scholarly journals Quantitative Detection of Borrelia burgdorferi by Real-Time PCR

1999 ◽  
Vol 37 (6) ◽  
pp. 1958-1963 ◽  
Author(s):  
Andreas Pahl ◽  
Uta Kühlbrandt ◽  
Kay Brune ◽  
Martin Röllinghoff ◽  
André Gessner
Keyword(s):  
Borrelia Burgdorferi ◽  
Real Time ◽  
Real Time Pcr ◽  
Inbred Mice ◽  
Penicillin G ◽  
Quantitative Detection ◽  
Target Sequence ◽  
Detection Range ◽  
The Real ◽  
Specificity And Sensitivity

Currently, no easy and reliable methods allowing for the quantification of Borrelia burgdorferi in tissues of infected humans or animals are available. Due to the lack of suitable assays to detect B. burgdorferi CFU and the qualitative nature of the currently performed PCR assays, we decided to exploit the recently developed real-time PCR. This technology measures the release of fluorescent oligonucleotides during the PCR. Flagellin of B. burgdorferi was chosen as the target sequence. A linear quantitative detection range of 5 logs with a calculated detection limit of one to three spirochetes per assay reaction mixture was observed. The fact that no signals were obtained with closely related organisms such as Borrelia hermsii argues for a high specificity of this newly developed method. A similar method was developed to quantify mouse actin genomic sequences to allow for the standardization of spirochete load. The specificity and sensitivity of the B. burgdorferi and the actin real-time PCR were not altered when samples were spiked with mouse cells or spirochetes, respectively. To evaluate the applicability of the real-time PCR, we used the mouse model of Lyme disease. The fate of B. burgdorferi was monitored in different tissues from inbred mice and from mice treated with antibiotics. Susceptible C3H/HeJ mice had markedly higher burdens of bacterial DNA than resistant BALB/c mice, and penicillin G treatment significantly reduced the numbers of spirochetes. Since these results show a close correlation between clinical symptoms and bacterial burden of tissues, we are currently analyzing human biopsy specimens to evaluate the real-time PCR in a diagnostic setting.

Multiplex Quantitative Real-Time PCR for the Detection of t(14;18) Translocations with Breakpoints within 5 Different Regions of the BCL-2 Gene: MBR, 3′MBR, mcr, 5′mcr, icr.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2826-2826
Author(s):  
Frank Schüler ◽  
Sandra C. Dölken ◽  
Carsten Hirt ◽  
Gottfried Dolken
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Mononuclear Cells ◽  
Consensus Sequence ◽  
Degraded Dna ◽  
Quantitative Detection ◽  
Target Sequence ◽  
Quantitative Real Time Pcr ◽  
Consensus Primer ◽  
Target Sequences

Abstract Follicular lymphomas (FL) are associated with the chromosomal translocation t(14;18)(q32;q21). Most breakpoints of chromosome 18 (60%) occur in the major breakpoint region (MBR) of the BCL-2 gene. Further breakpoints have been detected in the minor cluster region (mcr), less frequent breakpoints are found in regions called 3′-MBR, 5′-mcr and icr. On chromosome 14 most breakpoints are located within one of the six JH-genes. Therefore, BCL-2 translocations with breakpoints within the MBR and mcr are generally detected by PCR using combinations of different BCL-2 primers with one JH-consensus primer. We have developed a multiplex quantitative real-time PCR strategy that that can be used to detect t(14;18) translocations with breakpoints located within all regions mentioned above. To minimize the costs for expensive probes we used the JH-consensus sequence as a target for one “consensus probe” (fluorescent labelled minor groove binder probe) for all assays in combination with 6 different JH intron primers. To reduce the size of amplified PCR fragments 12 BCL-2 primers were chosen in combination with 6 JH intron primers for the detection of all 5 breakpoint regions. It is very important to choose short DNA target sequences for amplification: (a) to establish a real-time PCR with a high amplification efficacy; (b) to be able to amplify target sequences also from partially degraded DNA isolated from formaldehyde-fixed paraffin-embedded tissue sections; (c) to achieve a high sensitivity to detect 1–3 copies per assay. Peripheral bood mononuclear cells (PBMNC) and formalin fixed, paraffin embedded lymph node tissue obtained from 139 FL patients at the time of diagnosis (LN and PBMNC, n = 54; LN only, n = 3; PBMNC only, n = 82) were tested by multiplex quantitative real-time PCR. 80 breakpoints were identified within the MBR (61%) region. For comparison, 78/80 breakpoints were also detected by our standard real-time PCR assay with one BCL-2-MBR- primer and one JH consensus primer in combination with a fluorescent probe located within the BCL-2 sequence [Doelken et al., BioTechniques, 1998]. Two additional translocations with breakpoints located 5′ of the target sequence of the standard PCR were found by using two additional MBR primers. In addition, five mcr breakpoints (5%), one breakpoint in the 3′MBR region and one breakpoint in the icr region were found. Based on these results the prevalence of breakpoints in various regions of the BCL-2 gene in FL patients is: MBR = 61% (80/139); mcr = 5% (5/139); 3′MBR = 1% (1/139); icr = 1% (1/139); 5′mcr = 0%). Furthermore, based on quantitative PCR results the t(14;18) translocations detected in this study were undoubtedly lymphoma associated and did not belong to t(14;18)-positive non-lymphoma B cell clones found in healthy persons. By applying this multiplex quantitative real-time PCR strategy t(14;18) translocations with breakpoints in five different breakpoint clusters can be detected in about 70% of patients with follicular lymphoma. The assays can be used for a fast and reliable quantitative detection of t(14;18) translocations on DNA isolated from fresh lymph nodes or pathological specimens as well as blood samples at the time of diagnosis. In almost all cases quantitative results will allow a distinction whether the translocation found is lymphoma associated or not, which will in turn allow a quantitative MRD analysis on follow-up samples during and after treatment.

scholarly journals An improved rapid quantitative detection and identification method for a wide range of fungi

2009 ◽  
Vol 58 (8) ◽  
pp. 1037-1044 ◽  
Author(s):  
Nobutoshi Soeta ◽  
Masanori Terashima ◽  
Mitsukazu Gotoh ◽  
Shuichi Mori ◽  
Kyoko Nishiyama ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Technique ◽  
Fungal Species ◽  
Quantitative Detection ◽  
Its2 Region ◽  
The Real ◽  
Detection And Identification ◽  
Wide Range ◽  
Pcr Products

To develop a rapid and quantitative diagnostic technique for the detection and identification of a wide range of fungi, an improved molecular method based on real-time PCR and the analysis of its products that targets the internal transcribed spacer (ITS) 2 region was established. The real-time PCR could quantitatively and specifically detect the ITS2 region from all 24 tested pathogenic fungal species at between 101 and 107 copies per test without amplification of bacterial or human DNA. The sequences of the primer-binding sites are conserved in the registered sequences of 34 other pathogenic fungal species, suggesting that the PCR would also detect these species. The hyperpolymorphic nature of the ITS2 region between fungal species in terms of length and nucleotide sequence provided valuable information for the determination of species. By labelling the 5′ end of the reverse primer with NED fluorescent dye, the fragment lengths of the real-time PCR products and their 3′-terminal fragments, derived using restriction enzyme ScrFI digestion, were easily evaluated by capillary electrophoresis. Using this analysis, the number and species of fungi present in samples could be estimated. Moreover, sequence analysis of the real-time PCR products could accurately determine species in samples containing a single species. This diagnostic technique can estimate a wide range of fungi from various clinical samples within 1 day and accurately identify them in 2 days. Quantitative results for fungal titre in samples can also provide useful information for understanding the progression of disease and the efficacy of antifungal chemotherapy.

scholarly journals Quantitative Detection of Listeria monocytogenes and Listeria innocua by Real-Time PCR: Assessment of hly, iap, and lin02483 Targets and AmpliFluor Technology

2004 ◽  
Vol 70 (3) ◽  
pp. 1366-1377 ◽  
Author(s):  
David Rodr�guez-L�zaro ◽  
Marta Hern�ndez ◽  
Mariela Scortti ◽  
Teresa Esteve ◽  
Jos� A. V�zquez-Boland ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Target Genes ◽  
Dynamic Range ◽  
Significant Proportion ◽  
Quantitative Detection ◽  
Target Sequence ◽  
Food Borne ◽  
Highly Sensitive

ABSTRACT We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne pathogen Listeria monocytogenes and the closely related nonpathogenic species L. innocua. The target genes were hly and iap for L. monocytogenes and lin02483 for L. innocua. The assays were 100% specific, as determined with 100 Listeria strains and 45 non-Listeria strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions. Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and R 2 values of >0.996. There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%). The hly-based assay accurately quantified L. monocytogenes in all of the samples tested. The iap-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability. The combination of the two assays enabled us to classify L. monocytogenes isolates into one of the two major phylogenetic divisions of the species, I and II. We also assessed the new AmpliFluor technology for the quantitative detection of L. monocytogenes by RTi-PCR. The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules).

scholarly journals Direct Quantitative Detection and Identification of Lactococcal Bacteriophages from Milk and Whey by Real-Time PCR: Application for the Detection of Lactococcal Bacteriophages in Goat's Raw Milk Whey in France

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Mai Huong Ly-Chatain ◽  
Loïc Durand ◽  
Véronique Rigobello ◽  
Annabelle Vera ◽  
Yann Demarigny
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Raw Milk ◽  
Quantitative Detection ◽  
Amplification Efficiency ◽  
The Real ◽  
Milk Whey ◽  
Detection And Identification ◽  
Milk Fermentation

The presence ofLactococcusbacteriophages in milk can partly or completely inhibit milk fermentation. To prevent the problems associated with the bacteriophages, the real-time PCR was developed in this study for direct detection from whey and milk of three main groups ofLactococcusbacteriophages, c2, 936, and P335. The optimization of DNA extraction protocol from complex matrices such as whey and milk was optimized allowed the amplification of PCR without any matrix and nontarget contaminant interference. The real-time PCR program was specific and with the detection limit of 102PFU/mL. The curve slopes were −3.49, −3.69, and −3.45 with the amplification efficiency estimated at 94%, 94%, and 98% and the correlation coefficient () of 0.999, 0.999, and 0.998 for c2, 936 and P335 group, respectively. This method was then used to detect the bacteriophages in whey and goat's raw milk coming from three farms located in the Rhône-Alpes region (France).

Rapid and Simultaneous Quantitation of Escherichia coli O157:H7, Salmonella, and Shigella in Ground Beef by Multiplex Real-Time PCR and Immunomagnetic Separation†

2007 ◽  
Vol 70 (6) ◽  
pp. 1366-1372 ◽  
Author(s):  
LUXIN WANG ◽  
YONG LI ◽  
AZLIN MUSTAPHA
Keyword(s):  
Escherichia Coli ◽  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Quantitative Detection ◽  
Escherichia Coli O157 ◽  
The Real ◽  
E Coli

The objective of this study was to establish a multiplex real-time PCR for the simultaneous quantitation of Escherichia coli O157:H7, Salmonella, and Shigella. Genomic DNA for the real-time PCR was extracted by the boiling method. Three sets of primers and corresponding TaqMan probes were designed to target these three pathogenic bacteria. Multiplex real-time PCR was performed with TaqMan Universal PCR Master Mix in an ABI Prism 7700 Sequence Detection System. Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log CFU per milliliter) via linear regression. With optimized conditions, the quantitative detection range of the real-time multiplex PCR for pure cultures was 102 to 109 CFU/ml for E. coli O157:H7, 103 to 109 CFU/ml for Salmonella, and 101 to 108 CFU/ml for Shigella. When the established multiplex real-time PCR system was applied to artificially contaminated ground beef, the detection limit was 105 CFU/g for E. coli O157:H7, 103 CFU/g for Salmonella, and 104 CFU/g for Shigella. Immunomagnetic separation (IMS) was further used to separate E. coli O157:H7 and Salmonella from the beef samples. With the additional use of IMS, the detection limit was 103 CFU/g for both pathogens. Results from this study showed that TaqMan real-time PCR, combined with IMS, is potentially an effective method for the rapid and reliable quantitation of E. coli O157:H7, Salmonella, and Shigella in food.

Quantitative detection and monitoring of Colletotrichum siamense in rubber trees using real-time PCR

Plant Disease ◽  
2021 ◽  
Author(s):  
Yannan Du ◽  
Meng Wang ◽  
Mingteng Long ◽  
Ye Yang ◽  
Xiaoyu Liang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Disease Risk ◽  
Rubber Tree ◽  
Detection Methods ◽  
Quantitative Detection ◽  
The Real ◽  
Colletotrichum Siamense ◽  
Pcr Method ◽  
Detection And Quantification

Colletotrichum siamense is one of the most important pathogens of rubber trees in Asia. The proper detection and quantification of C. siamense populations in rubber trees are of importance for monitoring the epidemics of the disease. In this study, we developed an ITS-based real-time PCR method to efficiently detect C. siamense infecting rubber tree, which reliably detected as little as 100 fg genomic DNA, 100 copies of target DNA and 20 conidia. The real-time PCR protocol recognized all C. siamense isolates collected from three provinces in China, while no amplification was observed with rubber tree and its other pathogens. Detection and quantification of C. siamense were performed in artificially and naturally infected rubber leaves. We could still detect C. siamense in plant mixes of which only 0.0001% of the tissue infected. An accumulation of C. siamense DNA was observed during the whole infection process at all three leaf phenological stages, suggesting the real-time PCR method can be used to monitor C. siamense development in rubber trees. Finally, the method allowed the detection of C. siamense in naturally infected and symptomless leaves of rubber trees in the fields. Compared with earlier detection methods, the real-time PCR method is more specific and more sensitive, and will be of great use for studies aiming to gain a better understanding of the epidemiology of Colletotrichum leaf disease, as well as the prediction of disease risk and the control proposal.

Quantitative detection of human herpes virus 1 & 2 using the real time PCR STAT-NAT HSV1 & 2 assays

2019 ◽  
Vol 493 ◽  
pp. S565-S566
Author(s):  
M. Gramegna ◽  
G. Ferri ◽  
L. Bavagnoli ◽  
M.L. Incandela ◽  
D. Rigamonti ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Herpes Virus ◽  
Quantitative Detection ◽  
Human Herpes ◽  
The Real ◽  
Human Herpes Virus ◽  
Herpès Virus

scholarly journals Quantitative Detection of Borrelia burgdorferi sensu lato in Erythema Migrans Skin Lesions Using Internally Controlled Duplex Real Time PCR

PLoS ONE ◽  
2013 ◽  
Vol 8 (5) ◽  
pp. e63968 ◽  
Author(s):  
Maria O’Rourke ◽  
Andreas Traweger ◽  
Lara Lusa ◽  
Dasa Stupica ◽  
Vera Maraspin ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Real Time ◽  
Real Time Pcr ◽  
Erythema Migrans ◽  
Skin Lesions ◽  
Borrelia Burgdorferi Sensu Lato ◽  
Quantitative Detection

scholarly journals Development of Real-Time Polymerase Chain Reaction Assay with TaqMan Probe for the Quantitative Detection of Infectious Hypodermal and Hematopoietic Necrosis Virus from Shrimp

2006 ◽  
Vol 89 (1) ◽  
pp. 240-244 ◽  
Author(s):  
Zhi-Qin Yue ◽  
Hong Liu ◽  
Wei-Ji Wang ◽  
Zhi-Wen Lei ◽  
Cheng-Zhu Liang ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Taqman Probe ◽  
Quantitative Detection ◽  
Necrosis Virus ◽  
Chain Reaction ◽  
Pcr Assay ◽  
The Real ◽  
Polymerase Chain

Abstract An assay was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) based on real-time quantitative polymerase chain reaction (PCR). A pair of primers and a TaqMan probe were designed that are specific for the recognition of a conservative region in the IHHNV genome. The IHHNV real-time PCR assay had a detection limit of 9 DNA copies,with a dynamic range of detection between 9 106 and 9 DNA copies. The primer pairs and probe were specific to IHHNV and did not cross-reactwith shrimp genomic DNAor other shrimp viruses such as White Spot Syndrome Virus (WSSV), Monodon Baculovirus (MBV), and hepatopancreatic parvovirus (HPV). This assay has a broad application for basic and clinical investigations. For clinical samples, the real-time PCR assay detected all the positive samples screened by conventional PCR, which indicated the sensitivity of the real-time assay. The IHHNV real-time PCR assay with high sensitivity, specificity, wide range of detection ability, and simplicity is particularly useful for screening large numbers of specimens and measuring viral loads to monitor the broodstock.

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