigen tests (UAT). Concordance, specificity, sensitivity and positive and negative predictive values (PPV and NPV, respectively) were calculated. Results Overall PCR detected eight additional LD cases, six of which were due to Legionella pneumophila (Lp) non-serogroup 1. The two real-time PCR assays were concordant in 99.4% of the samples. Considering in-house real-time PCR as the reference method, specificity of culture and UAT was 100% and 97.9% (95% CI: 96.2–99.6), while the sensitivity was 63.6% (95%CI: 58.6–68.6) and 77.8% (95% CI: 72.9–82.7). PPV and NPV for culture were 100% and 93.7% (95% CI: 91.2-96.3). PPV and NPV for UAT were 87.5% (95% CI: 83.6-91.4) and 95.8% (95% CI: 93.5-98.2). Conclusion Regardless of the real-time PCR assay used, it was possible to diagnose LD cases with higher sensitivity than using culture or UAT. These data encourage the adoption of PCR as routine laboratory testing to diagnose LD and such methods should be eligible to define a confirmed LD case.

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Development and evaluation of duplex TaqMan real-time PCR assay for detection and differentiation of wide-type and MGF505-2R gene-deleted African swine fever viruses

10.21203/rs.3.rs-59710/v2 ◽

2020 ◽

Author(s):

zhenhua Guo ◽

Kunpeng Li ◽

Songlin Qiao ◽

Xinxin Chen ◽

Ruiguang Deng ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Large Scale ◽

Limit Of Detection ◽

African Swine Fever ◽

Economic Losses ◽

Clinical Samples ◽

Diagnosis Method ◽

Pcr Method ◽

And Control

Abstract Background: African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used.Results: In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent.Conclusion: We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

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Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

Archives of Pathology & Laboratory Medicine ◽

10.5858/134.3.444 ◽

2010 ◽

Vol 134 (3) ◽

pp. 444-448 ◽

Cited By ~ 1

Author(s):

Zhengming Gu ◽

Jianmin Pan ◽

Matthew J. Bankowski ◽

Randall T. Hayden

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Quantitative Polymerase Chain Reaction ◽

Bk Virus ◽

Clinical Samples ◽

Quantitative Detection ◽

Chain Reaction ◽

Pcr Method ◽

Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

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Novel SYBR Green Real Time PCR Method for Direct Quantification and Detection of Escherichia coli from Clinical Samples

ZANCO Journal of Pure and Applied Sciences ◽

10.21271/zjpas.29.2.20 ◽

2017 ◽

Vol 29 (2) ◽

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Real Time Pcr ◽

Sybr Green ◽

Clinical Samples ◽

Pcr Method ◽

Direct Quantification

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Absolute Quantitation of Different Genotypes of Hepatitis C Virus RNA in Clinical Samples by a Modified Real-Time PCR Method

Laboratory Medicine ◽

10.1309/lmhxo54sa8gypirh ◽

2011 ◽

Vol 42 (6) ◽

pp. 333-337

Author(s):

Karina del Carmen Trujillo-Murillo ◽

Diana Cristina Pérez-Ibave ◽

Clara Patricia Ríos-Ibarra ◽

Eda Guadalupe Ramirez-Valles ◽

Ana Rosa Rincón-Sánchez ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Real Time ◽

Real Time Pcr ◽

Clinical Samples ◽

Hepatitis C Virus Rna ◽

Absolute Quantitation ◽

Virus Rna ◽

Pcr Method

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Development and evaluation of duplex TaqMan real-time PCR assay for detection and differentiation of wide-type and MGF505-2R gene-deleted African swine fever viruses

BMC Veterinary Research ◽

10.1186/s12917-020-02639-2 ◽

2020 ◽

Vol 16 (1) ◽

Author(s):

Zhenhua Guo ◽

Kunpeng Li ◽

Songlin Qiao ◽

Xin-xin Chen ◽

Ruiguang Deng ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Large Scale ◽

Limit Of Detection ◽

African Swine Fever ◽

Economic Losses ◽

Clinical Samples ◽

Diagnosis Method ◽

Pcr Method ◽

And Control

Abstract Background African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used. Results In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent. Conclusion We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

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Development and Evaluation of Duplex TaqMan Real-Time PCR Assay for Detection and Differentiation of Wide-Type and MGF505 Gene-Deleted African Swine Fever Viruses

10.21203/rs.3.rs-59710/v1 ◽

2020 ◽

Author(s):

Zhenhua Guo ◽

Kunpeng Li ◽

Songlin Qiao ◽

Xinxin Chen ◽

Ruiguang Deng ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Large Scale ◽

Limit Of Detection ◽

African Swine Fever ◽

Economic Losses ◽

Clinical Samples ◽

Diagnosis Method ◽

Pcr Method ◽

And Control

Abstract Background: African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used. Results: In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent. Conclusion: We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

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Application of Legionella pneumophila-specific quantitative real-time PCR combined with direct amplification and sequence-based typing in the diagnosis and epidemiological investigation of Legionnaires’ disease

European Journal of Clinical Microbiology & Infectious Diseases ◽

10.1007/s10096-011-1535-0 ◽

2012 ◽

Vol 31 (8) ◽

pp. 2017-2028 ◽

Cited By ~ 40

Author(s):

M. Mentasti ◽

N. K. Fry ◽

B. Afshar ◽

C. Palepou-Foxley ◽

F. C. Naik ◽

...

Keyword(s):

Real Time ◽

Legionella Pneumophila ◽

Real Time Pcr ◽

Epidemiological Investigation ◽

Quantitative Real Time Pcr ◽

Legionnaires Disease ◽

Direct Amplification

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Molecular Diagnosis of Leishmaniasis: Quantification of Parasite Load by a Real-Time PCR Assay with High Sensitivity

Pathogens ◽

10.3390/pathogens10070865 ◽

2021 ◽

Vol 10 (7) ◽

pp. 865

Author(s):

Germano Castelli ◽

Federica Bruno ◽

Stefano Reale ◽

Simone Catanzaro ◽

Viviana Valenza ◽

...

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Real Time Pcr ◽

High Sensitivity ◽

Parasite Load ◽

Clinical Samples ◽

Pcr Assay ◽

Predictive Values ◽

Low Sensitivity

Real-time PCR was developed to quantify Leishmania infantum kinetoplast DNA and optimized to achieve a sensitivity of 1 parasite/mL. For this purpose, we cloned the conserved kDNA fragment of 120 bp into competent cells and correlated them with serial dilutions of DNA extracted from reference parasite cultures calculating that a parasite cell contains approximately 36 molecules of kDNA. This assay was applied to estimate parasite load in clinical samples from visceral, cutaneous leishmaniasis patients and infected dogs and cats comparing with conventional diagnosis. The study aimed to propose a real-time PCR for the detection of Leishmania DNA from clinical samples trying to solve the diagnostic problems due to the low sensitivity of microscopic examination or the low predictive values of serology and resolve problems related to in vitro culture. The quantitative PCR assay in this study allowed detection of Leishmania DNA and quantification of considerably low parasite loads in samples that had been diagnosed negative by conventional techniques. In conclusion, this quantitative PCR can be used for the diagnosis of both human, canine and feline Leishmaniasis with high sensitivity and specificity, but also for evaluating treatment and the endpoint determination of leishmaniasis.

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