Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India

PubMed Central

Dinoop, K.P.; Parija, Subhash Chandra; Mandal, Jharna; Swaminathan, R.P.; Narayanan, P.

2016-01-01

Background & objectives: Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Methods: Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. Results: In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated (P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Interpretation & conclusions: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods. PMID:26997014

Comparison of culture, single and multiplex real-time PCR for detection of Sabin poliovirus shedding in recently vaccinated Indian children.

PubMed

Giri, Sidhartha; Rajan, Anand K; Kumar, Nirmal; Dhanapal, Pavithra; Venkatesan, Jayalakshmi; Iturriza-Gomara, Miren; Taniuchi, Mami; John, Jacob; Abraham, Asha Mary; Kang, Gagandeep

2017-08-01

Although, culture is considered the gold standard for poliovirus detection from stool samples, real-time PCR has emerged as a faster and more sensitive alternative. Detection of poliovirus from the stool of recently vaccinated children by culture, single and multiplex real-time PCR was compared. Of the 80 samples tested, 55 (68.75%) were positive by culture compared to 61 (76.25%) and 60 (75%) samples by the single and one step multiplex real-time PCR assays respectively. Real-time PCR (singleplex and multiplex) is more sensitive than culture for poliovirus detection in stool, although the difference was not statistically significant. © 2017 Wiley Periodicals, Inc.

Optically-synchronized encoder and multiplexer scheme for interleaved photonics analog-to-digital conversion

NASA Astrophysics Data System (ADS)

Villa, Carlos; Kumavor, Patrick; Donkor, Eric

2008-04-01

Photonics Analog-to-Digital Converters (ADCs) utilize a train of optical pulses to sample an electrical input waveform applied to an electrooptic modulator or a reverse biased photodiode. In the former, the resulting train of amplitude-modulated optical pulses is detected (converter to electrical) and quantized using a conversional electronics ADC- as at present there are no practical, cost-effective optical quantizers available with performance that rival electronic quantizers. In the latter, the electrical samples are directly quantized by the electronics ADC. In both cases however, the sampling rate is limited by the speed with which the electronics ADC can quantize the electrical samples. One way to increase the sampling rate by a factor N is by using the time-interleaved technique which consists of a parallel array of N electrical ADC converters, which have the same sampling rate but different sampling phase. Each operating at a quantization rate of fs/N where fs is the aggregated sampling rate. In a system with no real-time operation, the N channels digital outputs are stored in memory, and then aggregated (multiplexed) to obtain the digital representation of the analog input waveform. Alternatively, for real-time operation systems the reduction of storing time in the multiplexing process is desired to improve the time response of the ADC. The complete elimination of memories come expenses of concurrent timing and synchronization in the aggregation of the digital signal that became critical for a good digital representation of the analog signal waveform. In this paper we propose and demonstrate a novel optically synchronized encoder and multiplexer scheme for interleaved photonics ADCs that utilize the N optical signals used to sample different phases of an analog input signal to synchronize the multiplexing of the resulting N digital output channels in a single digital output port. As a proof of concept, four 320 Megasamples/sec 12-bit of resolution digital signals were multiplexed to form an aggregated 1.28 Gigasamples/sec single digital output signal.

Simultaneous detection and serotyping of dengue infection using single tube multiplex CDC Dengue Real-Time RT-PCR from India.

PubMed

Sharma, Shashi; Tandel, Kundan; Danwe, Surabhi; Bhatt, Puneet; Dash, P K; Ranjan, Praveer; Rathi, K R; Gupta, Rajiv Mohan; Parida, M M

2018-03-01

Four antigenically different dengue virus serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) are known to cause infections in humans. Some of these are known to cause more severe disease than the others. Chances for developing Dengue hemorrhagic fever-dengue shock syndrome (DHF-DSS) increases significantly with history of previous infection with one of the four serotypes. Therefore, early diagnosis, serotyping and providing early warning of dengue fever epidemics to concerned authorities becomes very important for better patient outcome and to curb the rapid spread in the community. During the 2014 outbreak, a total of 100 samples from suspected cases of dengue were collected. NS1 antigen based rapid test was used for serological diagnosis. Dengue complex one step reverse transcription-polymerase chain reaction was performed to look for presence of viral RNA. Single tube multiplex RT-PCR was also performed to look for infecting serotype. CDC Dengue Multiplex Real Time PCR assay was performed for rapid diagnosis and simultaneous serotyping of the dengue virus. Out of the 100 samples screened, 69 were found to be positive by NS1Ag Rapid test. 34 samples were found positive by dengue consensus RT-PCR assay. 22 samples were found to be positive by single tube Dengue multiplex RT-PCR assay. Serotype DEN-2 was present in maximum numbers followed by DEN-3. 44 samples were found positive by DENV CDC Multiplex Real time PCR assay. DEN-2 was found in maximum numbers followed by DEN-1. Dengue remains to be an important health problem in India and across the globe. Few serotypes of dengue are more dangerous than the others. Rapid diagnosis and serotyping remains the key for better patient management and prevention of disease spreading in the community. Highly sensitive, specific and rapid CDC real time RT-PCR assay was found to be most promising tool among all available molecular diagnostic methods. This will serve a rapid and reliable simultaneous dengue virus detection as well serotyping assay in near future for rapid identification of dengue suspected sample screening.

Selective and reversible ammonia gas detection with nanoporous film functionalized silicon photonic micro-ring resonator.

PubMed

Yebo, Nebiyu A; Sree, Sreeprasanth Pulinthanathu; Levrau, Elisabeth; Detavernier, Christophe; Hens, Zeger; Martens, Johan A; Baets, Roel

2012-05-21

Portable, low cost and real-time gas sensors have a considerable potential in various biomedical and industrial applications. For such applications, nano-photonic gas sensors based on standard silicon fabrication technology offer attractive opportunities. Deposition of high surface area nano-porous coatings on silicon photonic sensors is a means to achieve selective, highly sensitive and multiplexed gas detection on an optical chip. Here we demonstrate selective and reversible ammonia gas detection with functionalized silicon-on-insulator optical micro-ring resonators. The micro-ring resonators are coated with acidic nano-porous aluminosilicate films for specific ammonia sensing, which results in a reversible response to NH(3)with selectivity relative to CO(2). The ammonia detection limit is estimated at about 5 ppm. The detectors reach a steady response to NH(3) within 30 and return to their base level within 60 to 90 seconds. The work opens perspectives on development of nano-photonic sensors for real-time, non-invasive, low cost and light weight biomedical and industrial sensing applications.

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides.

PubMed

Ito, Takao; Suzaki, Koichi

2017-01-01

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides

PubMed Central

Suzaki, Koichi

2017-01-01

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays. PMID:28957362

Multiplex real-time PCR assay for detection of Escherichia coli O157:H7 and screening for non-O157 Shiga toxin-producing E. coli.

PubMed

Li, Baoguang; Liu, Huanli; Wang, Weimin

2017-11-09

Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, are responsible for numerous foodborne outbreaks annually worldwide. E. coli O157:H7, as well as pathogenic non-O157:H7 STECs, can cause life-threating complications, such as bloody diarrhea (hemolytic colitis) and hemolytic-uremic syndrome (HUS). Previously, we developed a real-time PCR assay to detect E. coli O157:H7 in foods by targeting a unique putative fimbriae protein Z3276. To extend the detection spectrum of the assay, we report a multiplex real-time PCR assay to specifically detect E. coli O157:H7 and screen for non-O157 STEC by targeting Z3276 and Shiga toxin genes (stx1 and stx2). Also, an internal amplification control (IAC) was incorporated into the assay to monitor the amplification efficiency. The multiplex real-time PCR assay was developed using the Life Technology ABI 7500 System platform and the standard chemistry. The optimal amplification mixture of the assay contains 12.5 μl of 2 × Universal Master Mix (Life Technology), 200 nM forward and reverse primers, appropriate concentrations of four probes [(Z3276 (80 nM), stx1 (80 nM), stx2 (20 nM), and IAC (40 nM)], 2 μl of template DNA, and water (to make up to 25 μl in total volume). The amplification conditions of the assay were set as follows: activation of TaqMan at 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 60 s. The multiplex assay was optimized for amplification conditions. The limit of detection (LOD) for the multiplex assay was determined to be 200 fg of bacterial DNA, which is equivalent to 40 CFU per reaction which is similar to the LOD generated in single targeted PCRs. Inclusivity and exclusivity determinants were performed with 196 bacterial strains. All E. coli O157:H7 (n = 135) were detected as positive and all STEC strains (n = 33) were positive for stx1, or stx2, or stx1 and stx2 (Table 1). No cross reactivity was detected with Salmonella enterica, Shigella strains, or any other pathogenic strains tested. A multiplex real-time PCR assay that can rapidly and simultaneously detect E. coli O157:H7 and screen for non-O157 STEC strains has been developed and assessed for efficacy. The inclusivity and exclusivity tests demonstrated high sensitivity and specificity of the multiplex real-time PCR assay. In addition, this multiplex assay was shown to be effective for the detection of E. coli O157:H7 from two common food matrices, beef and spinach, and may be applied for detection of E. coli O157:H7 and screening for non-O157 STEC strains from other food matrices as well.

[Multiplex real-time PCR method for rapid detection of Marburg virus and Ebola virus].

PubMed

Yang, Yu; Bai, Lin; Hu, Kong-Xin; Yang, Zhi-Hong; Hu, Jian-Ping; Wang, Jing

2012-08-01

Marburg virus and Ebola virus are acute infections with high case fatality rates. A rapid, sensitive detection method was established to detect Marburg virus and Ebola virus by multiplex real-time fluorescence quantitative PCR. Designing primers and Taqman probes from highly conserved sequences of Marburg virus and Ebola virus through whole genome sequences alignment, Taqman probes labeled by FAM and Texas Red, the sensitivity of the multiplex real-time quantitative PCR assay was optimized by evaluating the different concentrations of primers and Probes. We have developed a real-time PCR method with the sensitivity of 30.5 copies/microl for Marburg virus positive plasmid and 28.6 copies/microl for Ebola virus positive plasmids, Japanese encephalitis virus, Yellow fever virus, Dengue virus were using to examine the specificity. The Multiplex real-time PCR assays provide a sensitive, reliable and efficient method to detect Marburg virus and Ebola virus simultaneously.

Time multiplexing for increased FOV and resolution in virtual reality

NASA Astrophysics Data System (ADS)

Miñano, Juan C.; Benitez, Pablo; Grabovičkić, Dejan; Zamora, Pablo; Buljan, Marina; Narasimhan, Bharathwaj

2017-06-01

We introduce a time multiplexing strategy to increase the total pixel count of the virtual image seen in a VR headset. This translates into an improvement of the pixel density or the Field of View FOV (or both) A given virtual image is displayed by generating a succession of partial real images, each representing part of the virtual image and together representing the virtual image. Each partial real image uses the full set of physical pixels available in the display. The partial real images are successively formed and combine spatially and temporally to form a virtual image viewable from the eye position. Partial real images are imaged through different optical channels depending of its time slot. Shutters or other schemes are used to avoid that a partial real image be imaged through the wrong optical channels or at the wrong time slot. This time multiplexing strategy needs real images be shown at high frame rates (>120fps). Available display and shutters technologies are discussed. Several optical designs for achieving this time multiplexing scheme in a compact format are shown. This time multiplexing scheme allows increasing the resolution/FOV of the virtual image not only by increasing the physical pixel density but also by decreasing the pixels switching time, a feature that may be simpler to achieve in certain circumstances.

Multiplex real-time PCR assays for the identification of the potato cyst and tobacco cyst nematodes

USDA-ARS?s Scientific Manuscript database

TaqMan primer-probe sets were developed for the detection and identification of potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis using two-tube, multiplex real-time PCR. One tube contained a primer-probe set specific for G. pallida (pale cyst nematode) multiplexed with another prim...

One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.

PubMed

Malandraki, Ioanna; Varveri, Christina; Olmos, Antonio; Vassilakos, Nikon

2015-03-01

A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys. Copyright © 2014 Elsevier B.V. All rights reserved.

Quantitation of Marek's disease and chicken anemia viruses in organs of experimentally infected chickens and commercial chickens by multiplex real-time PCR.

PubMed

Davidson, Irit; Raibshtein, I; Al-Touri, A

2013-06-01

The worldwide distribution of chicken anemia virus (CAV) and Marek's disease virus (MDV) is well documented. In addition to their economic significance in single- or dual-virus infections, the two viruses can often accompany various other pathogens and affect poultry health either directly, by causing tumors, anemia, and delayed growth, or indirectly, by aggravating other diseases, as a result of their immunosuppressive effects. After a decade of employing the molecular diagnosis of those viruses, which replaced conventional virus isolation, we present the development of a real-time multiplex PCR for the simultaneous detection of both viruses. The real-time PCRs for MDV and for CAV alone are more sensitive than the respective end-point PCRs. In addition, the multiplex real-time shows a similar sensitivity when compared to the single real-time PCR for each virus. The newly developed real-time multiplex PCR is of importance in terms of the diagnosis and detection of low copies of each virus, MDV and CAV in single- and in multiple-virus infections, and its applicability will be further evaluated.

Two Multiplex Real-Time PCR Assays to Detect and Differentiate Acinetobacter baumannii and Non- baumannii Acinetobacter spp. Carrying blaNDM, blaOXA-23-Like, blaOXA-40-Like, blaOXA-51-Like, and blaOXA-58-Like Genes

PubMed Central

Yang, Qiu; Rui, Yongyu

2016-01-01

Nosocomial infections caused by Acinetobacter spp. resistant to carbapenems are increasingly reported worldwide. Carbapenem-resistant Acinetobacter (CRA) is becoming a serious concern with increasing patient morbidity, mortality, and lengths of hospital stay. Therefore, the rapid detection of CRA is essential for epidemiological surveillance. Polymerase chain reaction (PCR) has been extensively used for the rapid identification of most pathogens. In this study, we have developed two multiplex real-time PCR assays to detect and differentiate A. baumannii and non-A. baumannii Acinetobacter spp, and common carbapenemase genes, including blaNDM, blaOXA-23-like, blaOXA-40-like, blaOXA-51-like, and blaOXA-58-like. We demonstrate the potential utility of these assays for the direct detection of blaNDM-, blaOXA-23-like-, blaOXA-40-like-, blaOXA-51-like-, and blaOXA-58-like-positive CRA in clinical specimens. Primers were specifically designed, and two multiplex real-time PCR assays were developed: multiplex real-time PCR assay1 for the detection of Acinetobacter baumannii 16S–23S rRNA internal transcribed spacer sequence, the Acinetobacter recA gene, and class-B-metalloenzyme-encoding gene blaNDM; and multiplex real-time PCR assay2 to detect class-D-oxacillinase-encoding genes (blaOXA-23-like, blaOXA-40-like, blaOXA-51-like,and blaOXA-58-like). The assays were performed on an ABI Prism 7500 FAST Real-Time PCR System. CRA isolates were used to compare the assays with conventional PCR and sequencing. Known amounts of CRA cells were added to sputum and fecal specimens and used to test the multiplex real-time PCR assays. The results for target and nontarget amplification showed that the multiplex real-time PCR assays were specific, the limit of detection for each target was 10 copies per 20 μL reaction volume, the assays were linear over six log dilutions of the target genes (r2 > 0.99), and the Ct values of the coefficients of variation for intra- and interassay reproducibility were less than 5%. The multiplex real-time PCR assays showed 100% concordance with conventional PCR when tested against 400 CRA isolates and their sensitivity for the target DNA in sputum and fecal specimens was 102 CFU/mL. Therefore, these novel multiplex real-time PCR assays allow the sensitive and specific characterization and differentiation of blaNDM-, blaOXA-23-like-, blaOXA-40-like-, blaOXA-51-like-, and blaOXA-58-like-positive CRA, making them potential tools for the direct detection of CRA in clinical specimens and the surveillance of nosocomial infections. PMID:27391234

Viruses causing severe acute respiratory infections (SARI) in children ≤5 years of age at a tertiary care hospital in Rajasthan, India.

PubMed

Malhotra, Bharti; Swamy, M Anjaneya; Janardhan Reddy, P V; Gupta, M L

2016-12-01

Severe acute respiratory infection (SARI) is one of the leading causes of death among children worldwide. As different respiratory viruses exhibit similar symptoms, simultaneous detection of these viruses in a single reaction mixture can save time and cost. The present study was done in a tertiary care children's hospital for rapid identification of viruses causing SARI among children less than or equal to five years of age using multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) kit. A total of 155 throat swabs were collected from equal number of children suspected to have SARI and processed for extraction of nucleic acids using automated extraction system. Multiplex real-time RT-PCR was done to identify the viruses in the samples. The overall positivity for viruses in the study was found to be 72.9 per cent with a co-infection rate of 19.5 per cent. Human metapneumovirus (HMPV) was the predominant virus detected in 25.7 per cent children followed by influenza A (H1N1)pdm09, human rhinovirus (HRV) and human adenovirus (HAdV) in 19.9, 11.0 and 8.8 per cent children, respectively. The HMPV was at its peak in February 2013, HAdV showed two peaks in March-April, 2012 and November 2012-March 2013 while HRV was detected throughout the year. Multiplex real-time PCR helped in rapid identification of viruses. Seventeen viruses were detected in SARI cases with overall positivity of 72.9 per cent. HMPV was the most predominant virus. However, for better clinico-virological correlation, studies are required with complete work up of all the aetiological agents, clinical profile of patients and treatment outcome.

Rapid and accurate identification of Mycobacterium tuberculosis complex and common non-tuberculous mycobacteria by multiplex real-time PCR targeting different housekeeping genes.

PubMed

Nasr Esfahani, Bahram; Rezaei Yazdi, Hadi; Moghim, Sharareh; Ghasemian Safaei, Hajieh; Zarkesh Esfahani, Hamid

2012-11-01

Rapid and accurate identification of mycobacteria isolates from primary culture is important due to timely and appropriate antibiotic therapy. Conventional methods for identification of Mycobacterium species based on biochemical tests needs several weeks and may remain inconclusive. In this study, a novel multiplex real-time PCR was developed for rapid identification of Mycobacterium genus, Mycobacterium tuberculosis complex (MTC) and the most common non-tuberculosis mycobacteria species including M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and the M. gordonae in three reaction tubes but under same PCR condition. Genetic targets for primer designing included the 16S rDNA gene, the dnaJ gene, the gyrB gene and internal transcribed spacer (ITS). Multiplex real-time PCR was setup with reference Mycobacterium strains and was subsequently tested with 66 clinical isolates. Results of multiplex real-time PCR were analyzed with melting curves and melting temperature (T (m)) of Mycobacterium genus, MTC, and each of non-tuberculosis Mycobacterium species were determined. Multiplex real-time PCR results were compared with amplification and sequencing of 16S-23S rDNA ITS for identification of Mycobacterium species. Sensitivity and specificity of designed primers were each 100 % for MTC, M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and M. gordonae. Sensitivity and specificity of designed primer for genus Mycobacterium was 96 and 100 %, respectively. According to the obtained results, we conclude that this multiplex real-time PCR with melting curve analysis and these novel primers can be used for rapid and accurate identification of genus Mycobacterium, MTC, and the most common non-tuberculosis Mycobacterium species.

Clinical Validation of Multiplex Real-Time PCR Assays for Detection of Bacterial Meningitis Pathogens

PubMed Central

Theodore, M. Jordan; Mair, Raydel; Trujillo-Lopez, Elizabeth; du Plessis, Mignon; Wolter, Nicole; Baughman, Andrew L.; Hatcher, Cynthia; Vuong, Jeni; Lott, Lisa; von Gottberg, Anne; Sacchi, Claudio; McDonald, J. Matthew; Messonnier, Nancy E.; Mayer, Leonard W.

2012-01-01

Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae are important causes of meningitis and other infections, and rapid, sensitive, and specific laboratory assays are critical for effective public health interventions. Singleplex real-time PCR assays have been developed to detect N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA and serogroup-specific genes in the cap locus for N. meningitidis serogroups A, B, C, W135, X, and Y. However, the assay sensitivity for serogroups B, W135, and Y is low. We aimed to improve assay sensitivity and develop multiplex assays to reduce time and cost. New singleplex real-time PCR assays for serogroup B synD, W135 synG, and Y synF showed 100% specificity for detecting N. meningitidis species, with high sensitivity (serogroup B synD, 99% [75/76]; W135 synG, 97% [38/39]; and Y synF, 100% [66/66]). The lower limits of detection (LLD) were 9, 43, and 10 copies/reaction for serogroup B synD, W135 synG, and Y synF assays, respectively, a significant improvement compared to results for the previous singleplex assays. We developed three multiplex real-time PCR assays for detection of (i) N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA (NHS assay); (ii) N. meningitidis serogroups A, W135, and X (AWX assay); and (iii) N. meningitidis serogroups B, C, and Y (BCY assay). Each multiplex assay was 100% specific for detecting its target organisms or serogroups, and the LLD was similar to that for the singleplex assay. Pairwise comparison of real-time PCR between multiplex and singleplex assays showed that cycle threshold values of the multiplex assay were similar to those for the singleplex assay. There were no substantial differences in sensitivity and specificity between these multiplex and singleplex real-time PCR assays. PMID:22170919

Clinical validation of multiplex real-time PCR assays for detection of bacterial meningitis pathogens.

PubMed

Wang, Xin; Theodore, M Jordan; Mair, Raydel; Trujillo-Lopez, Elizabeth; du Plessis, Mignon; Wolter, Nicole; Baughman, Andrew L; Hatcher, Cynthia; Vuong, Jeni; Lott, Lisa; von Gottberg, Anne; Sacchi, Claudio; McDonald, J Matthew; Messonnier, Nancy E; Mayer, Leonard W

2012-03-01

Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae are important causes of meningitis and other infections, and rapid, sensitive, and specific laboratory assays are critical for effective public health interventions. Singleplex real-time PCR assays have been developed to detect N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA and serogroup-specific genes in the cap locus for N. meningitidis serogroups A, B, C, W135, X, and Y. However, the assay sensitivity for serogroups B, W135, and Y is low. We aimed to improve assay sensitivity and develop multiplex assays to reduce time and cost. New singleplex real-time PCR assays for serogroup B synD, W135 synG, and Y synF showed 100% specificity for detecting N. meningitidis species, with high sensitivity (serogroup B synD, 99% [75/76]; W135 synG, 97% [38/39]; and Y synF, 100% [66/66]). The lower limits of detection (LLD) were 9, 43, and 10 copies/reaction for serogroup B synD, W135 synG, and Y synF assays, respectively, a significant improvement compared to results for the previous singleplex assays. We developed three multiplex real-time PCR assays for detection of (i) N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA (NHS assay); (ii) N. meningitidis serogroups A, W135, and X (AWX assay); and (iii) N. meningitidis serogroups B, C, and Y (BCY assay). Each multiplex assay was 100% specific for detecting its target organisms or serogroups, and the LLD was similar to that for the singleplex assay. Pairwise comparison of real-time PCR between multiplex and singleplex assays showed that cycle threshold values of the multiplex assay were similar to those for the singleplex assay. There were no substantial differences in sensitivity and specificity between these multiplex and singleplex real-time PCR assays.

Multiplex qRT-PCR for the Detection of Western Equine Encephalomyelitis, St. Louis Encephalitis, and West Nile Viral RNA in Mosquito Pools (Diptera: Culicidae)

PubMed Central

Brault, Aaron C.; Fang, Ying; Reisen, William K.

2015-01-01

Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer–probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk. PMID:26334826

Detection of selected intestinal helminths and protozoa at Hospital Universiti Sains Malaysia using multiplex real-time PCR.

PubMed

Basuni, M; Mohamed, Z; Ahmad, M; Zakaria, N Z; Noordin, R

2012-09-01

Intestinal parasites are the causative agents of a number of important human infections in developing countries. The objective of this study was to determine the prevalence of selected helminths and protozoan infections among patients admitted with gastrointestinal disorders at Hospital Universiti Sains Malaysia, Kelantan, Malaysia using multiplex real-time PCR. In addition microscopic examination was also performed following direct smear, zinc sulphate concentration and Kato-Katz thick smear techniques; and the presence of protozoan parasites was confirmed using trichrome and acid-fast stains. Of the 225 faecal samples analysed, 26.2% were positive for intestinal parasites by the multiplex real-time PCR, while 5.3% were positive by microscopy. As compared to microscopy, the multiplex real-time PCR detected 5.8 and 4.5 times more positives for the selected helminth and protozoan infections respectively. Among the selected helminths detected in this study, hookworm was the most prevalent by real-time PCR, while Ascaris lumbricoides was detected the most by microscopy. Meanwhile, among the selected protozoa detected in this study, Entamoeba histolytica was the most prevalent by real-time PCR, however microscopy detected equal number of cases with E. histolytica and Giardia lamblia. This study showed that real-time PCR can be used to obtain a more accurate prevalence data on intestinal helminths and protozoa.

Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains.

PubMed

He, Peiyan; Chen, Zhongwen; Luo, Jianyong; Wang, Henghui; Yan, Yong; Chen, Lixia; Gao, Wenjie

2014-01-01

Foodborne disease caused by pathogenic Vibrio parahaemolyticus has become a serious public health problem in many countries. Rapid diagnosis and the identification of pathogenic V. parahaemolyticus are very important in the context of public health. In this study, an EvaGreen-based multiplex real-time PCR assay was established for the detection of pathogenic V. parahaemolyticus. This assay targeted three genetic markers of V. parahaemolyticus (species-specific gene toxR and virulence genes tdh and trh). The assay could unambiguously identify pathogenic V. parahaemolyticus with a minimum detection limit of 1.4 pg genomic DNA per reaction (concentration giving a positive multiplex real-time PCR result in 95% of samples). The specificity of the assay was evaluated using 72 strains of V. parahaemolyticus and other bacteria. A validation of the assay with clinical samples confirmed its sensitivity and specificity. Our data suggest the newly established multiplex real-time PCR assay is practical, cost-effective, specific, sensitive and capable of high-throughput detection of pathogenic V. parahaemolyticus. Copyright © 2014. Published by Elsevier Ltd.

Fluorescence-Raman Dual Modal Endoscopic System for Multiplexed Molecular Diagnostics

NASA Astrophysics Data System (ADS)

Jeong, Sinyoung; Kim, Yong-Il; Kang, Homan; Kim, Gunsung; Cha, Myeong Geun; Chang, Hyejin; Jung, Kyung Oh; Kim, Young-Hwa; Jun, Bong-Hyun; Hwang, Do Won; Lee, Yun-Sang; Youn, Hyewon; Lee, Yoon-Sik; Kang, Keon Wook; Lee, Dong Soo; Jeong, Dae Hong

2015-03-01

Optical endoscopic imaging, which was recently equipped with bioluminescence, fluorescence, and Raman scattering, allows minimally invasive real-time detection of pathologies on the surface of hollow organs. To characterize pathologic lesions in a multiplexed way, we developed a dual modal fluorescence-Raman endomicroscopic system (FRES), which used fluorescence and surface-enhanced Raman scattering nanoprobes (F-SERS dots). Real-time, in vivo, and multiple target detection of a specific cancer was successful, based on the fast imaging capability of fluorescence signals and the multiplex capability of simultaneously detected SERS signals using an optical fiber bundle for intraoperative endoscopic system. Human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) on the breast cancer xenografts in a mouse orthotopic model were successfully detected in a multiplexed way, illustrating the potential of FRES as a molecular diagnostic instrument that enables real-time tumor characterization of receptors during routine endoscopic procedures.

Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense.

PubMed

Janzen, Timothy W; Thomas, Matthew C; Goji, Noriko; Shields, Michael J; Hahn, Kristen R; Amoako, Kingsley K

2015-02-01

Bacillus anthracis, the causative agent of anthrax, has the capacity to form highly resilient spores as part of its life cycle. The potential for the dissemination of these spores using food as a vehicle is a huge public health concern and, hence, requires the development of a foodborne bioterrorism response approach. In this work, we address a critical gap in food biodefense by presenting a novel, combined, sequential method involving the use of real-time PCR and pyrosequencing for the rapid, specific detection of B. anthracis spores in three food matrices: milk, apple juice, and bottled water. The food samples were experimentally inoculated with 40 CFU ml(-1), and DNA was extracted from the spores and analyzed after immunomagnetic separation. Applying the combination of multiplex real-time PCR and pyrosequencing, we successfully detected the presence of targets on both of the virulence plasmids and the chromosome. The results showed that DNA amplicons generated from a five-target multiplexed real-time PCR detection using biotin-labeled primers can be used for single-plex pyrosequencing detection. The combined use of multiplexed real-time PCR and pyrosequencing is a novel, rapid detection method for B. anthracis from food and provides a tool for accurate, quantitative identification with potential biodefense applications.

Establishment of a multiplex real-time RT-PCR assay for rapid identification of H6 subtype avian influenza viruses.

PubMed

Yang, Fan; Wu, Haibo; Liu, Fumin; Lu, Xiangyun; Peng, Xiuming; Wu, Nanping

2018-06-01

The H6 subtype avian influenza viruses (AIVs) possess the capacity for zoonotic transmission from avian species to humans. Establishment of a specific, rapid and sensitive method to screen H6 AIVs is necessary. Based on the conserved domain of the matrix and H6 AIV hemagglutinin genes, two TaqMan minor-groove-binder probes and multiplex real-time RT-PCR primers were designed in this study. The multiplex real-time RT-PCR assay developed in this study had high specificity and repeatability and a detection limit of 30 copies per reaction. This rapid diagnostic method will be useful for clinical detection and surveillance of H6 AIVs in China.

Multiplex real-time PCR for identification of canine parvovirus antigenic types.

PubMed

Kaur, Gurpreet; Chandra, Mudit; Dwivedi, P N; Narang, Deepti

2016-07-01

Canine parvovirus (CPV) is an important disease causing gastroenteritis and/or haemorrhagic gastroenteritis in dogs. There are four antigenic types of CPV reported worldwide viz. CPV 2, CPV 2a, CPV 2b and CPV 2c. The diagnosis of CPV with the identification of the antigen type responsible remains problematic. In the present study, identification as well as antigenic typing of CPV was done using a de novo multiplex real time PCR to combat the problem of antigenic type identification. From the study it could be concluded that the here developed multiplex real time PCR assay could be used for rapid detection of CPV as well as typing of its three antigenic types. Copyright © 2016 Elsevier B.V. All rights reserved.

Zika Virus and Chikungunya Virus CoInfections: A Series of Three Cases from a Single Center in Ecuador

PubMed Central

Zambrano, Hector; Waggoner, Jesse J.; Almeida, Cristina; Rivera, Lisette; Benjamin, Juan Quintana; Pinsky, Benjamin A.

2016-01-01

Zika virus (ZIKV) and chikungunya virus (CHIKV) cocirculate throughout much of the tropical Western Hemisphere; however, few cases of coinfection with these two pathogens have been reported. Herein, we describe three cases of ZIKV–CHIKV coinfection detected at a single center in Ecuador: a patient who developed symptoms on postoperative day 5 from an orthopedic procedure, a woman who had traveled to Ecuador for fertility treatment, and a woman who was admitted for Guillain–Barré syndrome and had ZIKV and CHIKV detected in serum and cerebrospinal fluid. All cases were diagnosed using a multiplex real-time reverse transcription polymerase chain reaction, and ZIKV viremia was detected as late as 16 days after symptom onset. These cases demonstrate the varied clinical presentation of ZIKV–CHIKV coinfections as well as the importance of multiplexed arboviral testing for these pathogens. PMID:27402518

Zika Virus and Chikungunya Virus CoInfections: A Series of Three Cases from a Single Center in Ecuador.

PubMed

Zambrano, Hector; Waggoner, Jesse J; Almeida, Cristina; Rivera, Lisette; Benjamin, Juan Quintana; Pinsky, Benjamin A

2016-10-05

Zika virus (ZIKV) and chikungunya virus (CHIKV) cocirculate throughout much of the tropical Western Hemisphere; however, few cases of coinfection with these two pathogens have been reported. Herein, we describe three cases of ZIKV-CHIKV coinfection detected at a single center in Ecuador: a patient who developed symptoms on postoperative day 5 from an orthopedic procedure, a woman who had traveled to Ecuador for fertility treatment, and a woman who was admitted for Guillain-Barré syndrome and had ZIKV and CHIKV detected in serum and cerebrospinal fluid. All cases were diagnosed using a multiplex real-time reverse transcription polymerase chain reaction, and ZIKV viremia was detected as late as 16 days after symptom onset. These cases demonstrate the varied clinical presentation of ZIKV-CHIKV coinfections as well as the importance of multiplexed arboviral testing for these pathogens. © The American Society of Tropical Medicine and Hygiene.

Simultaneous detection and differentiation of three Potyviridae viruses by a multiplex TaqMan real time RT-PCR assay

USDA-ARS?s Scientific Manuscript database

A multiplex TaqMan real time RT-PCR was developed for detection and differentiation of Sweet potato virus G, Sweet potato latent virus and Sweet potato mild mottle virus in one tube. Amplification and detection of a fluorogenic cytochrome oxidase gene was included as an internal control. The assay w...

Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels.

PubMed

Agardh, Elisabet; Gustavsson, Carin; Hagert, Per; Nilsson, Marie; Agardh, Carl-David

2006-02-01

The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.

Twisted Acoustics: Metasurface-Enabled Multiplexing and Demultiplexing.

PubMed

Jiang, Xue; Liang, Bin; Cheng, Jian-Chun; Qiu, Cheng-Wei

2018-05-01

Metasurfaces are used to enable acoustic orbital angular momentum (a-OAM)-based multiplexing in real-time, postprocess-free, and sensor-scanning-free fashions to improve the bandwidth of acoustic communication, with intrinsic compatibility and expandability to cooperate with other multiplexing schemes. The metasurface-based communication relying on encoding information onto twisted beams is numerically and experimentally demonstrated by realizing real-time picture transfer, which differs from existing static data transfer by encoding data onto OAM states. With the advantages of real-time transmission, passive and instantaneous data decoding, vanishingly low loss, compact size, and high transmitting accuracy, the study of a-OAM-based information transfer with metasurfaces offers new route to boost the capacity of acoustic communication and great potential to profoundly advance relevant fields. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Clinical evaluation of a new single-tube multiplex reverse transcription PCR assay for simultaneous detection of 11 respiratory viruses, Mycoplasma pneumoniae and Chlamydia in hospitalized children with acute respiratory infections.

PubMed

Zhao, Meng-Chuan; Li, Gui-Xia; Zhang, Dan; Zhou, Hang-Yu; Wang, Hao; Yang, Shuo; Wang, Le; Feng, Zhi-Shan; Ma, Xue-Jun

2017-06-01

Respiratory Pathogen 13 Detection Kit (13× kit) is able to simultaneously detect 11 respiratory viruses, Mycoplasma pneumoniae (MP) and Chlamydia in a single reaction. Using 572 Nasopharyngeal aspirates collected from hospitalized children, the clinical performance of 13× kit for detecting 11 respiratory viruses was evaluated in comparison with a routinely used 2-tube multiplex reverse transcription PCR assay (2-tube assay) at provincial Centers for Disease Control and Prevention in China. The clinical performance of 13× kit for detecting MP and Chlamydia was evaluated by commercial real-time quantitative PCR (qPCR) kits or sequencing. For tested viruses, the assay concordance was 95.98% and the kappa coefficient was 0.89. All the MP and Chlamydia positive samples detected by 13× kit were confirmed as true positives. The utilization of the 13× kit in clinical settings will be helpful for doctors to assess clinical outcome according to virus type or multiple infections, and to limit the use of antibiotics. Copyright © 2017 Elsevier Inc. All rights reserved.

Design of primers and probes for quantitative real-time PCR methods.

PubMed

Rodríguez, Alicia; Rodríguez, Mar; Córdoba, Juan J; Andrade, María J

2015-01-01

Design of primers and probes is one of the most crucial factors affecting the success and quality of quantitative real-time PCR (qPCR) analyses, since an accurate and reliable quantification depends on using efficient primers and probes. Design of primers and probes should meet several criteria to find potential primers and probes for specific qPCR assays. The formation of primer-dimers and other non-specific products should be avoided or reduced. This factor is especially important when designing primers for SYBR(®) Green protocols but also in designing probes to ensure specificity of the developed qPCR protocol. To design primers and probes for qPCR, multiple software programs and websites are available being numerous of them free. These tools often consider the default requirements for primers and probes, although new research advances in primer and probe design should be progressively added to different algorithm programs. After a proper design, a precise validation of the primers and probes is necessary. Specific consideration should be taken into account when designing primers and probes for multiplex qPCR and reverse transcription qPCR (RT-qPCR). This chapter provides guidelines for the design of suitable primers and probes and their subsequent validation through the development of singlex qPCR, multiplex qPCR, and RT-qPCR protocols.

Failure to detect Xenotropic murine leukaemia virus-related virus in Chinese patients with chronic fatigue syndrome

PubMed Central

2010-01-01

Background Recent controversy has surrounded the question of whether xenotropic murine leukaemia virus-related virus (XMRV) contributes to the pathogenesis of chronic fatigue syndrome (CFS). To investigate the question in a Chinese population, 65 CFS patients and 85 blood donor controls were enrolled and multiplex real-time PCR or reverse transcriptase PCR (RT-PCR) was developed to analyze the XMRV infection status of the study participants. The assay was standardized by constructing plasmid DNAs and armored RNAs as XMRV standards and competitive internal controls (CICs), respectively. Results The sensitivities of the multiplex real-time PCR and RT-PCR assays were 20 copies/reaction and 10 IU/ml, respectively, with 100% specificity. The within-run precision coefficient of variation (CV) ranged from 1.76% to 2.80% and 1.70% to 2.59%, while the between-run CV ranged from 1.07% to 2.56% and 1.06% to 2.74%. XMRV was not detected in the 65 CFS patients and 65 normal individuals out of 85 controls. Conclusions This study failed to show XMRV in peripheral blood mononuclear cells (PBMCs) and plasma of Chinese patients with CFS. The absence of XMRV nucleic acids does not support an association between XMRV infection and the development of CFS in Chinese. PMID:20836869

Detection of Anaplasma phagocytophilum, Babesia microti, Borrelia burgdorferi, Borrelia miyamotoi, and Powassan Virus in Ticks by a Multiplex Real-Time Reverse Transcription-PCR Assay

PubMed Central

Tagliafierro, Teresa; Cucura, D. Moses; Rochlin, Ilia; Sameroff, Stephen; Lipkin, W. Ian

2017-01-01

ABSTRACT Ixodes scapularis ticks are implicated in transmission of Anaplasma phagocytophilum, Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, and Powassan virus. We describe the establishment and implementation of the first multiplex real-time PCR assay with the capability to simultaneously detect and differentiate all five pathogens in a single reaction. The application of this assay for analysis of ticks at sites in New York and Connecticut revealed a high prevalence of B. microti in ticks from Suffolk County, NY. These findings are consistent with reports of a higher incidence of babesiosis from clinicians managing the care of patients with tick-borne diseases in this region. IMPORTANCE The understanding of pathogen prevalence is an important factor in the determination of human risks for tick-borne diseases and can help guide diagnosis and treatment. The implementation of our assay addresses a critical need in surveillance of tick-borne diseases, through generation of a comprehensive assessment of pathogen prevalence in I. scapularis. Our finding of a high frequency of ticks infected with Babesia microti in Suffolk County, NY, implicates this agent as a probable frequent cause of non-Lyme tick-borne disease in this area. PMID:28435891

Detection of Anaplasma phagocytophilum, Babesia microti, Borrelia burgdorferi, Borrelia miyamotoi, and Powassan Virus in Ticks by a Multiplex Real-Time Reverse Transcription-PCR Assay.

PubMed

Tokarz, Rafal; Tagliafierro, Teresa; Cucura, D Moses; Rochlin, Ilia; Sameroff, Stephen; Lipkin, W Ian

2017-01-01

Ixodes scapularis ticks are implicated in transmission of Anaplasma phagocytophilum , Borrelia burgdorferi , Borrelia miyamotoi , Babesia microti , and Powassan virus. We describe the establishment and implementation of the first multiplex real-time PCR assay with the capability to simultaneously detect and differentiate all five pathogens in a single reaction. The application of this assay for analysis of ticks at sites in New York and Connecticut revealed a high prevalence of B. microti in ticks from Suffolk County, NY. These findings are consistent with reports of a higher incidence of babesiosis from clinicians managing the care of patients with tick-borne diseases in this region. IMPORTANCE The understanding of pathogen prevalence is an important factor in the determination of human risks for tick-borne diseases and can help guide diagnosis and treatment. The implementation of our assay addresses a critical need in surveillance of tick-borne diseases, through generation of a comprehensive assessment of pathogen prevalence in I. scapularis . Our finding of a high frequency of ticks infected with Babesia microti in Suffolk County, NY, implicates this agent as a probable frequent cause of non-Lyme tick-borne disease in this area.

An experimental study for rapid detection and quantification of endodontic microbiota following photo-activated disinfection via new multiplex real-time PCR assay.

PubMed

Pourhajibagher, Maryam; Raoofian, Reza; Ghorbanzadeh, Roghayeh; Bahador, Abbas

2018-03-01

The infected root canal system harbors one of the highest accumulations of polymicrobial infections. Since the eradication of endopathogenic microbiota is a major goal in endodontic infection therapy, photo-activated disinfection (PAD) can be used as an alternative therapeutic method in endodontic treatment. Compared to cultivation-based approaches, molecular techniques are more reliable for identifying microbial agents associated with endodontic infections. The purpose of this study was to evaluate the ability of designed multiplex real-time PCR protocol for the rapid detection and quantification of six common microorganisms involved in endodontic infection before and after the PAD. Samples were taken from the root canals of 50 patients with primary and secondary/persistent endodontic infections using sterile paper points. PAD with toluidine blue O (TBO) plus diode laser was performed on root canals. Resampling was then performed, and the samples were transferred to transport medium. Then, six target microorganisms were detected using multiplex real-time PCR before and after the PAD. Veillonella parvula was found using multiplex real-time PCR to have the highest frequency among samples collected before the PAD (29.4%), followed by Porphyromonas gingivalis (23.1%), Aggregatibacter actinomycetemcomitans (13.6%), Actinomyces naeslundii (13.0%), Enterococcus faecalis (11.5%), and Lactobacillus rhamnosus (9.4%). After TBO-mediated PAD, P. gingivalis strains, the most resistance microorganisms, were recovered in 41.7% of the samples using molecular approach (P > 0.05). As the results shown, multiplex real-time PCR as an accurate detection approach with high-throughput and TBO-mediated PAD as an efficient antimicrobial strategy due to the significant reduction of the endopathogenic count can be used for detection and treatment of microbiota involved in infected root canals, respectively. Copyright © 2018 Elsevier B.V. All rights reserved.

A novel multiplex real-time PCR assay for the detection and quantification of HPV16/18 and HSV1/2 in cervical cancer screening.

PubMed

Zhao, Youyun; Cao, Xuan; Tang, Jingfeng; Zhou, Li; Gao, Yinglin; Wang, Jiangping; Zheng, Yi; Yin, Shanshan; Wang, Yefu

2012-04-01

Infection with human papillomavirus (HPV), particularly HPV16 and HPV18, is the main cause of invasive cervical cancer, although other factors such as herpes simplex virus (HSV) may act in conjunction with HPV in this context. To explore the possibility of developing a system for rapid diagnosis and clinical screening of cervical cancer, we developed a multiplex real-time PCR assay that can simultaneously detect and quantify HPV16/18 and HSV1/2. To evaluate its possibilities and practical uses, 177 samples collected from patients with suspected HPV and HSV infection in exfoliated cervical cells, genital herpes or labial herpes were tested by multiplex real-time PCR and compared with results obtained by DNA sequencing. Each virus was detected over a range from 1.0 × 10(1) to 1.0 × 10(7) copies/reaction. The clinical sensitivity was 100% for HPV16/18 and HSV1/2. The clinical specificity was 97.1% for HPV16, 98.1% for HPV18, 97.0% for HSV1 and 96.0% for HSV2. The kappa value was 0.96 for HPV16, 0.92 for HPV18, 0.94 for HSV1 and 0.93 for HSV2, when DNA sequencing was used as the reference standard. In summary, this novel multiplex real-time PCR allows the rapid and specific detection of HPV16/18 and HSV1/2, as well as coinfection with HPV and HSV, in clinical samples. In the future, this multiplex real-time PCR assay will assist in cervical cancer screening, viral treatment evaluation and epidemiological studies in which high throughput analysis is required. Copyright © 2012 Elsevier Ltd. All rights reserved.

Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction.

PubMed

Grau, Frederic R; Schroeder, Megan E; Mulhern, Erin L; McIntosh, Michael T; Bounpheng, Mangkey A

2015-03-01

African swine fever (ASF), classical swine fever (CSF), and foot-and-mouth disease (FMD) are highly contagious animal diseases of significant economic importance. Pigs infected with ASF and CSF viruses (ASFV and CSFV) develop clinical signs that may be indistinguishable from other diseases. Likewise, various causes of vesicular disease can mimic clinical signs caused by the FMD virus (FMDV). Early detection is critical to limiting the impact and spread of these disease outbreaks, and the ability to perform herd-level surveillance for all 3 diseases rapidly and cost effectively using a single diagnostic sample and test is highly desirable. This study assessed the feasibility of simultaneous ASFV, CSFV, and FMDV detection by multiplex reverse transcription real-time polymerase chain reaction (mRT-qPCR) in swine oral fluids collected through the use of chewing ropes. Animal groups were experimentally infected independently with each virus, observed for clinical signs, and oral fluids collected and tested throughout the course of infection. All animal groups chewed on the ropes readily before and after onset of clinical signs and before onset of lameness or serious clinical signs. ASFV was detected as early as 3 days postinoculation (dpi), 2-3 days before onset of clinical disease; CSFV was detected at 5 dpi, coincident with onset of clinical disease; and FMDV was detected as early as 1 dpi, 1 day before the onset of clinical disease. Equivalent results were observed in 4 independent studies and demonstrate the feasibility of oral fluids and mRT-qPCR for surveillance of ASF, CSF, and FMD in swine populations. © 2015 The Author(s).

A new multiplex real-time polymerase chain reaction assay for the diagnosis of periprosthetic joint infection.

PubMed

Kawamura, Masaki; Kobayashi, Naomi; Inaba, Yutaka; Choe, Hyonmin; Tezuka, Taro; Kubota, So; Saito, Tomoyuki

2017-11-01

A new multiplex real-time polymerase chain reaction (PCR) assay was developed to detect methicillin-resistant Staphylococcus (MRS) and to distinguish between gram-positive and gram-negative bacteria. In this study, we validated the sensitivity and specificity of this assay with periprosthetic joint infections (PJIs) and evaluated the utility of PCR for culture-negative PJI. Forty-five samples from 23 infectious PJI cases and 106 samples from 64 non-infectious control cases were analyzed by real-time PCR using a LightCycler Nano ® system. Twenty-eight clinical samples, comprising bacteria of known species isolated consecutively in the microbiological laboratory of our hospital, were used to determine the spectrum of bacterial species that could be detected using the new multiplex primers and probes. The sensitivity and specificity of the MRS- and universal-PCR assays were 92% and 99%, and 91% and 88%, respectively. Twenty-eight species of clinically isolated bacteria were detected using this method and the concordance rate for the identification of gram-positive or gram-negative organisms was 96%. Eight samples were identified as PCR-positive despite a culture-negative result. This novel multiplex real-time PCR system has acceptable sensitivity and specificity and several advantages; therefore, it has potential use for the diagnosis of PJIs, particularly in culture-negative cases.

Assessment of the Usefulness of Multiplex Real-Time PCR Tests in the Diagnostic and Therapeutic Process of Pneumonia in Hospitalized Children: A Single-Center Experience.

PubMed

Gowin, Ewelina; Bartkowska-Śniatkowska, Alicja; Jończyk-Potoczna, Katarzyna; Wysocka-Leszczyńska, Joanna; Bobkowski, Waldemar; Fichna, Piotr; Sobkowiak, Paulina; Mazur-Melewska, Katarzyna; Bręborowicz, Anna; Wysocki, Jacek; Januszkiewicz-Lewandowska, Danuta

2017-01-01

The aim of the study was assessment of the usefulness of multiplex real-time PCR tests in the diagnostic and therapeutic process in children hospitalized due to pneumonia and burdened with comorbidities. Methods . The study group included 97 children hospitalized due to pneumonia at the Karol Jonscher Teaching Hospital in Poznań, in whom multiplex real-time PCR tests (FTD respiratory pathogens 33; fast-track diagnostics) were used. Results . Positive test results of the test were achieved in 74 patients (76.3%). The average age in the group was 56 months. Viruses were detected in 61 samples (82% of all positive results); bacterial factors were found in 29 samples (39% of all positive results). The presence of comorbidities was established in 90 children (92.78%). On the basis of the obtained results, 5 groups of patients were established: viral etiology of infection, 34 patients; bacterial etiology, 7 patients; mixed etiology, 23 patients; pneumocystis, 9 patients; and no etiology diagnosed, 24 patients. Conclusions . Our analysis demonstrated that the participation of viruses in causing severe lung infections is significant in children with comorbidities. Multiplex real-time PCR tests proved to be more useful in establishing the etiology of pneumonia in hospitalized children than the traditional microbiological examinations.

Ultrafast, sensitive and large-volume on-chip real-time PCR for the molecular diagnosis of bacterial and viral infections.

PubMed

Houssin, Timothée; Cramer, Jérémy; Grojsman, Rébecca; Bellahsene, Lyes; Colas, Guillaume; Moulet, Hélène; Minnella, Walter; Pannetier, Christophe; Leberre, Maël; Plecis, Adrien; Chen, Yong

2016-04-21

To control future infectious disease outbreaks, like the 2014 Ebola epidemic, it is necessary to develop ultrafast molecular assays enabling rapid and sensitive diagnoses. To that end, several ultrafast real-time PCR systems have been previously developed, but they present issues that hinder their wide adoption, notably regarding their sensitivity and detection volume. An ultrafast, sensitive and large-volume real-time PCR system based on microfluidic thermalization is presented herein. The method is based on the circulation of pre-heated liquids in a microfluidic chip that thermalize the PCR chamber by diffusion and ultrafast flow switches. The system can achieve up to 30 real-time PCR cycles in around 2 minutes, which makes it the fastest PCR thermalization system for regular sample volume to the best of our knowledge. After biochemical optimization, anthrax and Ebola simulating agents could be respectively detected by a real-time PCR in 7 minutes and a reverse transcription real-time PCR in 7.5 minutes. These detections are respectively 6.4 and 7.2 times faster than with an off-the-shelf apparatus, while conserving real-time PCR sample volume, efficiency, selectivity and sensitivity. The high-speed thermalization also enabled us to perform sharp melting curve analyses in only 20 s and to discriminate amplicons of different lengths by rapid real-time PCR. This real-time PCR microfluidic thermalization system is cost-effective, versatile and can be then further developed for point-of-care, multiplexed, ultrafast and highly sensitive molecular diagnoses of bacterial and viral diseases.

One-step multiplex RT-qPCR detects three citrus viroids from different genera in a wide range of hosts.

PubMed

Osman, Fatima; Dang, Tyler; Bodaghi, Sohrab; Vidalakis, Georgios

2017-07-01

A one-step multiplex reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) based on species-specific minor groove binding (MGB) probes, was developed for the simultaneous detection, identification, and quantification of three citrus viroids belonging to different genera. Citrus exocortis viroid (Pospiviroid), Hop stunt viroid (Hostuviroid), and Citrus bark cracking viroid (Cocadviroid) cause a variety of maladies in agriculturally significant crops. Therefore, reliable assays for their detection are essential tools for various government and industry organizations implementing disease management programs. Singleplex qPCR primers and MGB probes were designed individually for the detection of the three targeted viroids, and subsequently combined in a one-step multiplex RT-qPCR reaction. A wide host range of woody plants, including citrus, grapevines, apricots, plums and herbaceous plants such as tomato, cucumber, eggplant and chrysanthemum different world regions were used to validate the assay. Single, double and triple viroid infections were identified in the tested samples. The developed multiplex RT-qPCR assay was compared with a previously reported SYBR Green I RT-qPCR for the universal detection of citrus viroids. Both assays accurately identified all citrus viroid infected samples. The multiplex assay complemented the SYBR Green I universal detection assay by differentiating among citrus viroid species in the positive samples. The developed multiplex RT-qPCR assay has the potential to simultaneously detect each targeted viroid and could be used in high throughput screenings for citrus viroids in field surveys, germplasm banks, nurseries and other viroid disease management programs. Copyright © 2017. Published by Elsevier B.V.

Development and clinical validation of a multiplex real-time PCR assay for herpes simplex and varicella zoster virus.

PubMed

Tan, Thean Yen; Zou, Hao; Ong, Danny Chee Tiong; Ker, Khor Jia; Chio, Martin Tze Wei; Teo, Rachael Yu Lin; Koh, Mark Jean Aan

2013-12-01

Herpes simplex virus (HSV) and varicella zoster virus (VZV) are related members of the Herpesviridae family and are well-documented human pathogens causing a spectrum of diseases, from mucocutaneous disease to infections of the central nervous system. This study was carried out to evaluate and validate the performance of a multiplex real-time polymerase chain reaction (PCR) assay in detecting and differentiating HSV1, HSV2, and VZV from clinical samples. Consensus PCR primers for HSV were designed from the UL30 component of the DNA polymerase gene of HSV, with 2 separate hydrolysis probes designed to differentiate HSV1 and HSV2. Separate primers and a probe were also designed against the DNA polymerase gene of VZV. A total of 104 clinical samples were available for testing by real-time PCR, conventional PCR, and viral culture. The sensitivity and specificity of the real-time assay was calculated by comparing the multiplex PCR result with that of a combined standard of virus culture and conventional PCR. The sensitivity of the real-time assay was 100%, with specificity ranging from 98% to 100% depending on the target gene. Both PCR methods detected more positive samples for HSV or VZV, compared with conventional virus culture. This multiplex PCR assay provides accurate and rapid diagnostic capabilities for the diagnosis and differentiation of HSV1, HSV2, and VZV infections, with the presence of an internal control to monitor for inhibition of the PCR reaction.

Multiplex qRT-PCR for the Detection of Western Equine Encephalomyelitis, St. Louis Encephalitis, and West Nile Viral RNA in Mosquito Pools (Diptera: Culicidae).

PubMed

Brault, Aaron C; Fang, Ying; Reisen, William K

2015-05-01

Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer-probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Development a of multiplex TaqMan real-time RT-PCR assay for simultaneous detection of Asian prunus viruses, plum bark necrosis stem pitting associated virus, and peach latent mosaic virus

USDA-ARS?s Scientific Manuscript database

Asian prunus viruses (APV 1, APV 2 and APV 3) and Plum bark necrosis stem pitting associated virus (PBNSPaV) are two recently described viruses infecting Prunus spp., and Peach latent mosaic viroid (PLMVd) is a viroid that infects the same species. A single-tube multiplex, TaqMan real-time RT-PCR as...

Development of single-step multiplex real-time RT-PCR assays for rapid diagnosis of enterovirus 71, coxsackievirus A6, and A16 in patients with hand, foot, and mouth disease.

PubMed

Puenpa, Jiratchaya; Suwannakarn, Kamol; Chansaenroj, Jira; Vongpunsawad, Sompong; Poovorawan, Yong

2017-10-01

Real-time reverse-transcription polymerase chain reaction (rRT-PCR) to detect enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) has facilitated the rapid and accurate identification of the two most common etiological agents underlying hand, foot, and mouth disease (HFMD). However, the worldwide emergence of CV-A6 infection in HFMD necessitates development of an improved multiplex rRT-PCR method. To rapidly determine the etiology of HFMD, two rRT-PCR assays using TaqMan probes were developed to differentiate among three selected common enteroviruses (EV-A71, CV-A16 and CV-A6) and to enable broad detection of enteroviruses (pan-enterovirus assay). No cross-reactions were observed with other RNA viruses examined. The detection limits of both assays were 10 copies per microliter for EV-A71, CV-A6 and CV-A16, and pan-enterovirus. The methods showed high accuracy (EV-A71, 90.6%; CV-A6, 92.0%; CV-A16, 100%), sensitivity (EV-A71, 96.5%; CV-A6, 95.8%; CV-A16, 99.0%), and specificity (EV-A71, 100%; CV-A6, 99.9%; CV-A16, 99.9%) in testing clinical specimens (n=1049) during 2014-2016, superior to those of conventional RT-PCR. Overall, the multiplex rRT-PCR assays enabled highly sensitive detection and rapid simultaneous typing of EV-A71, CV-A6 and CV-A16, and enteroviruses, rendering them feasible and attractive methods for large-scale surveillance of enteroviruses associated with HFMD outbreaks. Copyright © 2017 Elsevier B.V. All rights reserved.

A multiplex real-time PCR assay for the identification and differentiation of Salmonella enterica serovar Typhimurium and monophasic serovar 4,[5],12:i:-.

PubMed

Prendergast, Deirdre M; Hand, Darren; Nί Ghallchóir, Eadaoin; McCabe, Evonne; Fanning, Seamus; Griffin, Margaret; Egan, John; Gutierrez, Montserrat

2013-08-16

Salmonella enterica subsp. enterica serovar 4,[5],12:i:- is considered to be a monophasic variant of Salmonella Typhimurium and is increasingly associated with human infections. The use of PCR for the unequivocal identification of strains identified by conventional serotyping as 4,[5],12:i:- has been recommended by the European Food Safety Authority (EFSA), in particular the conventional multiplex PCR developed by Tennant et al. (2010). An alternative protocol for the identification and differentiation of S. Typhimurium and S. Typhimurium-like strains, including its monophasic variants, based on a multiplex real-time PCR assay was developed in our laboratory. A panel of 206 Salmonella strains was used to validate our multiplex real-time PCR against the conventional multiplex PCR recommended by EFSA, i.e. 43 Salmonella strains of serovars other than Typhimurium and 163 routine isolates determined by slide agglutination serotyping to have an incomplete antigenic formula compatible with the S. Typhimurium formula 4,[5],12:i:1,2. Both methods correctly identified the 43 Salmonella strains as non S. Typhimurium. Among the 163 isolates of undetermined serovar by conventional serotyping, both PCR protocols identified 54 isolates as S. Typhimurium, 101 as monophasic S. Typhimurium and 8 as non-S. Typhimurium. Twenty isolates phenotypically lacking the phase-2 H antigen were positive for the fljB.1,2 gene. These strains have been recently described in the literature by other workers and have been referred to as "inconsistent" variants of S. Typhimurium. Antimicrobial resistance and phage typing were also performed on the S. Typhimurium isolates, including monophasic variants, and approximately half of the isolates identified as monophasic S. Typhimurium by our multiplex real-time PCR protocol were DT193 with the resistance pattern ASSuT. There was 100% concordance between the conventional PCR and the multiplex real-time PCR method developed in this study which proved that our protocol is equivalent to the one recommended by EFSA. In comparison to the conventional PCR, this new protocol is faster and is currently being applied routinely in our laboratory to all isolates that could potentially be S. Typhimurium. Copyright © 2013 Elsevier B.V. All rights reserved.

Real-Time Reverse Transcription-PCR Assay for Detection of Mumps Virus RNA in Clinical Specimens▿

PubMed Central

Boddicker, Jennifer D.; Rota, Paul A.; Kreman, Trisha; Wangeman, Andrea; Lowe, Louis; Hummel, Kimberly B.; Thompson, Robert; Bellini, William J.; Pentella, Michael; DesJardin, Lucy E.

2007-01-01

The mumps virus is a negative-strand RNA virus in the family Paramyxoviridae. Mumps infection results in an acute illness with symptoms including fever, headache, and myalgia, followed by swelling of the salivary glands. Complications of mumps can include meningitis, deafness, pancreatitis, orchitis, and first-trimester abortion. Laboratory confirmation of mumps infection can be made by the detection of immunoglobulin M-specific antibodies to mumps virus in acute-phase serum samples, the isolation of mumps virus in cell culture, or by detection of the RNA of the mumps virus by reverse transcription (RT)-PCR. We developed and validated a multiplex real-time RT-PCR assay for rapid mumps diagnosis in a clinical setting. This assay used oligonucleotide primers and a TaqMan probe targeting the mumps SH gene, as well as primers and a probe that targeted the human RNase P gene to assess the presence of PCR inhibitors and as a measure of specimen quality. The test was specific, since it did not amplify a product from near-neighbor viruses, as well as sensitive and accurate. Real-time RT-PCR results showed 100% correlation with results from viral culture, the gold standard for mumps diagnostic testing. Assay efficiency was over 90% and displayed good precision after performing inter- and intraassay replicates. Thus, we have developed and validated a molecular method for rapidly diagnosing mumps infection that may be used to complement existing techniques. PMID:17652480

Optical chaos and hybrid WDM/TDM based large capacity quasi-distributed sensing network with real-time fiber fault monitoring.

PubMed

Luo, Yiyang; Xia, Li; Xu, Zhilin; Yu, Can; Sun, Qizhen; Li, Wei; Huang, Di; Liu, Deming

2015-02-09

An optical chaos and hybrid wavelength division multiplexing/time division multiplexing (WDM/TDM) based large capacity quasi-distributed sensing network with real-time fiber fault monitoring is proposed. Chirped fiber Bragg grating (CFBG) intensity demodulation is adopted to improve the dynamic range of the measurements. Compared with the traditional sensing interrogation methods in time, radio frequency and optical wavelength domains, the measurand sensing and the precise locating of the proposed sensing network can be simultaneously interrogated by the relative amplitude change (RAC) and the time delay of the correlation peak in the cross-correlation spectrum. Assisted with the WDM/TDM technology, hundreds of sensing units could be potentially multiplexed in the multiple sensing fiber lines. Based on the proof-of-concept experiment for axial strain measurement with three sensing fiber lines, the strain sensitivity up to 0.14% RAC/με and the precise locating of the sensors are achieved. Significantly, real-time fiber fault monitoring in the three sensing fiber lines is also implemented with a spatial resolution of 2.8 cm.

A Multiplex RT-PCR Assay for S. aureus, L. monocytogenes, and Salmonella spp. Detection in Raw Milk with Pre-enrichment.

PubMed

Ding, Tian; Suo, Yuanjie; Zhang, Zhaohuan; Liu, Donghong; Ye, Xingqian; Chen, Shiguo; Zhao, Yong

2017-01-01

This study firstly developed a multiplex real-time PCR (RT-PCR) technique combined with a pre-enrichment step to simultaneously detect Staphylococcus aureus ( S. aureus ), Listeria monocytogenes ( L. monocytogenes ) and Salmonella spp. in raw milk and the dairy farm environment (feces, soil, feed, water) in one reaction. Brain heart infusion (BHI) broth was selected for the enrichment step to increase the density of the target bacteria by using an incubation of 4 h before multiplex RT-PCR. The results showed that the detection limit of the multiplex real-time assay was approximately 10 2 CFU/mL for pure cultures and artificially contaminated milk without enrichment, while 12, 14, and 10 CFU/25 mL, respectively, for S. aureus, L. monocytogenes , and Salmonella spp. after pre-enrichment. The newly developed multiplex RT-PCR assay was applied to 46 dairy farm environmental samples and raw milk samples covering a wide variety of sample types. The results demonstrated that the multiplex RT-PCR assay coupled with the BHI enrichment broth was suitable for the simultaneous screening of S. aureus, L. monocytogenes , and Salmonella spp. in the pasture environment and in raw milk. The multiplex RT-PCR assay clearly and successfully shortened the total detection time and reduced labor compared to conventional culture-based methods for testing natural samples.

A Multiplex RT-PCR Assay for S. aureus, L. monocytogenes, and Salmonella spp. Detection in Raw Milk with Pre-enrichment

PubMed Central

Ding, Tian; Suo, Yuanjie; Zhang, Zhaohuan; Liu, Donghong; Ye, Xingqian; Chen, Shiguo; Zhao, Yong

2017-01-01

This study firstly developed a multiplex real-time PCR (RT-PCR) technique combined with a pre-enrichment step to simultaneously detect Staphylococcus aureus (S. aureus), Listeria monocytogenes (L. monocytogenes) and Salmonella spp. in raw milk and the dairy farm environment (feces, soil, feed, water) in one reaction. Brain heart infusion (BHI) broth was selected for the enrichment step to increase the density of the target bacteria by using an incubation of 4 h before multiplex RT-PCR. The results showed that the detection limit of the multiplex real-time assay was approximately 102 CFU/mL for pure cultures and artificially contaminated milk without enrichment, while 12, 14, and 10 CFU/25 mL, respectively, for S. aureus, L. monocytogenes, and Salmonella spp. after pre-enrichment. The newly developed multiplex RT-PCR assay was applied to 46 dairy farm environmental samples and raw milk samples covering a wide variety of sample types. The results demonstrated that the multiplex RT-PCR assay coupled with the BHI enrichment broth was suitable for the simultaneous screening of S. aureus, L. monocytogenes, and Salmonella spp. in the pasture environment and in raw milk. The multiplex RT-PCR assay clearly and successfully shortened the total detection time and reduced labor compared to conventional culture-based methods for testing natural samples. PMID:28620364

Usefulness of Multiplex Real-Time PCR for Simultaneous Pathogen Detection and Resistance Profiling of Staphylococcal Bacteremia

PubMed Central

Chung, Yousun; Kim, Taek Soo; Min, Young Gi; Hong, Yun Ji; Park, Jeong Su; Hwang, Sang Mee; Song, Kyoung-Ho; Kim, Eu Suk; Kim, Hong Bin; Song, Junghan; Kim, Eui-Chong

2016-01-01

Staphylococci are the leading cause of nosocomial blood stream infections. Fast and accurate identification of staphylococci and confirmation of their methicillin resistance are crucial for immediate treatment with effective antibiotics. A multiplex real-time PCR assay that targets mecA, femA specific for S. aureus, femA specific for S. epidermidis, 16S rRNA for universal bacteria, and 16S rRNA specific for staphylococci was developed and evaluated with 290 clinical blood culture samples containing Gram-positive cocci in clusters (GPCC). For the 262 blood cultures identified to the species level with the MicroScan WalkAway system (Siemens Healthcare Diagnostics, USA), the direct real-time PCR assay of positive blood cultures showed very good agreement for the categorization of staphylococci into methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. epidermidis (MRSE), methicillin-susceptible S. epidermidis (MSSE), methicillin-resistant non-S. epidermidis CoNS (MRCoNS), and methicillin-susceptible non-S. epidermidis CoNS (MSCoNS) (κ = 0.9313). The direct multiplex real-time PCR assay of positive blood cultures containing GPCC can provide essential information at the critical point of infection with a turnaround time of no more than 4 h. Further studies should evaluate the clinical outcome of using this rapid real-time PCR assay in glycopeptide antibiotic therapy in clinical settings. PMID:27403436

Multiplex real-time PCR assay for Legionella species.

PubMed

Kim, Seung Min; Jeong, Yoojung; Sohn, Jang Wook; Kim, Min Ja

2015-12-01

Legionella pneumophila serogroup 1 (sg1) accounts for the majority of infections in humans, but other Legionella species are also associated with human disease. In this study, a new SYBR Green I-based multiplex real-time PCR assay in a single reaction was developed to allow the rapid detection and differentiation of Legionella species by targeting specific gene sequences. Candidate target genes were selected, and primer sets were designed by referring to comparative genomic hybridization data of Legionella species. The Legionella species-specific groES primer set successfully detected all 30 Legionella strains tested. The xcpX and rfbA primers specifically detected L. pneumophila sg1-15 and L. pneumophila sg1, respectively. In addition, this assay was validated by testing clinical samples and isolates. In conclusion, this novel multiplex real-time PCR assay might be a useful diagnostic tool for the rapid detection and differentiation of Legionella species in both clinical and epidemiological studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Diagnosis of ocular toxoplasmosis by two polymerase chain reaction (PCR) examinations: qualitative multiplex and quantitative real-time.

PubMed

Sugita, Sunao; Ogawa, Manabu; Inoue, Shizu; Shimizu, Norio; Mochizuki, Manabu

2011-09-01

To establish a two-step polymerase chain reaction (PCR) diagnostic system for ocular toxoplasmosis. A total of 13 ocular fluid samples (11 aqueous humor and 2 vitreous fluid) were collected from 13 patients with clinically suspected ocular toxoplasmosis. Ten ocular samples from other uveitis patients and 20 samples from subjects without ocular inflammation were used as controls. Two polymerase chain reaction (PCR) methods, i.e., qualitative multiplex PCR and quantitative real-time PCR, were used to measure the toxoplasma genome (T. gondii B1 gene). Qualitative multiplex PCR detected T. gondii B1 gene in the ocular fluids of 11 out of 13 patients with clinically suspected ocular toxoplasmosis. In real-time PCR, we detected high copy numbers of T. gondii DNA (5.1 × 10(2)-2.1 × 10(6) copies/mL) in a total of 10 patients (10/13, 77%). Only ocular toxoplasmosis scar lesions were observed in the three real-time PCR-negative patients. PCR assay results for the samples from the two control groups were all negative. The two-step PCR examination to detect toxoplasma DNA is a useful tool for diagnosing ocular toxoplasmosis.

Development of a multiplex real-time PCR for the simultaneous detection of herpes simplex and varicella zoster viruses in cerebrospinal fluid and lesion swab specimens.

PubMed

Wong, Anita A; Pabbaraju, Kanti; Wong, Sallene; Tellier, Raymond

2016-03-01

Herpes simplex viruses (HSV) and varicella zoster virus (VZV) can have very similar and wide-ranging clinical presentations. Rapid identification is necessary for timely antiviral therapy, especially with infections involving the central nervous system, neonates, and immunocompromised individuals. Detection of HSV-1, HSV-2 and VZV was combined into one real-time PCR reaction utilizing hydrolysis probes. The assay was validated on the LightCycler(®) (Roche) and Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific Inc.) to detect alphaherpesviruses in cerebral spinal fluid (CSF) and lesion swab specimens, respectively. Validation data on blood and tissue samples are also presented. The multiplex assay showed excellent sensitivity, specificity and reproducibility when compared to two singleplex real-time PCR assays for CSF samples and direct fluorescent antigen/culture for lesion swab samples. Implementation of the multiplex assay has facilitated improved sensitivity and accuracy as well as reduced turn-around-times and costs. The results from a large data set of 16,622 prospective samples tested between August 16, 2012 to February 1, 2014 at the Provincial Laboratory for Public Health (Alberta, Canada) are presented here. Copyright © 2015 Elsevier B.V. All rights reserved.

Multiplex Real-Time PCR for Monitoring Heterobasidion annosum Colonization in Norway Spruce Clones That Differ in Disease Resistance

PubMed Central

Hietala, Ari M.; Eikenes, Morten; Kvaalen, Harald; Solheim, Halvor; Fossdal, Carl G.

2003-01-01

A multiplex real-time PCR assay was developed to monitor the dynamics of the Picea abies-Heterobasidion annosum pathosystem. Tissue cultures and 32-year-old trees with low or high resistance to this pathogen were used as the host material. Probes and primers were based on a laccase gene for the pathogen and a polyubiquitin gene for the host. The real-time PCR procedure was compared to an ergosterol-based quantification method in a tissue culture experiment, and there was a strong correlation (product moment correlation coefficient, 0.908) between the data sets. The multiplex real-time PCR procedure had higher resolution and sensitivity during the early stages of colonization and also could be used to monitor the host. In the tissue culture experiment, host DNA was degraded more rapidly in the clone with low resistance than in the clone with high resistance. In the field experiment, the lesions elicited were not strictly proportional to the area colonized by the pathogen. Fungal colonization was more restricted and localized in the lesion in the clone with high resistance, whereas in the clone with low resistance, the fungus could be detected until the visible end of the lesion. Thus, the real-time PCR assay gives better resolution than does the traditionally used lesion length measurement when screening host clones for resistance. PMID:12902224

Rapid detection of Wuchereria bancrofti and Brugia malayi in mosquito vectors (Diptera: Culicidae) using a real-time fluorescence resonance energy transfer multiplex PCR and melting curve analysis.

PubMed

Intapan, Pewpan M; Thanchomnang, Tongjit; Lulitanond, Viraphong; Maleewong, Wanchai

2009-01-01

We developed a single-step real-time fluorescence resonance energy transfer (FRET) multiplex polymerase chain reaction (PCR) merged with melting curve analysis for the detection of Wuchereria bancrofti and Brugia malayi DNA in blood-fed mosquitoes. Real-time FRET multiplex PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two families of repeated DNA elements: the 188 bp SspI repeated sequence, specific to W. bancrofti, and the 153-bp HhaI repeated sequence, specific to the genus Brugia and two pairs of specific fluorophore-labeled probes. Both W. bancrofti and B. malayi can be differentially detected in infected vectors by this process through their different fluorescence channel and melting temperatures. The assay could distinguish both human filarial DNAs in infected vectors from the DNAs of Dirofilaria immitis- and Plasmodium falciparum-infected human red blood cells and noninfected mosquitoes and human leukocytes. The technique showed 100% sensitivity and specificity and offers a rapid and reliable procedure for differentially identifying lymphatic filariasis. The introduced real-time FRET multiplex PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The test can be used to screen mosquito vectors in endemic areas and therefore should be a useful diagnostic tool for the evaluation of infection rate of the mosquito populations and for xenomonitoring in the community after eradication programs such as the Global Program to Eliminate Lymphatic Filariasis.

High-speed real-time OFDM transmission based on FPGA

NASA Astrophysics Data System (ADS)

Xiao, Xin; Li, Fan; Yu, Jianjun

2016-02-01

In this paper, we review our recent research progresses on real-time orthogonal frequency division multiplexing (OFDM) transmission based on FPGA. We successfully demonstrated four-channel wavelength-division multiplexing (WDM) 256.51Gb/s 16-ary quadrature amplitude modulation (16QAM)-OFDM signal transmission system for short-reach optical amplifier free inter-connection with real-time reception. Four optical carriers are modulated by four different 16QAM-OFDM signals via 10G-class direct modulation lasers (DMLs). We achieved highest capacity real-time reception optical OFDM signal transmission over 2.4-km SMF with the bit-error ratio (BER) under soft-decision forward error correction (SD-FEC) limitation of 2.4×10-2. In order to achieve higher spectrum efficiency (SE), we demonstrate 4-channel high level QAM-OFDM transmission over 20-km SMF-28 with real-time reception. 58.72-Gb/s 256QAM-OFDM and 56.4-Gb/s 128QAM-OFDM signal transmission within 25-GHz grid is achieved with the BER under 2.4×10-2 and real-time reception.

On the Application of Time-Reversed Space-Time Block Code to Aeronautical Telemetry

DTIC Science & Technology

2014-06-01

Keying (SOQPSK), bit error rate (BER), Orthogonal Frequency Division Multiplexing ( OFDM ), Generalized time-reversed space-time block codes (GTR-STBC) 16...Alamouti code [4]) is optimum [2]. Although OFDM is generally applied on a per subcarrier basis in frequency selective fading, it is not a viable...Calderbank, “Finite-length MIMO decision feedback equal- ization for space-time block-coded signals over multipath-fading channels,” IEEE Transac- tions on

Real-time multiplexed digital cavity-enhanced spectroscopy

DOE PAGES

Boyson, Toby K.; Dagdigian, Paul J.; Pavey, Karl D.; ...

2015-10-01

Cavity-enhanced spectroscopy is a sensitive optical absorption technique but one where the practical applications have been limited to studying small wavelength ranges. In addition, this Letter shows that wideband operation can be achieved by combining techniques usually reserved for the communications community with that of cavity-enhanced spectroscopy, producing a multiplexed real-time cavity-enhanced spectrometer. We use multiple collinear laser sources operating asynchronously and simultaneously while being detected on a single photodetector. This is synonymous with radio frequency (RF) cellular systems in which signals are detected on a single antenna but decoded uniquely. Here, we demonstrate results with spectra of methyl salicylatemore » and show parts-per-billion per root hertz sensitivity measured in real-time.« less

Metric projection for dynamic multiplex networks.

PubMed

Jurman, Giuseppe

2016-08-01

Evolving multiplex networks are a powerful model for representing the dynamics along time of different phenomena, such as social networks, power grids, biological pathways. However, exploring the structure of the multiplex network time series is still an open problem. Here we propose a two-step strategy to tackle this problem based on the concept of distance (metric) between networks. Given a multiplex graph, first a network of networks is built for each time step, and then a real valued time series is obtained by the sequence of (simple) networks by evaluating the distance from the first element of the series. The effectiveness of this approach in detecting the occurring changes along the original time series is shown on a synthetic example first, and then on the Gulf dataset of political events.

Rapid detection and typing of pathogenic nonpneumophila Legionella spp. isolates using a multiplex real-time PCR assay.

PubMed

Benitez, Alvaro J; Winchell, Jonas M

2016-04-01

We developed a single tube multiplex real-time PCR assay that allows for the rapid detection and typing of 9 nonpneumophila Legionella spp. isolates that are clinically relevant. The multiplex assay is capable of simultaneously detecting and discriminating L. micdadei, L. bozemanii, L. dumoffii, L. longbeachae, L. feeleii, L. anisa, L. parisiensis, L. tucsonensis serogroup (sg) 1 and 3, and L. sainthelensis sg 1 and 2 isolates. Evaluation of the assay with nucleic acid from each of these species derived from both clinical and environmental isolates and typing strains demonstrated 100% sensitivity and 100% specificity when tested against 43 other Legionella spp. Typing of L. anisa, L. parisiensis, and L. tucsonensis sg 1 and 3 isolates was accomplished by developing a real-time PCR assay followed by high-resolution melt (HRM) analysis targeting the ssrA gene. Further typing of L. bozemanii, L. longbeachae, and L. feeleii isolates to the serogroup level was accomplished by developing a real-time PCR assay followed by HRM analysis targeting the mip gene. When used in conjunction with other currently available diagnostic tests, these assays may aid in rapidly identifying specific etiologies associated with Legionella outbreaks, clusters, sporadic cases, and potential environmental sources. Published by Elsevier Inc.

Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hindson, B J; Baker, B R; Bentley Tammero, L F

2007-09-18

A high-throughput multiplexed assay (Multiplex Version 1.0) was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspectmore » cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRTPCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.« less

Interactive MPEG-4 low-bit-rate speech/audio transmission over the Internet

NASA Astrophysics Data System (ADS)

Liu, Fang; Kim, JongWon; Kuo, C.-C. Jay

1999-11-01

The recently developed MPEG-4 technology enables the coding and transmission of natural and synthetic audio-visual data in the form of objects. In an effort to extend the object-based functionality of MPEG-4 to real-time Internet applications, architectural prototypes of multiplex layer and transport layer tailored for transmission of MPEG-4 data over IP are under debate among Internet Engineering Task Force (IETF), and MPEG-4 systems Ad Hoc group. In this paper, we present an architecture for interactive MPEG-4 speech/audio transmission system over the Internet. It utilities a framework of Real Time Streaming Protocol (RTSP) over Real-time Transport Protocol (RTP) to provide controlled, on-demand delivery of real time speech/audio data. Based on a client-server model, a couple of low bit-rate bit streams (real-time speech/audio, pre- encoded speech/audio) are multiplexed and transmitted via a single RTP channel to the receiver. The MPEG-4 Scene Description (SD) and Object Descriptor (OD) bit streams are securely sent through the RTSP control channel. Upon receiving, an initial MPEG-4 audio- visual scene is constructed after de-multiplexing, decoding of bit streams, and scene composition. A receiver is allowed to manipulate the initial audio-visual scene presentation locally, or interactively arrange scene changes by sending requests to the server. A server may also choose to update the client with new streams and list of contents for user selection.

One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples.

PubMed

Gautam, Rashi; Mijatovic-Rustempasic, Slavica; Esona, Mathew D; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D

2016-01-01

Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8-100% sensitivity, 99.7-100% specificity, 85-95% efficiency and a limit of detection of 4-60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81-92% efficiency and limit of detection of 150-600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8-100% sensitivity, 100% specificity, 86-89% efficiency and a limit of detection of 12-400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82-90% efficiency and limit of detection of 120-4000 copies in multiplex reaction. Discussion. The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.

One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

PubMed Central

Mijatovic-Rustempasic, Slavica; Esona, Mathew D.; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D.

2016-01-01

Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction. Discussion. The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains. PMID:26839745

Methods for detection of GMOs in food and feed.

PubMed

Marmiroli, Nelson; Maestri, Elena; Gullì, Mariolina; Malcevschi, Alessio; Peano, Clelia; Bordoni, Roberta; De Bellis, Gianluca

2008-10-01

This paper reviews aspects relevant to detection and quantification of genetically modified (GM) material within the feed/food chain. The GM crop regulatory framework at the international level is evaluated with reference to traceability and labelling. Current analytical methods for the detection, identification, and quantification of transgenic DNA in food and feed are reviewed. These methods include quantitative real-time PCR, multiplex PCR, and multiplex real-time PCR. Particular attention is paid to methods able to identify multiple GM events in a single reaction and to the development of microdevices and microsensors, though they have not been fully validated for application.

Method and apparatus for high speed data acquisition and processing

DOEpatents

Ferron, J.R.

1997-02-11

A method and apparatus are disclosed for high speed digital data acquisition. The apparatus includes one or more multiplexers for receiving multiple channels of digital data at a low data rate and asserting a multiplexed data stream at a high data rate, and one or more FIFO memories for receiving data from the multiplexers and asserting the data to a real time processor. Preferably, the invention includes two multiplexers, two FIFO memories, and a 64-bit bus connecting the FIFO memories with the processor. Each multiplexer receives four channels of 14-bit digital data at a rate of up to 5 MHz per channel, and outputs a data stream to one of the FIFO memories at a rate of 20 MHz. The FIFO memories assert output data in parallel to the 64-bit bus, thus transferring 14-bit data values to the processor at a combined rate of 40 MHz. The real time processor is preferably a floating-point processor which processes 32-bit floating-point words. A set of mask bits is prestored in each 32-bit storage location of the processor memory into which a 14-bit data value is to be written. After data transfer from the FIFO memories, mask bits are concatenated with each stored 14-bit data value to define a valid 32-bit floating-point word. Preferably, a user can select any of several modes for starting and stopping direct memory transfers of data from the FIFO memories to memory within the real time processor, by setting the content of a control and status register. 15 figs.

Method and apparatus for high speed data acquisition and processing

DOEpatents

Ferron, John R.

1997-01-01

A method and apparatus for high speed digital data acquisition. The apparatus includes one or more multiplexers for receiving multiple channels of digital data at a low data rate and asserting a multiplexed data stream at a high data rate, and one or more FIFO memories for receiving data from the multiplexers and asserting the data to a real time processor. Preferably, the invention includes two multiplexers, two FIFO memories, and a 64-bit bus connecting the FIFO memories with the processor. Each multiplexer receives four channels of 14-bit digital data at a rate of up to 5 MHz per channel, and outputs a data stream to one of the FIFO memories at a rate of 20 MHz. The FIFO memories assert output data in parallel to the 64-bit bus, thus transferring 14-bit data values to the processor at a combined rate of 40 MHz. The real time processor is preferably a floating-point processor which processes 32-bit floating-point words. A set of mask bits is prestored in each 32-bit storage location of the processor memory into which a 14-bit data value is to be written. After data transfer from the FIFO memories, mask bits are concatenated with each stored 14-bit data value to define a valid 32-bit floating-point word. Preferably, a user can select any of several modes for starting and stopping direct memory transfers of data from the FIFO memories to memory within the real time processor, by setting the content of a control and status register.

High Throughput Multiplex PCR and Probe-based Detection with Luminex Beads for Seven Intestinal Parasites

PubMed Central

Taniuchi, Mami; Verweij, Jaco J.; Noor, Zannatun; Sobuz, Shihab U.; van Lieshout, Lisette; Petri, William A.; Haque, Rashidul; Houpt, Eric R.

2011-01-01

Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and copro-antigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites—Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis—were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites. PMID:21292910

Development of a multiplex real-time PCR assay for phylogenetic analysis of Uropathogenic Escherichia coli.

PubMed

Hasanpour, Mojtaba; Najafi, Akram

2017-06-01

Uropathogenic Escherichia coli (UPEC) is among major pathogens causing 80-90% of all episodes of urinary tract infections (UTIs). Recently, E. coli strains are divided into eight main phylogenetic groups including A, B1, B2, C, D, E, F, and clade I. This study was aimed to develop a rapid, sensitive, and specific multiplex real time PCR method capable of detecting phylogenetic groups of E. coli strains. This study was carried out on E. coli strains (isolated from the patient with UTI) in which the presence of all seven target genes had been confirmed in our previous phylogenetic study. An EvaGreen-based singleplex and multiplex real-time PCR with melting curve analysis was designed for simultaneous detection and differentiation of these genes. The primers were selected mainly based on the production of amplicons with melting temperatures (T m ) ranging from 82°C to 93°C and temperature difference of more than 1.5°C between each peak.The multiplex real-time PCR assays that have been developed in the present study were successful in detecting the eight main phylogenetic groups. Seven distinct melting peaks were discriminated, with Tm value of 93±0.8 for arpA, 89.2±0.1for chuA, 86.5±0.1 for yjaA, 82.3±0.2 for TspE4C2, 87.8±0.1for trpAgpC, 85.4±0.6 for arpAgpE genes, and 91±0.5 for the internal control. To our knowledge, this study is the first melting curve-based real-time PCR assay developed for simultaneous and discrete detection of these seven target genes. Our findings showed that this assay has the potential to be a rapid, reliable and cost-effective alternative for routine phylotyping of E. coli strains. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of Staphylococcus aureus enterotoxigenic strains in bovine raw milk by reversed passive latex agglutination and multiplex polymerase chain reaction.

PubMed

Mansour, Asmaa Samy; Wagih, Gad El-Said; Morgan, Sabry D; Elhariri, Mahmoud; El-Shabrawy, Mona A; Abuelnaga, Azza S M; Elgabry, E A

2017-08-01

This review gives an outline of the assessment of enterotoxigenic Staphylococcus aureus tainting levels in raw milk from different sources in Egypt and characterization of enterotoxigenic strains utilizing a technique in light of PCR to identify genes coding for the production of staphylococcal enterotoxin (SE). The obtained data were compared with results from the application of the reversed passive latex. Multiplex PCR and reversed passive latex agglutination (RPLA) were used. A total of 141 samples of raw milk (cow's milk=33, buffalo's milk=58, and bulk tank milk=50) were investigated for S. aureus contamination and tested for enterotoxin genes presence and toxin production. S. aureus was detected in 23 (16.3%) samples phenotypically and genotypically by amplification of nuc gene. The S. aureus isolates were investigated for SEs genes ( sea to see ) by multiplex PCR and the toxin production by these isolates was screened by RPLA. SEs genes were detected in six isolates (26.1%) molecularly; see was the most observed gene where detected in all isolates, two isolates harbored seb , and two isolates harbored sec . According to RPLA, three isolates produced SEB and SEC. The study revealed the widespread of S. aureus strains caring genes coding for toxins. The real significance of the presence of these strains or its toxins in raw milk and their possible impact a potential hazard for staphylococcal food poisoning by raw milk consumption. Therefore, detection of enterotoxigenic S. aureus strains in raw milk is necessary for consumer safety.

Detection of Rickettsia rickettsii, Rickettsia parkeri, and Rickettsia akari in Skin Biopsy Specimens Using a Multiplex Real-time Polymerase Chain Reaction Assay

PubMed Central

Denison, Amy M.; Amin, Bijal D.; Nicholson, William L.; Paddock, Christopher D.

2015-01-01

Background Rickettsia rickettsii, Rickettsia parkeri, and Rickettsia akari are the most common causes of spotted fever group rickettsioses indigenous to the United States. Infected patients characteristically present with a maculopapular rash, often accompanied by an inoculation eschar. Skin biopsy specimens are often obtained from these lesions for diagnostic evaluation. However, a species-specific diagnosis is achieved infrequently from pathologic specimens because immunohistochemical stains do not differentiate among the causative agents of spotted fever group rickettsiae, and existing polymerase chain reaction (PCR) assays generally target large gene segments that may be difficult or impossible to obtain from formalin-fixed tissues. Methods This work describes the development and evaluation of a multiplex real-time PCR assay for the detection of these 3 Rickettsia species from formalin-fixed, paraffin-embedded (FFPE) skin biopsy specimens. Results The multiplex PCR assay was specific at discriminating each species from FFPE controls of unrelated bacterial, viral, protozoan, and fungal pathogens that cause skin lesions, as well as other closely related spotted fever group Rickettsia species. Conclusions This multiplex real-time PCR demonstrates greater sensitivity than nested PCR assays in FFPE tissues and provides an effective method to specifically identify cases of Rocky Mountain spotted fever, rickettsialpox, and R. parkeri rickettsiosis by using skin biopsy specimens. PMID:24829214

A Versatile Multichannel Digital Signal Processing Module for Microcalorimeter Arrays

NASA Astrophysics Data System (ADS)

Tan, H.; Collins, J. W.; Walby, M.; Hennig, W.; Warburton, W. K.; Grudberg, P.

2012-06-01

Different techniques have been developed for reading out microcalorimeter sensor arrays: individual outputs for small arrays, and time-division or frequency-division or code-division multiplexing for large arrays. Typically, raw waveform data are first read out from the arrays using one of these techniques and then stored on computer hard drives for offline optimum filtering, leading not only to requirements for large storage space but also limitations on achievable count rate. Thus, a read-out module that is capable of processing microcalorimeter signals in real time will be highly desirable. We have developed multichannel digital signal processing electronics that are capable of on-board, real time processing of microcalorimeter sensor signals from multiplexed or individual pixel arrays. It is a 3U PXI module consisting of a standardized core processor board and a set of daughter boards. Each daughter board is designed to interface a specific type of microcalorimeter array to the core processor. The combination of the standardized core plus this set of easily designed and modified daughter boards results in a versatile data acquisition module that not only can easily expand to future detector systems, but is also low cost. In this paper, we first present the core processor/daughter board architecture, and then report the performance of an 8-channel daughter board, which digitizes individual pixel outputs at 1 MSPS with 16-bit precision. We will also introduce a time-division multiplexing type daughter board, which takes in time-division multiplexing signals through fiber-optic cables and then processes the digital signals to generate energy spectra in real time.

Characterization, adaptive traffic shaping, and multiplexing of real-time MPEG II video

NASA Astrophysics Data System (ADS)

Agrawal, Sanjay; Barry, Charles F.; Binnai, Vinay; Kazovsky, Leonid G.

1997-01-01

We obtain network traffic model for real-time MPEG-II encoded digital video by analyzing video stream samples from real-time encoders from NUKO Information Systems. MPEG-II sample streams include a resolution intensive movie, City of Joy, an action intensive movie, Aliens, a luminance intensive (black and white) movie, Road To Utopia, and a chrominance intensive (color) movie, Dick Tracy. From our analysis we obtain a heuristic model for the encoded video traffic which uses a 15-stage Markov process to model the I,B,P frame sequences within a group of pictures (GOP). A jointly-correlated Gaussian process is used to model the individual frame sizes. Scene change arrivals are modeled according to a gamma process. Simulations show that our MPEG-II traffic model generates, I,B,P frame sequences and frame sizes that closely match the sample MPEG-II stream traffic characteristics as they relate to latency and buffer occupancy in network queues. To achieve high multiplexing efficiency we propose a traffic shaping scheme which sets preferred 1-frame generation times among a group of encoders so as to minimize the overall variation in total offered traffic while still allowing the individual encoders to react to scene changes. Simulations show that our scheme results in multiplexing gains of up to 10% enabling us to multiplex twenty 6 Mbps MPEG-II video streams instead of 18 streams over an ATM/SONET OC3 link without latency or cell loss penalty. This scheme is due for a patent.

A new multiplex real-time PCR test for HSV1/2 and syphilis: an evaluation of its impact in the laboratory and clinical setting.

PubMed

Scott, Laura Jane; Gunson, Rory N; Carman, William F; Winter, Andrew J

2010-12-01

To develop, evaluate and implement a new multiplex real-time PCR test for the detection of herpes simplex virus (HSV)1, HSV2 and syphilis in a single sample using a single test. A multiplex real-time PCR test detecting HSV1, HSV2 and Treponema pallidum was designed, validated and evaluated for a period of 6 months on patients attending the Sandyford Initiative (a series of genitourinary medicine clinics in and around Glasgow). A total of 692 samples were tested, and T pallidum PCR positives were confirmed by a second PCR at the Scottish Reference Laboratory (SBSTIRL). All PCR results were aligned with dark ground microscopy findings and serological results where available and compared. The laboratory validation of the multiplex assay showed the test to be sensitive, specific and robust. Of the 692 samples, 139 were positive for HSV1, 136 for HSV2, 15 for syphilis, one for both syphilis and HSV1, and 401 were negative; the reference laboratory confirmed all T pallidum PCR-positive samples. The PCR test was more sensitive than both dark ground microscopy and serological testing for the diagnosis of primary syphilis. The introduction of this new test has led to a better turnaround time for the diagnosis of genital ulcer disease, better detection of primary syphilis infection, and the detection of unexpected cases of syphilis where the aetiological agent suspected was HSV.

Detection of α-thalassemia-1 Southeast Asian and Thai Type Deletions and β-thalassemia 3.5-kb Deletion by Single-tube Multiplex Real-time PCR with SYBR Green1 and High-resolution Melting Analysis

PubMed Central

Wiengkum, Thanatcha; Srithep, Sarinee; Chainoi, Isarapong; Singboottra, Panthong; Wongwiwatthananukit, Sanchai

2011-01-01

Background Prevention and control of thalassemia requires simple, rapid, and accurate screening tests for carrier couples who are at risk of conceiving fetuses with severe thalassemia. Methods Single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting (HRM) analysis were used for the identification of α-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and β-thalassemia 3.5-kb gene deletion. The results were compared with those obtained using conventional gap-PCR. DNA samples were derived from 28 normal individuals, 11 individuals with α-thalassemia-1 SEA type deletion, 2 with α-thalassemia-1 Thai type deletion, and 2 with heterozygous β-thalassemia 3.5-kb gene deletion. Results HRM analysis indicated that the amplified fragments from α-thalassemia-1 SEA type deletion, α-thalassemia-1 Thai type deletion, β-thalassemia 3.5-kb gene deletion, and the wild-type β-globin gene had specific peak heights at mean melting temperature (Tm) values of 86.89℃, 85.66℃, 77.24℃, and 74.92℃, respectively. The results obtained using single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis showed 100% consistency with those obtained using conventional gap-PCR. Conclusions Single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis is a potential alternative for routine clinical screening of the common types of α- and β-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate. PMID:21779184

A centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria.

PubMed

Choi, Goro; Jung, Jae Hwan; Park, Byung Hyun; Oh, Seung Jun; Seo, Ji Hyun; Choi, Jong Seob; Kim, Do Hyun; Seo, Tae Seok

2016-06-21

In this study, we developed a centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria contaminated milk samples. The microdevice was designed to contain identical triplicate functional units and each unit has four reaction chambers, thereby making it possible to perform twelve direct-RPA reactions simultaneously. The integrated microdevice consisted of two layers: RPA reagents were injected in the top layer, while spiked milk samples with food poisoning bacteria were loaded into sample reservoirs in the bottom layer. For multiplex bacterial detection, the target gene-specific primers and probes were dried in each reaction chamber. The introduced samples and reagents could be equally aliquoted and dispensed into each reaction chamber by centrifugal force, and then the multiplex direct-RPA reaction was executed. The target genes of bacteria spiked in milk could be amplified at 39 °C without a DNA extraction step by using the direct-RPA cocktails, which were a combination of a direct PCR buffer and RPA enzymes. As the target gene amplification proceeded, the increased fluorescence signals coming from the reaction chambers were recorded in real-time at an interval of 2 min. The entire process, including the sample distribution, the direct-RPA reaction, and the real-time analysis, was accomplished with a custom-made portable genetic analyzer and a miniaturized optical detector. Monoplex, duplex, and triplex food poisoning bacteria (Salmonella enterica, Escherichia coli O157:H7, and Vibrio parahaemolyticus) detection was successfully performed with a detection sensitivity of 4 cells per 3.2 μL of milk samples within 30 min. By implementing the direct-PRA on the miniaturized centrifugal microsystem, the on-site food poisoning bacteria analysis would be feasible with high speed, sensitivity, and multiplicity.

Single-Tube Multiplexed Molecular Detection of Endemic Porcine Viruses in Combination with Background Screening for Transboundary Diseases

PubMed Central

Wernike, Kerstin; Hoffmann, Bernd

2013-01-01

Detection of several pathogens with multiplexed real-time quantitative PCR (qPCR) assays in a one-step setup allows the simultaneous detection of two endemic porcine and four different selected transboundary viruses. Reverse transcription (RT)-qPCR systems for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2), two of the most economically important pathogens of swine worldwide, were combined with a screening system for diseases notifiable to the World Organization of Animal Health, namely, classical and African swine fever, foot-and-mouth disease, and Aujeszky's disease. Background screening was implemented using the identical fluorophore for all four different RT-qPCR assays. The novel multiplex RT-qPCR system was validated with a large panel of different body fluids and tissues from pigs and other animal species. Both reference samples and clinical specimens were used for a complete evaluation. It could be demonstrated that a highly sensitive and specific parallel detection of the different viruses was possible. The assays for the notifiable diseases were even not affected by the simultaneous amplification of very high loads of PRRSV- and PCV2-specific sequences. The novel broad-spectrum multiplex assay allows in a unique form the routine investigation for endemic porcine pathogens with exclusion diagnostics of the most important transboundary diseases in samples from pigs with unspecific clinical signs, such as fever or hemorrhages. The new system could significantly improve early detection of the most important notifiable diseases of swine and could lead to a new approach in syndromic surveillance. PMID:23303496

Real-time RT-PCR high-resolution melting curve analysis and multiplex RT-PCR to detect and differentiate grapevine leafroll-associated virus 3 variant groups I, II, III and VI.

PubMed

Bester, Rachelle; Jooste, Anna E C; Maree, Hans J; Burger, Johan T

2012-09-27

Grapevine leafroll-associated virus 3 (GLRaV-3) is the main contributing agent of leafroll disease worldwide. Four of the six GLRaV-3 variant groups known have been found in South Africa, but their individual contribution to leafroll disease is unknown. In order to study the pathogenesis of leafroll disease, a sensitive and accurate diagnostic assay is required that can detect different variant groups of GLRaV-3. In this study, a one-step real-time RT-PCR, followed by high-resolution melting (HRM) curve analysis for the simultaneous detection and identification of GLRaV-3 variants of groups I, II, III and VI, was developed. A melting point confidence interval for each variant group was calculated to include at least 90% of all melting points observed. A multiplex RT-PCR protocol was developed to these four variant groups in order to assess the efficacy of the real-time RT-PCR HRM assay. A universal primer set for GLRaV-3 targeting the heat shock protein 70 homologue (Hsp70h) gene of GLRaV-3 was designed that is able to detect GLRaV-3 variant groups I, II, III and VI and differentiate between them with high-resolution melting curve analysis. The real-time RT-PCR HRM and the multiplex RT-PCR were optimized using 121 GLRaV-3 positive samples. Due to a considerable variation in melting profile observed within each GLRaV-3 group, a confidence interval of above 90% was calculated for each variant group, based on the range and distribution of melting points. The intervals of groups I and II could not be distinguished and a 95% joint confidence interval was calculated for simultaneous detection of group I and II variants. An additional primer pair targeting GLRaV-3 ORF1a was developed that can be used in a subsequent real-time RT-PCR HRM to differentiate between variants of groups I and II. Additionally, the multiplex RT-PCR successfully validated 94.64% of the infections detected with the real-time RT-PCR HRM. The real-time RT-PCR HRM provides a sensitive, automated and rapid tool to detect and differentiate different variant groups in order to study the epidemiology of leafroll disease.

Navigability of multiplex temporal network

NASA Astrophysics Data System (ADS)

Wang, Yan; Song, Qiao-Zhen

2017-01-01

Real world complex systems have multiple levels of relationships and in many cases, they need to be modeled as multiplex networks where the same nodes can interact with each other in different layers, such as social networks. However, social relationships only appear at prescribed times so the temporal structures of edge activations can also affect the dynamical processes located above them. To consider both factors are simultaneously, we introduce multiplex temporal networks and propose three different walk strategies to investigate the concurrent dynamics of random walks and the temporal structure of multiplex networks. Thus, we derive analytical results for the multiplex centrality and coverage function in multiplex temporal networks. By comparing them with the numerical results, we show how the underlying topology of the layers and the walk strategy affect the efficiency when exploring the networks. In particular, the most interesting result is the emergence of a super-diffusion process, where the time scale of the multiplex is faster than that of both layers acting separately.

The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting Rotavirus A and Norovirus

PubMed Central

Dung, Tran Thi Ngoc; Phat, Voong Vinh; Nga, Tran Vu Thieu; My, Phan Vu Tra; Duy, Pham Thanh; Campbell, James I.; Thuy, Cao Thu; Hoang, Nguyen Van Minh; Van Minh, Pham; Le Phuc, Hoang; Tuyet, Pham Thi Ngoc; Vinh, Ha; Kien, Duong Thi Hue; Huy, Huynh Le Anh; Vinh, Nguyen Thanh; Nga, Tran Thi Thu; Hau, Nguyen Thi Thu; Chinh, Nguyen Tran; Thuong, Tang Chi; Tuan, Ha Manh; Simmons, Cameron; Farrar, Jeremy J.; Baker, Stephen

2013-01-01

Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits. PMID:23046990

A novel, multiplex, real-time PCR-based approach for the detection of the commonly occurring pathogenic fungi and bacteria.

PubMed

Horváth, Ádám; Pető, Zoltán; Urbán, Edit; Vágvölgyi, Csaba; Somogyvári, Ferenc

2013-12-23

Polymerase chain reaction (PCR)-based techniques are widely used to identify fungal and bacterial infections. There have been numerous reports of different, new, real-time PCR-based pathogen identification methods although the clinical practicability of such techniques is not yet fully clarified.The present study focuses on a novel, multiplex, real-time PCR-based pathogen identification system developed for rapid differentiation of the commonly occurring bacterial and fungal causative pathogens of bloodstream infections. A multiplex, real-time PCR approach is introduced for the detection and differentiation of fungi, Gram-positive (G+) and Gram-negative (G-) bacteria. The Gram classification is performed with the specific fluorescence resonance energy transfer (FRET) probes recommended for LightCycler capillary real-time PCR. The novelty of our system is the use of a non-specific SYBR Green dye instead of labelled anchor probes or primers, to excite the acceptor dyes on the FRET probes. In conjunction with this, the use of an intercalating dye allows the detection of fungal amplicons.With the novel pathogen detection system, fungi, G + and G- bacteria in the same reaction tube can be differentiated within an hour after the DNA preparation via the melting temperatures of the amplicons and probes in the same tube. This modified FRET technique is specific and more rapid than the gold-standard culture-based methods. The fact that fungi, G + and G- bacteria were successfully identified in the same tube within an hour after the DNA preparation permits rapid and early evidence-based management of bloodstream infections in clinical practice.

Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens

PubMed Central

Clancy, Eoin; Cormican, Martin; Boo, Teck Wee; Cunney, Robert

2018-01-01

Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae, N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology. PMID:29425124

Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens.

PubMed

Higgins, Owen; Clancy, Eoin; Cormican, Martin; Boo, Teck Wee; Cunney, Robert; Smith, Terry J

2018-02-09

Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae , Neisseria meningitidis and Haemophilus influenzae . Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae , N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology.

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye

PubMed Central

Ahberg, Christian D.; Manz, Andreas; Neuzil, Pavel

2015-01-01

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio. PMID:26088868

Identification of Streptococcus pneumoniae lytA, plyA and psaA genes in pleural fluid by multiplex real-time PCR.

PubMed

Sanz, Juan Carlos; Ríos, Esther; Rodríguez-Avial, Iciar; Ramos, Belén; Marín, Mercedes; Cercenado, Emilia

2017-08-14

The aim was to evaluate the utility of a multiplex real-time PCR to detect Streptococcus pneumoniae lytA, plyA and psaA genes in pleural fluid (PF). A collection of 81 PF samples was used. Sixty were considered positive for S. pneumoniae according to previous results (54 by an in-house lytA gene PCR and eight by universal rRNA PCR). The sensitivity for detection of the lytA, plyA and psaA genes by multiplex PCR was 100% (60/60), 98.3% (59/60) and 91.7% (55/60), respectively. The detection of all three genes was negative in 21 samples formerly confirmed as negative for S. pneumoniae (100% specificity) by the other procedures (9 by in-house lytA PCR and 12 by rRNA PCR). The use of this multiplex PCR may be a useful option to identify S. pneumoniae directly in PF samples. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum.

PubMed

Rubio, Marcela da Silva; Penha Filho, Rafael Antonio Casarin; Almeida, Adriana Maria de; Berchieri, Angelo

2017-12-01

Currently there are 2659 Salmonella serovars. The host-specific biovars Salmonella Pullorum and Salmonella Gallinarum cause systemic infections in food-producing and wild birds. Fast diagnosis is crucial to control the dissemination in avian environments. The present work describes the development of a multiplex qPCR in real time using a low-cost DNA dye (SYBr Green) to identify and quantify these biovars. Primers were chosen based on genomic regions of difference (RoD) and optimized to control dimers. Primers pSGP detect both host-specific biovars but not other serovars and pSG and pSP differentiate biovars. Three amplicons showed different melting temperatures (Tm), allowing differentiation. The pSGP amplicon (97 bp) showed Tm of 78°C for both biovars. The pSG amplicon (273 bp) showed a Tm of 86.2°C for S. Gallinarum and pSP amplicon (260 bp) dissociated at 84.8°C for S. Pullorum identification. The multiplex qPCR in real time showed high sensitivity and was capable of quantifying 10 8 -10 1 CFU of these biovars.

Comparison of the performance in detection of HPV infections between the high-risk HPV genotyping real time PCR and the PCR-reverse dot blot assays.

PubMed

Zhang, Lahong; Dai, Yibei; Chen, Jiahuan; Hong, Liquan; Liu, Yuhua; Ke, Qiang; Chen, Yiwen; Cai, Chengsong; Liu, Xia; Chen, Zhaojun

2018-01-01

A new multiplex real-time PCR assay, the high-risk HPV genotyping real time PCR assay (HR HPV RT-PCR), has been developed to detect 15 high-risk HPV types with respective viral loads. In this report, a total of 684 cervical specimens from women diagnosed with vaginitis were assessed by the HR HPV RT-PCR and the PCR reaction and reverse dot blot (PCR-RDB) assays, using a PCR-sequencing method as a reference standard. A total coincidence of 97.7% between the HR HPV RT PCR and the PCR-RDB assays was determined with a Kappa value of 0.953. The HR HPV RT PCR assay had sensitivity, specificity, and concordance rates (accuracy) of 99.7%, 99.7%, and 99.7%, respectively, as confirmed by PCR-sequencing, while the PCR-RDB assay had respective rates of 98.8%, 97.1%, and 98.0%. The overall rate of HPV infection, determined by PCR-sequencing, in women diagnosed with vaginitis was 49.85%, including 36.26% of single infection and 13.6% of multiple infections. The most common infections among the 15 high-risk HPV types in women diagnosed with vaginitis were HPV-52, HPV-16, and HPV-58, with a total detection rate of 10.23%, 7.75%, and 5.85%, respectively. We conclude that the HR HPV RT PCR assay exhibits better clinical performance than the PCR-RDB assay, and is an ideal alternative method for HPV genotyping. In addition, the HR HPV RT PCR assay provides HPV DNA viral loads, and could serve as a quantitative marker in the diagnosis and treatment of single and multiple HPV infections. © 2017 Wiley Periodicals, Inc.

The genotyping of infectious bronchitis virus in Taiwan by a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction.

PubMed

Huang, Shr-Wei; Ho, Chia-Fang; Chan, Kun-Wei; Cheng, Min-Chung; Shien, Jui-Hung; Liu, Hung-Jen; Wang, Chi-Young

2014-11-01

Infectious bronchitis virus (IBV; Avian coronavirus) causes acute respiratory and reproductive and urogenital diseases in chickens. Following sequence alignment of IBV strains, a combination of selective primer sets was designed to individually amplify the IBV wild-type and vaccine strains using a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) approach. This system was shown to discriminate the IBV wild-type and vaccine strains. Moreover, an ARMS real-time RT-PCR (ARMS qRT-PCR) was combined with a high-resolution analysis (HRMA) to establish a melt curve analysis program. The specificity of the ARMS RT-PCR and the ARMS qRT-PCR was verified using unrelated avian viruses. Different melting temperatures and distinct normalized and shifted melting curve patterns for the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were detected. The new assays were used on samples of lung and trachea as well as virus from allantoic fluid and cell culture. In addition to being able to detect the presence of IBV vaccine and wild-type strains by ARMS RT-PCR, the IBV Mass, IBV H120, IBV TW-I, and IBV TW-II strains were distinguished using ARMS qRT-PCR by their melting temperatures and by HRMA. These approaches have acceptable sensitivities and specificities and therefore should be able to serve as options when carrying out differential diagnosis of IBV in Taiwan and China. © 2014 The Author(s).

Designing robust watermark barcodes for multiplex long-read sequencing.

PubMed

Ezpeleta, Joaquín; Krsticevic, Flavia J; Bulacio, Pilar; Tapia, Elizabeth

2017-03-15

To attain acceptable sample misassignment rates, current approaches to multiplex single-molecule real-time sequencing require upstream quality improvement, which is obtained from multiple passes over the sequenced insert and significantly reduces the effective read length. In order to fully exploit the raw read length on multiplex applications, robust barcodes capable of dealing with the full single-pass error rates are needed. We present a method for designing sequencing barcodes that can withstand a large number of insertion, deletion and substitution errors and are suitable for use in multiplex single-molecule real-time sequencing. The manuscript focuses on the design of barcodes for full-length single-pass reads, impaired by challenging error rates in the order of 11%. The proposed barcodes can multiplex hundreds or thousands of samples while achieving sample misassignment probabilities as low as 10-7 under the above conditions, and are designed to be compatible with chemical constraints imposed by the sequencing process. Software tools for constructing watermark barcode sets and demultiplexing barcoded reads, together with example sets of barcodes and synthetic barcoded reads, are freely available at www.cifasis-conicet.gov.ar/ezpeleta/NS-watermark . ezpeleta@cifasis-conicet.gov.ar. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Detection of Rickettsia rickettsii, Rickettsia parkeri, and Rickettsia akari in skin biopsy specimens using a multiplex real-time polymerase chain reaction assay.

PubMed

Denison, Amy M; Amin, Bijal D; Nicholson, William L; Paddock, Christopher D

2014-09-01

Rickettsia rickettsii, Rickettsia parkeri, and Rickettsia akari are the most common causes of spotted fever group rickettsioses indigenous to the United States. Infected patients characteristically present with a maculopapular rash, often accompanied by an inoculation eschar. Skin biopsy specimens are often obtained from these lesions for diagnostic evaluation. However, a species-specific diagnosis is achieved infrequently from pathologic specimens because immunohistochemical stains do not differentiate among the causative agents of spotted fever group rickettsiae, and existing polymerase chain reaction (PCR) assays generally target large gene segments that may be difficult or impossible to obtain from formalin-fixed tissues. This work describes the development and evaluation of a multiplex real-time PCR assay for the detection of these 3 Rickettsia species from formalin-fixed, paraffin-embedded (FFPE) skin biopsy specimens. The multiplex PCR assay was specific at discriminating each species from FFPE controls of unrelated bacterial, viral, protozoan, and fungal pathogens that cause skin lesions, as well as other closely related spotted fever group Rickettsia species. This multiplex real-time PCR demonstrates greater sensitivity than nested PCR assays in FFPE tissues and provides an effective method to specifically identify cases of Rocky Mountain spotted fever, rickettsialpox, and R. parkeri rickettsiosis by using skin biopsy specimens. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

PubMed Central

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M.; Specht, Christian G.; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-01

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling. PMID:26711992

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

PubMed

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M; Specht, Christian G; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-19

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

Large-memory real-time multichannel multiplexed pattern recognition

NASA Technical Reports Server (NTRS)

Gregory, D. A.; Liu, H. K.

1984-01-01

The principle and experimental design of a real-time multichannel multiplexed optical pattern recognition system via use of a 25-focus dichromated gelatin holographic lens (hololens) are described. Each of the 25 foci of the hololens may have a storage and matched filtering capability approaching that of a single-lens correlator. If the space-bandwidth product of an input image is limited, as is true in most practical cases, the 25-focus hololens system has 25 times the capability of a single lens. Experimental results have shown that the interfilter noise is not serious. The system has already demonstrated the storage and recognition of over 70 matched filters - which is a larger capacity than any optical pattern recognition system reported to date.

Multiplex Real-Time PCR for Detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL) Genes from Selective Enrichments from Animals and Retail Meat

PubMed Central

Velasco, Valeria; Sherwood, Julie S.; Rojas-García, Pedro P.; Logue, Catherine M.

2014-01-01

The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68–0.88 (from substantial to almost perfect agreement) and 0.29–0.77 (from fair to substantial agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0–0.49 (from no agreement beyond that expected by chance to moderate agreement) for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA) using the standard culture method. PMID:24849624

Multiplex real-time PCR for detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL) genes from selective enrichments from animals and retail meat.

PubMed

Velasco, Valeria; Sherwood, Julie S; Rojas-García, Pedro P; Logue, Catherine M

2014-01-01

The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68-0.88 (from substantial to almost perfect agreement) and 0.29-0.77 (from fair to substantial agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0-0.49 (from no agreement beyond that expected by chance to moderate agreement) for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA) using the standard culture method.

Clinical utility of an optimised multiplex real-time PCR assay for the identification of pathogens causing sepsis in Vietnamese patients.

PubMed

Tat Trung, Ngo; Van Tong, Hoang; Lien, Tran Thi; Van Son, Trinh; Thanh Huyen, Tran Thi; Quyen, Dao Thanh; Hoan, Phan Quoc; Meyer, Christian G; Song, Le Huu

2018-02-01

For the identification of bacterial pathogens, blood culture is still the gold standard diagnostic method. However, several disadvantages apply to blood cultures, such as time and rather large volumes of blood sample required. We have previously established an optimised multiplex real-time PCR method in order to diagnose bloodstream infections. In the present study, we evaluated the diagnostic performance of this optimised multiplex RT-PCR in blood samples collected from 110 septicaemia patients enrolled at the 108 Military Central Hospital, Hanoi, Vietnam. Positive results were obtained by blood culture, the Light Cylcler-based SeptiFast ® assay and our multiplex RT-PCR in 35 (32%), 31 (28%), and 31 (28%) samples, respectively. Combined use of the three methods confirmed 50 (45.5%) positive cases of bloodstream infection, a rate significantly higher compared to the exclusive use of one of the three methods (P=0.052, 0.012 and 0.012, respectively). The sensitivity, specificity and area under the curve (AUC) of our assay were higher compared to that of the SeptiFast ® assay (77.4%, 86.1% and 0.8 vs. 67.7%, 82.3% and 0.73, respectively). Combined use of blood culture and multiplex RT-PCR assay showed a superior diagnostic performance, as the sensitivity, specificity, and AUC reached 83.3%, 100%, and 0.95, respectively. The concordance between blood culture and the multiplex RT-PCR assay was highest for Klebsiella pneumonia (100%), followed by Streptococcus spp. (77.8%), Escherichia coli (66.7%), Staphylococcus spp. (50%) and Salmonella spp. (50%). In addition, the use of the newly established multiplex RT-PCR assay increased the spectrum of identifiable agents (Acintobacter baumannii, 1/32; Proteus mirabilis, 1/32). The combination of culture and the multiplex RT-PCR assay provided an excellent diagnostic accomplishment and significantly supported the identification of causative pathogens in clinical samples obtained from septic patients. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

A multiplex real-time PCR assay for identification of Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii in samples from AIDS patients with opportunistic pneumonia.

PubMed

Gago, Sara; Esteban, Cristina; Valero, Clara; Zaragoza, Oscar; Puig de la Bellacasa, Jorge; Buitrago, María José

2014-04-01

A molecular diagnostic technique based on real-time PCR was developed for the simultaneous detection of three of the most frequent causative agents of fungal opportunistic pneumonia in AIDS patients: Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii. This technique was tested in cultured strains and in clinical samples from HIV-positive patients. The methodology used involved species-specific molecular beacon probes targeted to the internal transcribed spacer regions of the rDNA. An internal control was also included in each assay. The multiplex real-time PCR assay was tested in 24 clinical strains and 43 clinical samples from AIDS patients with proven fungal infection. The technique developed showed high reproducibility (r(2) of >0.98) and specificity (100%). For H. capsulatum and Cryptococcus spp., the detection limits of the method were 20 and 2 fg of genomic DNA/20 μl reaction mixture, respectively, while for P. jirovecii the detection limit was 2.92 log10 copies/20 μl reaction mixture. The sensitivity in vitro was 100% for clinical strains and 90.7% for clinical samples. The assay was positive for 92.5% of the patients. For one of the patients with proven histoplasmosis, P. jirovecii was also detected in a bronchoalveolar lavage sample. No PCR inhibition was detected. This multiplex real-time PCR technique is fast, sensitive, and specific and may have clinical applications.

Rapid and simultaneous detection of Mycobacterium tuberculosis complex and Beijing/W genotype in sputum by an optimized DNA extraction protocol and a novel multiplex real-time PCR.

PubMed

Leung, Eric T Y; Zheng, L; Wong, Rity Y K; Chan, Edward W C; Au, T K; Chan, Raphael C Y; Lui, Grace; Lee, Nelson; Ip, Margaret

2011-07-01

Rapid diagnosis and genotyping of Mycobacterium tuberculosis by molecular methods are often limited by the amount and purity of DNA extracted from body fluids. In this study, we evaluated 12 DNA extraction methods and developed a highly sensitive protocol for mycobacterial DNA extraction directly from sputa using surface-coated magnetic particles. We have also developed a novel multiplex real-time PCR for simultaneous identification of M. tuberculosis complex and the Beijing/W genotype (a hypervirulent sublineage of M. tuberculosis) by using multiple fluorogenic probes targeting both the M. tuberculosis IS6110 and the Rv0927c-pstS3 intergenic region. With reference strains and clinical isolates, our real-time PCR accurately identified 20 non-Beijing/W and 20 Beijing/W M. tuberculosis strains from 17 different species of nontuberculosis Mycobacterium (NTM). Further assessment of our DNA extraction protocol and real-time PCR with 335 nonduplicate sputum specimens correctly identified all 74 M. tuberculosis culture-positive specimens. In addition, 15 culture-negative specimens from patients with confirmed tuberculosis were also identified. No cross-reactivity was detected with NTM specimens (n = 31). The detection limit of the assay is 10 M. tuberculosis bacilli, as determined by endpoint dilution analysis. In conclusion, an optimized DNA extraction protocol coupled with a novel multiprobe multiplex real-time PCR for the direct detection of M. tuberculosis, including Beijing/W M. tuberculosis, was found to confer high sensitivity and specificity. The combined procedure has the potential to compensate for the drawbacks of conventional mycobacterial culture in routine clinical laboratory setting, such as the lengthy incubation period and the limitation to viable organisms.

A multiplex real-time polymerase chain reaction assay differentiates between Bolbphorus damnificus and Bolbophorus type II sp

USDA-ARS?s Scientific Manuscript database

A duplex quantitative real-time polymerase chain reaction (qPCR) assay was developed to differentiate between Bolbophorus damnificus and Bolbophorus type II species cercariae. Both trematode species are prevalent throughout the commercial catfish industry,.as both infect the ram’s horn snail, Plano...

Multipurpose assessment for the quantification of Vibrio spp. and total bacteria in fish and seawater using multiplex real-time polymerase chain reaction.

PubMed

Kim, Ji Yeun; Lee, Jung-Lim

2014-10-01

This study describes the first multiplex real-time polymerase chain reaction assay developed, as a multipurpose assessment, for the simultaneous quantification of total bacteria and three Vibrio spp. (V. parahaemolyticus, V. vulnificus and V. anguillarum) in fish and seawater. The consumption of raw finfish as sushi or sashimi has been increasing the chance of Vibrio outbreaks in consumers. Freshness and quality of fishery products also depend on the total bacterial populations present. The detection sensitivity of the specific targets for the multiplex assay was 1 CFU mL⁻¹ in pure culture and seawater, and 10 CFU g⁻¹ in fish. While total bacterial counts by the multiplex assay were similar to those obtained by cultural methods, the levels of Vibrio detected by the multiplex assay were generally higher than by cultural methods of the same populations. Among the natural samples without Vibrio spp. inoculation, eight out of 10 seawater and three out of 20 fish samples were determined to contain Vibrio spp. Our data demonstrate that this multiplex assay could be useful for the rapid detection and quantification of Vibrio spp. and total bacteria as a multipurpose tool for surveillance of fish and water quality as well as diagnostic method. © 2014 The Authors. Journal of the Science of Food and Agriculture published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry.

Multipurpose assessment for the quantification of Vibrio spp. and total bacteria in fish and seawater using multiplex real-time polymerase chain reaction

PubMed Central

Kim, Ji Yeun; Lee, Jung-Lim

2014-01-01

Background This study describes the first multiplex real-time polymerase chain reaction assay developed, as a multipurpose assessment, for the simultaneous quantification of total bacteria and three Vibrio spp. (V. parahaemolyticus, V. vulnificus and V. anguillarum) in fish and seawater. The consumption of raw finfish as sushi or sashimi has been increasing the chance of Vibrio outbreaks in consumers. Freshness and quality of fishery products also depend on the total bacterial populations present. Results The detection sensitivity of the specific targets for the multiplex assay was 1 CFU mL−1 in pure culture and seawater, and 10 CFU g−1 in fish. While total bacterial counts by the multiplex assay were similar to those obtained by cultural methods, the levels of Vibrio detected by the multiplex assay were generally higher than by cultural methods of the same populations. Among the natural samples without Vibrio spp. inoculation, eight out of 10 seawater and three out of 20 fish samples were determined to contain Vibrio spp. Conclusion Our data demonstrate that this multiplex assay could be useful for the rapid detection and quantification of Vibrio spp. and total bacteria as a multipurpose tool for surveillance of fish and water quality as well as diagnostic method. © 2014 The Authors. Journal of the Science of Food and Agriculture published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:24752974

Real-Time, Fast Neutron Coincidence Assay of Plutonium With a 4-Channel Multiplexed Analyzer and Organic Scintillators

NASA Astrophysics Data System (ADS)

Joyce, Malcolm J.; Gamage, Kelum A. A.; Aspinall, M. D.; Cave, F. D.; Lavietes, A.

2014-06-01

The design, principle of operation and the results of measurements made with a four-channel organic scintillator system are described. The system comprises four detectors and a multiplexed analyzer for the real-time parallel processing of fast neutron events. The function of the real-time, digital multiple-channel pulse-shape discrimination analyzer is described together with the results of laboratory-based measurements with 252Cf, 241Am-Li and plutonium. The analyzer is based on a single-board solution with integrated high-voltage supplies and graphical user interface. It has been developed to meet the requirements of nuclear materials assay of relevance to safeguards and security. Data are presented for the real-time coincidence assay of plutonium in terms of doubles count rate versus mass. This includes an assessment of the limiting mass uncertainty for coincidence assay based on a 100 s measurement period and samples in the range 0-50 g. Measurements of count rate versus order of multiplicity for 252Cf and 241Am-Li and combinations of both are also presented.

Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hindson, B J; Reid, S M; Baker, B R

2007-07-26

A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouthmore » disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.« less

Development of an Internal Positive Control for Rapid Diagnosis of Avian Influenza Virus Infections by Real-Time Reverse Transcription-PCR with Lyophilized Reagents

PubMed Central

Das, Amaresh; Spackman, Erica; Senne, Dennis; Pedersen, Jan; Suarez, David L.

2006-01-01

We developed an internal positive control (IPC) RNA to help ensure the accuracy of the detection of avian influenza virus (AIV) RNA by reverse transcription (RT)-PCR and real-time RT-PCR (RRT-PCR). The IPC was designed to have the same binding sites for the forward and reverse primers of the AIV matrix gene as the target amplicon, but it had a unique internal sequence used for the probe site. The amplification of the viral RNA and the IPC by RRT-PCR were monitored with two different fluorescent probes in a multiplex format, one specific for the AIV matrix gene and the other for the IPC. The RRT-PCR test was further simplified with the use of lyophilized bead reagents for the detection of AIV RNA. The RRT-PCR with the bead reagents was more sensitive than the conventional wet reagents for the detection of AIV RNA. The IPC-based RRT-PCR detected inhibitors in blood, kidney, lungs, spleen, intestine, and cloacal swabs, but not allantoic fluid, serum, or tracheal swabs The accuracy of RRT-PCR test results with the lyophilized beads was tested on cloacal and tracheal swabs from experimental birds inoculated with AIV and compared with virus isolation (VI) on embryonating chicken eggs. There was 97 to 100% agreement of the RRT-PCR test results with VI for tracheal swabs and 81% agreement with VI for cloacal swabs, indicating a high level of accuracy of the RRT-PCR assay. The same IPC in the form of armored RNA was also used to monitor the extraction of viral RNA and subsequent detection by RRT-PCR. PMID:16954228

Rapid and simultaneous detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes by magnetic capture hybridization and multiplex real-time PCR.

PubMed

Carloni, Elisa; Rotundo, Luca; Brandi, Giorgio; Amagliani, Giulia

2018-05-25

The application of rapid, specific, and sensitive methods for pathogen detection and quantification is very advantageous in diagnosis of human pathogens in several applications, including food analysis. The aim of this study was the evaluation of a method for the multiplexed detection and quantification of three significant foodborne pathogenic species (Escherichia coli O157, Salmonella spp., and Listeria monocytogenes). The assay combines specific DNA extraction by multiplex magnetic capture hybridization (mMCH) with multiplex real-time PCR. The amplification assay showed linearity in the range 10 6 -10 genomic units (GU)/PCR for each co-amplified species. The sensitivity corresponded to 1 GU/PCR for E. coli O157 and L. monocytogenes, and 10 GU/PCR for Salmonella spp. The immobilization process and the hybrid capture of the MCH showed good efficiency and reproducibility for all targets, allowing the combination in equal amounts of the different nanoparticle types in mMCH. MCH and mMCH efficiencies were similar. The detection limit of the method was 10 CFU in samples with individual pathogens and 10 2  CFU in samples with combination of the three pathogens in unequal amounts (amount's differences of 2 or 3 log). In conclusion, this multiplex molecular platform can be applied to determine the presence of target species in food samples after culture enrichment. In this way, this method could be a time-saving and sensitive tool to be used in routine diagnosis.

A real-time multi-gases detection and concentration measurements based-on time-division multiplexed-lasers

NASA Astrophysics Data System (ADS)

Yazdandoust, Fatemeh; Tatenguem Fankem, Hervé; Milde, Tobias; Jimenez, Alvaro; Sacher, Joachim

2018-02-01

We report the development of a platform, based-on a Field-Programmable Gate Arrays (FPGAs) and suitable for Time-Division-Multiplexed DFB lasers. The designed platform is subsequently combined with a spectroscopy setup, for detection and quantification of species in a gas mixture. The experimental results show a detection limit of 460 ppm, an uncertainty of 0.1% and a computation time of less than 1000 clock cycles. The proposed system offers a high level of flexibility and is applicable to arbitrary types of gas-mixtures.

Comparison between culture and a multiplex quantitative real-time polymerase chain reaction assay detecting Ureaplasma urealyticum and U. parvum.

PubMed

Frølund, Maria; Björnelius, Eva; Lidbrink, Peter; Ahrens, Peter; Jensen, Jørgen Skov

2014-01-01

A novel multiplex quantitative real-time polymerase chain reaction (qPCR) for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard's 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR.

Detection of hepatitis C virus subtypes 6a, 6n, 6w and mixed infections using a modified multiplex real-time polymerase chain reaction protocol.

PubMed

Lee, Yuan-Ming; Chen, Yen-Ju; Lee, Cheng-Ming; Kuo, Lou-Hui; Wong, Wing-Wai; Chen, Yi-Ming Arthur

2011-12-01

In the past few years, many new subtypes in hepatitis C virus (HCV) genotype 6 have been identified. The aim of this study was to modify the multiplex real-time polymerase chain reaction (RT-PCR) protocol and use it to determine the HCV subtypes of a group of Taiwanese injection drug users (IDUs). We used 76 serum specimens collected in northern Taiwan in 2008. Multiplex RT-PCR was used for HCV subtyping among those serum samples having anti-HCV antibodies. Twenty cases were randomly selected for comparison with subtyping results from Inno-LiPa II tests and phylogenetic tree analysis using NS5B sequences. Multiplex RT-PCR assays showed that 60.5% (46/76) of IDUs had single HCV infection. Three out of 76 (3.9%) had double HCV infection (1b/6a, 2a/2b and 2b/6a). Besides this, 27.6% (21/76) had no HCV signal. One IDU had subtype 6n and two had subtype 6w infection. Inno-LiPa II tests misclassified all 6n and 6w cases as 1b subtype. Our modified multiplex RT-PCR protocol can be used to support molecular epidemiological studies and laboratory diagnoses of different HCV subtypes including genotype 6. Copyright © 2011. Published by Elsevier B.V.

Detection of diarrheagenic Escherichia coli by use of melting-curve analysis and real-time multiplex PCR.

PubMed

Guion, Chase E; Ochoa, Theresa J; Walker, Christopher M; Barletta, Francesca; Cleary, Thomas G

2008-05-01

Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli, stIa/stIb and lt for enterotoxigenic E. coli, eaeA for enteropathogenic E. coli and Shiga toxin-producing E. coli (STEC), stx(1) and stx(2) for STEC, ipaH for enteroinvasive E. coli, and daaD for diffusely adherent E. coli (DAEC). Eighty-nine of ninety diarrheagenic E. coli and 36/36 nonpathogenic E. coli strains were correctly identified using this approach (specificity, 1.00; sensitivity, 0.99). The single false negative was a DAEC strain. The total time between preparation of DNA from E. coli colonies on agar plates and completion of PCR and melting-curve analysis was less than 90 min. The cost of materials was low. Melting-point analysis of real-time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for detection of diarrheagenic E. coli.

Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Venkateswaran, K.S.; Nasarabadi, S.; Langlois, R.G.

2000-05-05

Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCRmore » amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.« less

A Droplet Digital PCR Method for Severe Combined Immunodeficiency Newborn Screening.

PubMed

Vidal-Folch, Noemi; Milosevic, Dragana; Majumdar, Ramanath; Gavrilov, Dimitar; Matern, Dietrich; Raymond, Kimiyo; Rinaldo, Piero; Tortorelli, Silvia; Abraham, Roshini S; Oglesbee, Devin

2017-09-01

Severe combined immunodeficiency (SCID) benefits from early intervention via hematopoietic cell transplantation to reverse T-cell lymphopenia (TCL). Newborn screening (NBS) programs use T-cell receptor excision circle (TREC) levels to detect SCID. Real-time quantitative PCR is often performed to quantify TRECs in dried blood spots (DBSs) for NBS. Yet, real-time quantitative PCR has inefficiencies necessitating normalization, repeat analyses, or standard curves. To address these issues, we developed a multiplex, droplet digital PCR (ddPCR) method for measuring absolute TREC amounts in one DBS punch. TREC and RPP30 levels were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. DBSs from 610 presumed-normal, 29 lymphocyte-profiled, and 10 clinically diagnosed infants (1 X-linked SCID, 1 RAG1 Omenn syndrome, and other conditions) were tested. Control infants showed 14 to 474 TREC copies/μL blood. SCID infants, and other TCL conditions, had ≤15 TREC copies/μL. The ddPCR lower limit of quantitation was 14 TREC copies/μL, and the limit of detection was 4 TREC copies/μL. Intra-assay and interassay imprecision was <20% CV for DBSs at 54 to 60 TREC copies/μL. Testing 29 infants with known lymphocyte profiles resulted in a sensitivity of 88.9% and a specificity of 100% at TRECs <20 copies/μL. We developed a multiplex ddPCR method for the absolute quantitation of DBS TRECs that can detect SCID and other TCL conditions associated with absent or low TRECs and validated this method for NBS. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Development and Validation of a Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Three Papaya Viruses

PubMed Central

Tuo, Decai; Shen, Wentao; Yang, Yong; Yan, Pu; Li, Xiaoying; Zhou, Peng

2014-01-01

Papaya ringspot virus (PRSV), Papaya leaf distortion mosaic virus (PLDMV), and Papaya mosaic virus (PapMV) produce similar symptoms in papaya. Each threatens commercial production of papaya on Hainan Island, China. In this study, a multiplex reverse transcription PCR assay was developed to detect simultaneously these three viruses by screening combinations of mixed primer pairs and optimizing the multiplex RT-PCR reaction conditions. A mixture of three specific primer pairs was used to amplify three distinct fragments of 613 bp from the P3 gene of PRSV, 355 bp from the CP gene of PLDMV, and 205 bp from the CP gene of PapMV, demonstrating the assay’s specificity. The sensitivity of the multiplex RT-PCR was evaluated by showing plasmids containing each of the viral target genes with 1.44 × 103, 1.79 × 103, and 1.91 × 102 copies for the three viruses could be detected successfully. The multiplex RT-PCR was applied successfully for detection of three viruses from 341 field samples collected from 18 counties of Hainan Island, China. Rates of single infections were 186/341 (54.5%), 93/341 (27.3%), and 3/341 (0.9%), for PRSV, PLDMV, and PapMV, respectively; 59/341 (17.3%) of the samples were co-infected with PRSV and PLDMV, which is the first time being reported in Hainan Island. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting multiple viruses in papaya and can be used for routine molecular diagnosis and epidemiological studies in papaya. PMID:25337891

Use of an Internal Positive Control in a Multiplex Reverse Transcription-PCR To Detect West Nile Virus RNA in Mosquito Pools

PubMed Central

Eisler, Diane L.; McNabb, Alan; Jorgensen, Danielle R.; Isaac-Renton, Judith L.

2004-01-01

We report on the use of West Nile virus Armored RNA as an internal positive control (IPC) for the extraction and reverse transcription-PCR (RT-PCR) of RNA extracted from field-collected mosquitoes and on a multiplex real-time Taqman RT-PCR to simultaneously detect the 3′ noncoding region of West Nile virus and the West Nile virus NS5-2 region comprising the IPC. Mosquito pools from the province of British Columbia, Canada (n = 635), were tested in duplicate and found to be negative for West Nile virus and positive for the IPC. Known West Nile virus-positive supernatants from mosquito pools from the provinces of Alberta and Manitoba were tested in duplicate and found to be positive for both regions of the West Nile virus genome. The mean cycle threshold (Ct) value for the IPC in batch extraction controls ± 2 standard deviations was found to be 36.43 ± 1.78 cycles. IPCs of 98.4% (624) of West Nile virus-negative pools fell within this range, indicating the reproducibility of RNA extraction and RT-PCR for pools varying in mosquito genus and number. A comparison of mosquito pool genera revealed no significant genus effect on the Ct value of the IPC. The incorporation of West Nile virus Armored RNA as an IPC allows monitoring of RNA extraction and RT-PCR and detection of false-negative results due to failures in these processes or to PCR inhibition, respectively. PMID:14766868

Multiplex Detection of Toxigenic Penicillium Species.

PubMed

Rodríguez, Alicia; Córdoba, Juan J; Rodríguez, Mar; Andrade, María J

2017-01-01

Multiplex PCR-based methods for simultaneous detection and quantification of different mycotoxin-producing Penicillia are useful tools to be used in food safety programs. These rapid and sensitive techniques allow taking corrective actions during food processing or storage for avoiding accumulation of mycotoxins in them. In this chapter, three multiplex PCR-based methods to detect at least patulin- and ochratoxin A-producing Penicillia are detailed. Two of them are different multiplex real-time PCR suitable for monitoring and quantifying toxigenic Penicillium using the nonspecific dye SYBR Green and specific hydrolysis probes (TaqMan). All of them successfully use the same target genes involved in the biosynthesis of such mycotoxins for designing primers and/or probes.

Multiplex Detection of Aspergillus Species.

PubMed

Martínez-Culebras, Pedro; Selma, María Victoria; Aznar, Rosa

2017-01-01

Multiplex real-time polymerase chain reaction (PCR) provides a fast and accurate DNA-based tool for the simultaneous amplification of more than one target sequence in a single reaction. Here a duplex real-time PCR assay is described for the simultaneous detection of Aspergillus carbonarius and members of the Aspergillus niger aggregate, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the beta-ketosynthase and the acyl transferase domains of the polyketide synthase of A. carbonarius and the A. niger aggregate, respectively.Besides, a rapid and efficient fungi DNA extraction procedure is described suitable to be applied in wine grapes. It includes a pulsifier equipment to remove conidia from grapes which prevents releasing of PCR inhibitors.

Species-specific differentiation of variola, monkeypox, and varicella-zoster viruses by multiplex real-time PCR assay.

PubMed

Maksyutov, Rinat A; Gavrilova, Elena V; Shchelkunov, Sergei N

2016-10-01

A method of one-stage rapid detection and differentiation of epidemiologically important variola virus (VARV), monkeypox virus (MPXV), and varicella-zoster virus (VZV) utilizing multiplex real-time TaqMan PCR assay was developed. Four hybridization probes with various fluorescent dyes and the corresponding fluorescence quenchers were simultaneously used for the assay. The hybridization probes specific for the VARV sequence contained FAM/BHQ1 as a dye/quencher pair; MPXV-specific, JOE/BHQ1; VZV-specific, TAMRA/BHQ2; and internal control-specific, Cy5/BHQ3. The specificity and sensitivity of the developed method were assessed by analyzing DNA of 32 strains belonging to orthopoxvirus and herpesvirus species. Copyright © 2016 Elsevier B.V. All rights reserved.

NAIMA as a solution for future GMO diagnostics challenges.

PubMed

Dobnik, David; Morisset, Dany; Gruden, Kristina

2010-03-01

In the field of genetically modified organism (GMO) diagnostics, real-time PCR has been the method of choice for target detection and quantification in most laboratories. Despite its numerous advantages, however, the lack of a true multiplexing option may render real-time PCR less practical in the face of future GMO detection challenges such as the multiplicity and increasing complexity of new transgenic events, as well as the repeated occurrence of unauthorized GMOs on the market. In this context, we recently reported the development of a novel multiplex quantitative DNA-based target amplification method, named NASBA implemented microarray analysis (NAIMA), which is suitable for sensitive, specific and quantitative detection of GMOs on a microarray. In this article, the performance of NAIMA is compared with that of real-time PCR, the focus being their performances in view of the upcoming challenge to detect/quantify an increasing number of possible GMOs at a sustainable cost and affordable staff effort. Finally, we present our conclusions concerning the applicability of NAIMA for future use in GMO diagnostics.

System Collects And Displays Demultiplexed Data

NASA Technical Reports Server (NTRS)

Reschke, Millard F.; Fariss, Julie L.; Kulecz, Walter B.; Paloski, William H.

1992-01-01

Electronic system collects, manipulates, and displays in real time results of manipulation of streams of data transmitted from remote scientific instrumentation. Interface circuit shifts data-and-clock signal from differential logic levels of multiplexer to single-ended logic levels of computer. System accommodates nonstandard data-transmission protocol. Software useful in applications where Macintosh computers used in real-time display and recording of data.

Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents.

PubMed

Aitichou, Mohamed; Saleh, Sharron; Kyusung, Park; Huggins, John; O'Guinn, Monica; Jahrling, Peter; Ibrahim, Sofi

2008-11-01

A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6-carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible.

High speed, long distance, data transmission multiplexing circuit

DOEpatents

Mariotti, Razvan

1991-01-01

A high speed serial data transmission multiplexing circuit, which is operable to accurately transmit data over long distances (up to 3 Km), and to multiplex, select and continuously display real time analog signals in a bandwidth from DC to 100 Khz. The circuit is made fault tolerant by use of a programmable flywheel algorithm, which enables the circuit to tolerate one transmission error before losing synchronization of the transmitted frames of data. A method of encoding and framing captured and transmitted data is used which has a low overhead and prevents some particular transmitted data patterns from locking an included detector/decoder circuit.

Evaluation of multiplex real-time PCR for detection of Haemophilus ducreyi, Treponema pallidum, herpes simplex virus type 1 and 2 in the diagnosis of genital ulcer disease in the Rakai District, Uganda.

PubMed

Suntoke, T R; Hardick, A; Tobian, A A R; Mpoza, B; Laeyendecker, O; Serwadda, D; Opendi, P; Gaydos, C A; Gray, R H; Wawer, M J; Quinn, T C; Reynolds, S J

2009-04-01

To develop a real-time PCR assay that reliably and accurately detects the predominant sexually transmitted aetiological agents of genital ulcer disease (GUD) (Haemophilus ducreyi, Treponema pallidum and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2)) and to assess the use of real-time PCR diagnostic testing in a rural African field site. Two multiplex real-time PCR reactions were used to detect H ducreyi/and HSV-1/HSV-2 in ulcer swabs from 100 people with symptomatic genital ulcers in rural Rakai, Uganda. Results were compared with syphilis, HSV-1 and HSV-2 serology. Of 100 GUD samples analysed from 43 HIV positive and 57 HIV negative individuals, 71% were positive for one or more sexually transmitted infection (STI) pathogens by real-time PCR (61% for HSV-2, 5% for T pallidum, 3% for HSV-1, 1% for H ducreyi and 1% for dual H ducreyi/HSV-2). The frequency of HSV in genital ulcers was 56% (32/57) in HIV negative individuals and 77% (33/43) in HIV positive individuals (p = 0.037). Assay reproducibility was evaluated by repeat PCR testing in the USA with 96% agreement (kappa = 0.85). STI pathogens were detected in the majority of GUD swab samples from symptomatic patients in Rakai, Uganda, by real-time PCR. HSV-2 was the predominant cause of genital ulcers. Real-time PCR technology can provide sensitive, rapid and reproducible evaluation of GUD aetiology in a resource-limited setting.

A multiplex allele-specific real-time PCR assay for screening of ESR1 mutations in metastatic breast cancer.

PubMed

Wang, Ting; Liu, Jin-Hui; Zhang, Jie; Wang, Le; Chen, Chao; Dai, Peng-Gao

2015-04-01

Acquired resistance to endocrine-based therapies occurs in virtually all estrogen receptor-α (ERα, encoded by ESR1) positive breast cancer patients. The underlying molecular mechanism is attributed to the activating mutations in ESR1. These mutations provide an exciting opportunity for the development of new antagonists that specifically inhibit the mutant proteins. Therefore, accurate detection of ESR1 mutations is of critical importance in clinical practice. We carried out a single tube, multiplex allele-specific real-time PCR assay for the detection of four ESR1 mutations (Y537S, Y537C, Y537N, and D538G). The assay was found to be highly specific and sensitive. With this assay, as low as 1% mutant DNA template in wild type DNA could be detected. Fifteen DNA samples were prepared from archived formalin-fixed paraffin-embedded metastatic breast cancer biopsies. They were further screened with this assay, and three samples were identified as ESR1 mutant. The results were validated with pyrosequencing and complete concordance was observed between the two assays. The multiplex allele-specific real-time PCR assay provides a rapid and reliable diagnostic tool for accurate detection of ESR1 mutations. This procedure may be used in the clinical treatment of breast cancer. Copyright © 2015 Elsevier Inc. All rights reserved.

Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer

PubMed Central

Vargas, Diana Y.; Kramer, Fred Russell; Tyagi, Sanjay; Marras, Salvatore A. E.

2016-01-01

We describe the use of “SuperSelective” primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long “5' anchor sequence” that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, “3' foot sequence” that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation’s abundance by comparing its threshold value to the threshold value of a reference gene present in the sample. PMID:27244445

Detection of intestinal protozoa in paediatric patients with gastrointestinal symptoms by multiplex real-time PCR.

PubMed

Maas, L; Dorigo-Zetsma, J W; de Groot, C J; Bouter, S; Plötz, F B; van Ewijk, B E

2014-06-01

The performance of a multiplex real-time PCR for the detection of Blastocystis, Dientamoeba fragilis, Giardia lamblia, Cryptosporidium species and Entamoeba species in faecal samples was evaluated in an observational prospective study. Paediatric patients (0-18 years) presenting with gastrointestinal symptoms and suspected of having enteroparasitic disease were included. A questionnaire on gastrointestinal symptoms and the chosen treatment was completed at the start of the study and after 6 weeks. Of 163 paediatric patients (mean age, 7.8 years), 114 (70%) had a PCR-positive faecal sample. D. fragilis was detected most frequently, in 101 patients, followed by Blastocystis in 49. In faecal samples of 47 patients, more than one protozoan was detected, mainly the combination of D. fragilis and Blastocystis. Reported gastrointestinal symptoms were abdominal pain (78%), nausea (30%), and altered bowel habits (28%). Eighty-nine of the PCR-positive patients were treated with antibiotics. A significant reduction in abdominal pain was observed both in treated and in untreated patients. This study demonstrated that multiplex real-time PCR detects a high percentage of intestinal protozoa in paediatric patients with gastrointestinal symptoms. However, interpretation and determination of the clinical relevance of a positive PCR result in this population are still difficult. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

Distinguishing body lice from head lice by multiplex real-time PCR analysis of the Phum_PHUM540560 gene.

PubMed

Drali, Rezak; Boutellis, Amina; Raoult, Didier; Rolain, Jean Marc; Brouqui, Philippe

2013-01-01

Body louse or head louse? Once removed from their environment, body and head lice are indistinguishable. Neither the morphological criteria used since the mid-18th century nor the various genetic studies conducted since the advent of molecular biology tools have allowed body lice and head lice to be differentiated. In this work, using a portion of the Phum_PHUM540560 gene from the body louse, we aimed to develop a multiplex real-time polymerase chain reaction (PCR) assay to differentiate between body and head lice in a single reaction. A total of 142 human lice were collected from mono-infested hosts from 13 countries on five continents. We first identified the louse clade using a cytochrome b (CYTB) PCR sequence alignment. We then aligned a fragment of the Phum_PHUM540560 gene amplified from head and body lice to design-specific TaqMan(©) FAM- and VIC-labeled probes. All the analyzed lice were Clade A lice. A total of 22 polymorphisms between the body and head lice were characterized. The multiplex real-time PCR analysis enabled the body and head lice to be distinguished in two hours. This method is simple, with 100% specificity and sensitivity. We confirmed that the Phum_PHUM540560 gene is a useful genetic marker for the study of lice.

Species Identification of Fox-, Mink-, Dog-, and Rabbit-Derived Ingredients by Multiplex PCR and Real-Time PCR Assay.

PubMed

Wu, Qingqing; Xiang, Shengnan; Wang, Wenjun; Zhao, Jinyan; Xia, Jinhua; Zhen, Yueran; Liu, Bang

2018-05-01

Various detection methods have been developed to date for identification of animal species. New techniques based on PCR approach have raised the hope of developing better identification methods, which can overcome the limitations of the existing methods. PCR-based methods used the mitochondrial DNA (mtDNA) as well as nuclear DNA sequences. In this study, by targeting nuclear DNA, multiplex PCR and real-time PCR methods were developed to assist with qualitative and quantitative analysis. The multiplex PCR was found to simultaneously and effectively distinguish four species (fox, dog, mink, and rabbit) ingredients by the different sizes of electrophoretic bands: 480, 317, 220, and 209 bp. Real-time fluorescent PCR's amplification profiles and standard curves showed good quantitative measurement responses and linearity, as indicated by good repeatability and coefficient of determination R 2  > 0.99. The quantitative results of quaternary DNA mixtures including mink, fox, dog, and rabbit DNA are in line with our expectations: R.D. (relative deviation) varied between 1.98 and 12.23% and R.S.D. (relative standard deviation) varied between 3.06 and 11.51%, both of which are well within the acceptance criterion of ≤ 25%. Combining the two methods is suitable for the rapid identification and accurate quantification of fox-, dog-, mink-, and rabbit-derived ingredients in the animal products.

Wireless Multiplexed Surface Acoustic Wave Sensors Project

NASA Technical Reports Server (NTRS)

Youngquist, Robert C.

2014-01-01

Wireless Surface Acoustic Wave (SAW) Sensor is a new technology for obtaining multiple, real-time measurements under extreme environmental conditions. This project plans to develop a wireless multiplexed sensor system that uses SAW sensors, with no batteries or semiconductors, that are passive and rugged, can operate down to cryogenic temperatures and up to hundreds of degrees C, and can be used to sense a wide variety of parameters over reasonable distances (meters).

New multiplex real-time PCR approach to detect gene mutations for spinal muscular atrophy.

PubMed

Liu, Zhidai; Zhang, Penghui; He, Xiaoyan; Liu, Shan; Tang, Shi; Zhang, Rong; Wang, Xinbin; Tan, Junjie; Peng, Bin; Jiang, Li; Hong, Siqi; Zou, Lin

2016-08-17

Spinal muscular atrophy (SMA) is the most common autosomal recessive disease in children, and the diagnosis is complicated and difficult, especially at early stage. Early diagnosis of SMA is able to improve the outcome of SMA patients. In our study, Real-time PCR was developed to measure the gene mutation or deletion of key genes for SMA and to further analyse genotype-phenotype correlation. The multiple real-time PCR for detecting the mutations of survival of motor neuron (SMN), apoptosis inhibitory protein (NAIP) and general transcription factor IIH, polypeptide 2 gene (GTF2H2) was established and confirmed by DNA sequencing and multiplex ligation-dependent probe amplification (MLPA). The diagnosis and prognosis of 141 hospitalized children, 100 normal children and further 2000 cases of dry blood spot (DBS) samples were analysed by this multiple real-time PCR. The multiple real-time PCR was established and the accuracy of it to detect the mutations of SMN, NAIP and GTF2H2 was at least 98.8 % comparing with DNA sequencing and MLPA. Among 141 limb movement disorders children, 75 cases were SMA. 71 cases of SMA (94.67 %) were with SMN c.840 mutation, 9 cases (12 %) with NAIP deletion and 3 cases (4 %) with GTF2H2 deletion. The multiple real-time PCR was able to diagnose and predict the prognosis of SMA patients. Simultaneously, the real-time PCR was applied to detect trace DNA from DBS and able to make an early diagnosis of SMA. The clinical and molecular characteristics of SMA in Southwest of China were presented. Our work provides a novel way for detecting SMA in children by using real-time PCR and the potential usage in newborn screening for early diagnosis of SMA.

Dual phase multiplex polymerase chain reaction

DOEpatents

Pemov, Alexander [Charlottesville, VA; Bavykin, Sergei [Darien, IL

2008-10-07

Highly specific and sensitive methods were developed for multiplex amplification of nucleic acids on supports such as microarrays. Based on a specific primer design, methods include five types of amplification that proceed in a reaction chamber simultaneously. These relate to four types of multiplex amplification of a target DNA on a solid support, directed by forward and reverse complex primers immobilized to the support and a fifth type--pseudo-monoplex polymerase chain reaction (PCR) of multiple targets in solution, directed by a single pair of unbound universal primers. The addition of the universal primers in the reaction mixture increases the yield over the traditional "bridge" amplification on a solid support by approximately ten times. Methods that provide multitarget amplification and detection of as little as 0.45-4.5.times.10.sup.-12 g (equivalent to 10.sup.2-10.sup.3 genomes) of a bacterial genomic DNA are disclosed.

Percolation in real multiplex networks

NASA Astrophysics Data System (ADS)

Bianconi, Ginestra; Radicchi, Filippo

2016-12-01

We present an exact mathematical framework able to describe site-percolation transitions in real multiplex networks. Specifically, we consider the average percolation diagram valid over an infinite number of random configurations where nodes are present in the system with given probability. The approach relies on the locally treelike ansatz, so that it is expected to accurately reproduce the true percolation diagram of sparse multiplex networks with negligible number of short loops. The performance of our theory is tested in social, biological, and transportation multiplex graphs. When compared against previously introduced methods, we observe improvements in the prediction of the percolation diagrams in all networks analyzed. Results from our method confirm previous claims about the robustness of real multiplex networks, in the sense that the average connectedness of the system does not exhibit any significant abrupt change as its individual components are randomly destroyed.

Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station.

PubMed

Parra, Macarena; Jung, Jimmy; Boone, Travis D; Tran, Luan; Blaber, Elizabeth A; Brown, Mark; Chin, Matthew; Chinn, Tori; Cohen, Jacob; Doebler, Robert; Hoang, Dzung; Hyde, Elizabeth; Lera, Matthew; Luzod, Louie T; Mallinson, Mark; Marcu, Oana; Mohamedaly, Youssef; Ricco, Antonio J; Rubins, Kathleen; Sgarlato, Gregory D; Talavera, Rafael O; Tong, Peter; Uribe, Eddie; Williams, Jeffrey; Wu, Diana; Yousuf, Rukhsana; Richey, Charles S; Schonfeld, Julie; Almeida, Eduardo A C

2017-01-01

The International Space Station (ISS) National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen Escherichia coli and mouse liver tissue. Cycle threshold (Ct) values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g) controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for molecular biology and demonstrates the feasibility of more complex wet bench experiments in the ISS National Lab environment.

Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station

PubMed Central

Boone, Travis D.; Tran, Luan; Blaber, Elizabeth A.; Brown, Mark; Chin, Matthew; Chinn, Tori; Cohen, Jacob; Doebler, Robert; Hoang, Dzung; Hyde, Elizabeth; Lera, Matthew; Luzod, Louie T.; Mallinson, Mark; Marcu, Oana; Mohamedaly, Youssef; Ricco, Antonio J.; Rubins, Kathleen; Sgarlato, Gregory D.; Talavera, Rafael O.; Tong, Peter; Uribe, Eddie; Williams, Jeffrey; Wu, Diana; Yousuf, Rukhsana; Richey, Charles S.; Schonfeld, Julie

2017-01-01

The International Space Station (ISS) National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen Escherichia coli and mouse liver tissue. Cycle threshold (Ct) values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g) controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for molecular biology and demonstrates the feasibility of more complex wet bench experiments in the ISS National Lab environment. PMID:28877184

Novel Multiplex Real-Time PCR Diagnostic Assay for Identification and Differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis Complex Strains▿†

PubMed Central

Reddington, Kate; O'Grady, Justin; Dorai-Raj, Siobhan; Maher, Majella; van Soolingen, Dick; Barry, Thomas

2011-01-01

Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC. PMID:21123525

Multiplexed immunosensing and kinetics monitoring in nanofluidic devices with highly enhanced target capture efficiency

PubMed Central

Lin, Yii-Lih; Huang, Yen-Jun; Teerapanich, Pattamon; Leïchlé, Thierry

2016-01-01

Nanofluidic devices promise high reaction efficiency and fast kinetic responses due to the spatial constriction of transported biomolecules with confined molecular diffusion. However, parallel detection of multiple biomolecules, particularly proteins, in highly confined space remains challenging. This study integrates extended nanofluidics with embedded protein microarray to achieve multiplexed real-time biosensing and kinetics monitoring. Implementation of embedded standard-sized antibody microarray is attained by epoxy-silane surface modification and a room-temperature low-aspect-ratio bonding technique. An effective sample transport is achieved by electrokinetic pumping via electroosmotic flow. Through the nanoslit-based spatial confinement, the antigen-antibody binding reaction is enhanced with ∼100% efficiency and may be directly observed with fluorescence microscopy without the requirement of intermediate washing steps. The image-based data provide numerous spatially distributed reaction kinetic curves and are collectively modeled using a simple one-dimensional convection-reaction model. This study represents an integrated nanofluidic solution for real-time multiplexed immunosensing and kinetics monitoring, starting from device fabrication, protein immobilization, device bonding, sample transport, to data analysis at Péclet number less than 1. PMID:27375819

Detection and Serotyping of Dengue Virus in Serum Samples by Multiplex Reverse Transcriptase PCR-Ligase Detection Reaction Assay▿ †

PubMed Central

Das, S.; Pingle, M. R.; Muñoz-Jordán, J.; Rundell, M. S.; Rondini, S.; Granger, K.; Chang, G.-J. J.; Kelly, E.; Spier, E. G.; Larone, D.; Spitzer, E.; Barany, F.; Golightly, L. M.

2008-01-01

The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV. PMID:18685000

The physiologic state of Escherichia coli O157:H7 does not affect its detection in two commercial real-time PCR-based tests

USDA-ARS?s Scientific Manuscript database

Multiplex real-time PCR detection of Escherichia coli O157:H7 is an efficient molecular tool with high sensitivity and specificity for meat safety and quality assurance in the beef industry. The Biocontrol GDS and the DuPont Qualicon BAX®-RT rapid detection systems are two commercial tests based on...

Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters▿

PubMed Central

Nordstrom, Jessica L.; Vickery, Michael C. L.; Blackstone, George M.; Murray, Shelley L.; DePaola, Angelo

2007-01-01

Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >104 CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh+ and trh+ strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus. PMID:17644647

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics.

PubMed

Zambenedetti, Miriam Ribas; Pavoni, Daniela Parada; Dallabona, Andreia Cristine; Dominguez, Alejandro Correa; Poersch, Celina de Oliveira; Fragoso, Stenio Perdigão; Krieger, Marco Aurélio

2017-05-01

Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

PubMed Central

Zambenedetti, Miriam Ribas; Pavoni, Daniela Parada; Dallabona, Andreia Cristine; Dominguez, Alejandro Correa; Poersch, Celina de Oliveira; Fragoso, Stenio Perdigão; Krieger, Marco Aurélio

2017-01-01

BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses. PMID:28403327

Gene expression in the rectus abdominus muscle of patients with and without pelvic organ prolapse.

PubMed

Hundley, Andrew F; Yuan, Lingwen; Visco, Anthony G

2008-02-01

The objective of the study was to compare gene expression in a group of actin and myosin-related proteins in the rectus muscle of 15 patients with pelvic organ prolapse and 13 controls. Six genes previously identified by microarray GeneChip analysis were examined using real-time quantitative reverse transcriptase-polymerase chain reaction analysis, including 2 genes showing differential expression in pubococcygeus muscle. Samples and controls were run in triplicate in multiplexed wells, and levels of gene expression were analyzed using the comparative critical threshold method. One gene, MYH3, was 3.2 times overexpressed in patients with prolapse (P = .032), but no significant differences in expression were seen for the other genes examined. An age-matched subset of 9 patients and controls showed that MYH3 gene expression was no longer significantly different (P = .058). Differential messenger ribonucleic acid levels of actin and myosin-related genes in patients with pelvic organ prolapse and controls may be limited to skeletal muscle from the pelvic floor.

Apollo experience report: Real-time display system

NASA Technical Reports Server (NTRS)

Sullivan, C. J.; Burbank, L. W.

1976-01-01

The real time display system used in the Apollo Program is described; the systematic organization of the system, which resulted from hardware/software trade-offs and the establishment of system criteria, is emphasized. Each basic requirement of the real time display system was met by a separate subsystem. The computer input multiplexer subsystem, the plotting display subsystem, the digital display subsystem, and the digital television subsystem are described. Also described are the automated display design and the generation of precision photographic reference slides required for the three display subsystems.

Photonic microwave waveforms generation based on pulse carving and superposition in time-domain

NASA Astrophysics Data System (ADS)

Xia, Yi; Jiang, Yang; Zi, Yuejiao; He, Yutong; Tian, Jing; Zhang, Xiaoyu; Luo, Hao; Dong, Ruyang

2018-05-01

A novel photonic approach for various microwave waveforms generation based on time-domain synthesis is theoretically analyzed and experimentally investigated. In this scheme, two single-drive Mach-Zehnder modulators are used for pulses shaping. After shifting the phase and implementing envelopes superposition of the pulses, desired waveforms can be achieved in time-domain. The theoretic analysis and simulations are presented. In the experimental demonstrations, a triangular waveform, square waveform, and half duty cycle sawtooth (or reversed-sawtooth) waveform are generated successfully. By utilizing time multiplexing technique, a frequency-doubled sawtooth (or reversed-sawtooth) waveform with 100% duty cycle can be obtained. In addition, a fundamental frequency sawtooth (or reversed-sawtooth) waveform with 100% duty cycle can also be achieved by the superposition of square waveform and frequency-doubled sawtooth waveform.

Simultaneous Detection of Rift Valley Fever, Bluetongue, Rinderpest, and Peste des Petits Ruminants Viruses by a Single-Tube Multiplex Reverse Transcriptase-PCR Assay Using a Dual-Priming Oligonucleotide System▿

PubMed Central

Yeh, Jung-Yong; Lee, Ji-Hye; Seo, Hyun-Ji; Park, Jee-Yong; Moon, Jin-San; Cho, In-Soo; Choi, In-Soo; Park, Seung-Yong; Song, Chang-Seon; Lee, Joong-Bok

2011-01-01

The aim of this study was to develop a highly sensitive and specific one-step multiplex reverse transcriptase PCR assay for the simultaneous and differential detection of Rift Valley Fever virus (RVFV), bluetongue virus (BTV), rinderpest virus (RPV), and Peste des petits ruminants virus (PPRV). These viruses cause mucosal lesions in cattle, sheep, and goats, and they are difficult to differentiate from one another based solely on their clinical presentation in suspected disease cases. In this study, we developed a multiplex reverse transcriptase PCR to detect these viruses using a novel dual-priming oligonucleotide (DPO). The DPO contains two separate priming regions joined by a polydeoxyinosine linker, which blocks extension of nonspecifically primed templates and consistently allows high PCR specificity even under less-than-optimal PCR conditions. A total of 19 DPO primers were designed to detect and discriminate between RVFV, BTV, RPV, and PPRV by the generation of 205-, 440-, 115-, and 243-bp cDNA products, respectively. The multiplex reverse transcriptase PCR described here enables the early diagnosis of these four viruses and may also be useful as part of a testing regime for cattle, sheep, or goats exhibiting similar clinical signs, including mucosal lesions. PMID:21307219

Development of a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction assay for the differential diagnosis of Feline leukemia virus vaccine and wild strains.

PubMed

Ho, Chia-Fang; Chan, Kun-Wei; Yang, Wei-Cheng; Chiang, Yu-Chung; Chung, Yang-Tsung; Kuo, James; Wang, Chi-Young

2014-07-01

A multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) was developed for the differential diagnosis of Feline leukemia virus (FeLV) vaccine and wild-type strains based on a point mutation between the vaccine strain (S) and the wild-type strain (T) located in the p27 gene. This system was further upgraded to obtain a real-time ARMS RT-PCR (ARMS qRT-PCR) with a high-resolution melt analysis (HRMA) platform. The genotyping of various strains of FeLV was determined by comparing the HRMA curves with the defined wild-type FeLV (strain TW1), and the results were expressed as a percentage confidence. The detection limits of ARMS RT-PCR and ARMS qRT-PCR combined with HRMA were 100 and 1 copies of transcribed FeLV RNA per 0.5 ml of sample, respectively. No false-positive results were obtained with 6 unrelated pathogens and 1 feline cell line. Twelve FeLV Taiwan strains were correctly identified using ARMS qRT-PCR combined with HRMA. The genotypes of the strains matched the defined FeLV wild-type strain genotype with at least 91.17% confidence. A higher degree of sequence polymorphism was found throughout the p27 gene compared with the long terminal repeat region. In conclusion, the current study describes the phylogenetic relationship of the FeLV Taiwan strains and demonstrates that the developed ARMS RT-PCR assay is able to be used to detect the replication of a vaccine strain that has not been properly inactivated, thus acting as a safety check for the quality of FeLV vaccines.

Simultaneous detection of Legionella species and L. anisa, L. bozemanii, L. longbeachae and L. micdadei using conserved primers and multiple probes in a multiplex real-time PCR assay.

PubMed

Cross, Kristen E; Mercante, Jeffrey W; Benitez, Alvaro J; Brown, Ellen W; Diaz, Maureen H; Winchell, Jonas M

2016-07-01

Legionnaires' disease is a severe respiratory disease that is estimated to cause between 8,000 and 18,000 hospitalizations each year, though the exact burden is unknown due to under-utilization of diagnostic testing. Although Legionella pneumophila is the most common species detected in clinical cases (80-90%), other species have also been reported to cause disease. However, little is known about Legionnaires' disease caused by these non-pneumophila species. We designed a multiplex real-time PCR assay for detection of all Legionella spp. and simultaneous specific identification of four clinically-relevant Legionella species, L. anisa, L. bozemanii, L. longbeachae, and L. micdadei, using 5'-hydrolysis probe real-time PCR. The analytical sensitivity for detection of nucleic acid from each target species was ≤50fg per reaction. We demonstrated the utility of this assay in spiked human sputum specimens. This assay could serve as a tool for understanding the scope and impact of non-pneumophila Legionella species in human disease. Published by Elsevier Inc.

Multiplex PCR method for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species.

PubMed

Trotta, Michele; Schönhuth, Susana; Pepe, Tiziana; Cortesi, M Luisa; Puyet, Antonio; Bautista, José M

2005-03-23

Mitochondrial 16S rRNA sequences from morphological validated grouper (Epinephelus aeneus, E. caninus, E. costae, and E. marginatus; Mycteroperca fusca and M. rubra), Nile perch (Lates niloticus), and wreck fish (Polyprion americanus) were used to develop an analytical system for group diagnosis based on two alternative Polymerase Chain Reaction (PCR) approaches. The first includes conventional multiplex PCR in which electrophoretic migration of different sizes of bands allowed identification of the fish species. The second approach, involving real-time PCR, produced a single amplicon from each species that showed different Tm values allowing the fish groups to be directly identified. Real-time PCR allows the quick differential diagnosis of the three groups of species and high-throughput screening of multiple samples. Neither PCR system cross-reacted with DNA samples from 41 common marketed fish species, thus conforming to standards for species validation. The use of these two PCR-based methods makes it now possible to discriminate grouper from substitute fish species.

Detection and identification of genetically modified EE-1 brinjal (Solanum melongena) by single, multiplex and SYBR® real-time PCR.

PubMed

Ballari, Rajashekhar V; Martin, Asha; Gowda, Lalitha R

2013-01-01

Brinjal is an important vegetable crop. Major crop loss of brinjal is due to insect attack. Insect-resistant EE-1 brinjal has been developed and is awaiting approval for commercial release. Consumer health concerns and implementation of international labelling legislation demand reliable analytical detection methods for genetically modified (GM) varieties. End-point and real-time polymerase chain reaction (PCR) methods were used to detect EE-1 brinjal. In end-point PCR, primer pairs specific to 35S CaMV promoter, NOS terminator and nptII gene common to other GM crops were used. Based on the revealed 3' transgene integration sequence, primers specific for the event EE-1 brinjal were designed. These primers were used for end-point single, multiplex and SYBR-based real-time PCR. End-point single PCR showed that the designed primers were highly specific to event EE-1 with a sensitivity of 20 pg of genomic DNA, corresponding to 20 copies of haploid EE-1 brinjal genomic DNA. The limits of detection and quantification for SYBR-based real-time PCR assay were 10 and 100 copies respectively. The prior development of detection methods for this important vegetable crop will facilitate compliance with any forthcoming labelling regulations. Copyright © 2012 Society of Chemical Industry.

Development of a multiplex real-time PCR assay for detection of human enteric viruses other than norovirus using samples collected from gastroenteritis patients in Fukui Prefecture, Japan.

PubMed

Kowada, Kazuaki; Takeuchi, Kenji; Hirano, Eiko; Toho, Miho; Sada, Kiyonao

2018-01-01

There are many varieties of gastroenteritis viruses, of which norovirus (NoV) accounts for over 90% of the viral food poisoning incidents in Japan. However, protocols for rapidly identifying other gastroenteritis viruses need to be established to investigate NoV-negative cases intensively. In this study, a multiplex real-time PCR assay targeting rotavirus A, rotavirus C, sapovirus, astrovirus, adenovirus, and enterovirus was developed using stool samples collected from gastroenteritis patients between 2010 and 2013 in Fukui Prefecture, Japan. Of the 126 samples collected sporadically from pediatric patients with suspected infectious gastroenteritis, 51 were positive for non-NoV target viruses, whereas 27 were positive for NoV, showing a high prevalence of non-NoV viruses in pediatric patients. In contrast, testing in 382 samples of 58 gastroenteritis outbreaks showed that non-NoV viruses were detected in 13 samples, with NoV in 267. Of the 267 NoV-positive patients, only two were co-infected with non-NoV target viruses, suggesting that testing for non-NoV gastroenteritis viruses in NoV-positive samples was mostly unnecessary in outbreak investigations. Given these results, multiplex real-time PCR testing for non-NoV gastroenteritis viruses, conducted separately from NoV testing, may be helpful to deal with two types of epidemiological investigations, regular surveillance of infectious gastroenteritis and urgent testing when gastroenteritis outbreaks occur. © 2017 Wiley Periodicals, Inc.

Prevalence of Sexually Transmitted Diseases in Asymptomatic Renal Transplant Recipients.

PubMed

Sarier, Mehmet; Sepin Ozen, Nevgun; Guler, Hicran; Duman, Ibrahim; Yüksel, Yücel; Tekin, Sabri; Yavuz, Asuman Havva; Yucetin, Levent; Erdogan Yilmaz, Mine

2018-04-04

Sexually transmitted diseases, which may be asymptomatic, have the potential to cause serious health problems in renal transplant recipients. The aim of this study was to determine the prevalence of sexually transmitted diseases in sexually active asymptomatic renal transplant patients by using real-time multiplex polymerase chain reaction assays. This prospective controlled study was conducted between November 2016 and January 2017 in our hospital. Our study group included 80 consecutive, sexually active asymptomatic patients (40 men and 40 women) who had undergone renal transplant in our hospital and who presented to our outpatient clinic for routine follow-up. We also included a control group of 80 consecutive, sexually active nontransplant patients (40 men and 40 women). All patient samples were tested for Gardnerella vaginalis and obligate anaerobes (Prevotella bivia, Porphyromonas species), Candida species, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma species, Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, herpes simplex virus 1 and 2, and Cytomegalovirus by real-time multiplex polymerase chain reaction. The prevalences of infection with Gardnerella vaginalis and obligate anaerobes (P = .043), Ureaplasma species (P = .02), and Cytomegalovirus (P = .016) were found to be significantly higher in the study group versus the control group. However, there was no difference between the 2 groups regarding the prevalence of Mycoplasma infection (P = .70). Sexually transmitted diseases may occur more frequently in sexually active asymptomatic renal transplant recipients than in nontransplanted individuals. Real-time multiplex polymerase chain reaction analysis may be a suitable method for determining these pathogens.

Pathogen Identification by Multiplex LightMix Real-Time PCR Assay in Patients with Meningitis and Culture-Negative Cerebrospinal Fluid Specimens

PubMed Central

Wagner, Karoline; Springer, Burkard; Pires, Valeria P.

2017-01-01

ABSTRACT Acute bacterial meningitis is a medical emergency, and delays in initiating effective antimicrobial therapy result in increased morbidity and mortality. Culture-based methods, thus far considered the “gold standard” for identifying bacterial microorganisms, require 24 to 48 h to provide a diagnosis. In addition, antimicrobial therapy is often started prior to clinical sample collection, thereby decreasing the probability of confirming the bacterial pathogen by culture-based methods. To enable a fast and accurate detection of the most important bacterial pathogens causing meningitis, namely, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Streptococcus agalactiae, and Listeria monocytogenes, we evaluated a commercially available multiplex LightMix real-time PCR (RT-PCR) in 220 cerebrospinal fluid (CSF) specimens. The majority of CSF samples were collected by lumbar puncture, but we also included some CSF samples from patients with symptoms of meningitis from the neurology department that were recovered from shunts. CSF samples were analyzed by multiplex RT-PCR enabling a first diagnosis within a few hours after sample arrival at our institute. In contrast, bacterial identification took between 24 and 48 h by culture. Overall, a high agreement of bacterial identification between culture and multiplex RT-PCR was observed (99%). Moreover, multiplex RT-PCR enabled the detection of pathogens, S. pneumoniae (n = 2), S. agalactiae (n = 1), and N. meningitidis (n = 1), in four culture-negative samples. As a complement to classical bacteriological CSF culture, the LightMix RT-PCR assay proved to be valuable by improving the rapidity and accuracy of the diagnosis of bacterial meningitis. PMID:29237781

Evaluation of Multiplex Type-Specific Real-Time PCR Assays Using the LightCycler and Joint Biological Agent Identification and Diagnostic System Platforms for Detection and Quantitation of Adult Human Respiratory Adenoviruses

DTIC Science & Technology

2010-04-01

53592), Escherichia coli, Klebsiella pneu- moniae (ATCC 13883), Pseudomonas aeruginosa (ATCC 97), Mycoplasma pneu- moniae, and Legionella pneumophila... Legionella pneumophila. Additionally, when we tested all samples with the multiplex assays, we did not see any cross- reactivity (data not shown...Chlamydophila pneumoniae Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Mycoplasma pneumoniae Legionella pneumophila VOL. 48, 2010

Simplified Real-Time Multiplex Detection of Loop-Mediated Isothermal Amplification Using Novel Mediator Displacement Probes with Universal Reporters.

PubMed

Becherer, Lisa; Bakheit, Mohammed; Frischmann, Sieghard; Stinco, Silvina; Borst, Nadine; Zengerle, Roland; von Stetten, Felix

2018-04-03

A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel mediator displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3-4 h), shorter times to positive (MD 4.1 ± 0.1 min shorter than MB, n = 36), improved signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, MB: 2.7 ± 0.4; n = 15), and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 and HTLV-1 and a biplex LAMP of Haemophilus ducreyi and Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels.

Rapid and reliable diagnostic method to detect Zika virus by real-time fluorescence reverse transcription loop-mediated isothermal amplification.

PubMed

Guo, Xu-Guang; Zhou, Yong-Zhuo; Li, Qin; Wang, Wei; Wen, Jin-Zhou; Zheng, Lei; Wang, Qian

2018-04-18

To detect Zika virus more rapidly and accurately, we developed a novel method that utilized a real-time fluorescence reverse transcription loop-mediated isothermal amplification (LAMP) technique. The NS5 gene was amplified by a set of six specific primers that recognized six distinct sequences. The amplification process, including 60 min of thermostatic reaction with Bst DNA polymerase following real-time fluorescence reverse transcriptase using genomic Zika virus standard strain (MR766), was conducted through fluorescent signaling. Among the six pairs of primers that we designate here, NS5 was the most efficient with a high sensitivity of up to 3.3 ng/μl and reproducible specificity on eight pathogen samples that were used as negative controls. The real-time fluorescence reverse transcription LAMP detection process can be completed within 35 min. Our study demonstrated that real-time fluorescence reverse transcription LAMP could be highly beneficial and convenient clinical application to detect Zika virus due to its high specificity and stability.

Finite-dimensional modeling of network-induced delays for real-time control systems

NASA Technical Reports Server (NTRS)

Ray, Asok; Halevi, Yoram

1988-01-01

In integrated control systems (ICS), a feedback loop is closed by the common communication channel, which multiplexes digital data from the sensor to the controller and from the controller to the actuator along with the data traffic from other control loops and management functions. Due to asynchronous time-division multiplexing in the network access protocols, time-varying delays are introduced in the control loop, which degrade the system dynamic performance and are a potential source of instability. The delayed control system is represented by a finite-dimensional, time-varying, discrete-time model which is less complex than the existing continuous-time models for time-varying delays; this approach allows for simpler schemes for analysis and simulation of the ICS.

Simultaneous detection of Zika, Chikungunya and Dengue viruses by a multiplex real-time RT-PCR assay.

PubMed

Pabbaraju, Kanti; Wong, Sallene; Gill, Kara; Fonseca, Kevin; Tipples, Graham A; Tellier, Raymond

2016-10-01

In the recent past, arboviruses such as Chikungunya (CHIKV) and Zika (ZIKV) have increased their area of endemicity and presented as an emerging global public health threat. To design an assay for the simultaneous detection of ZIKV, CHIKV and Dengue (DENV) 1-4 from patients with symptoms of arboviral infection. This would be advantageous because of the similar clinical presentation typically encountered with these viruses and their co-circulation in endemic areas. In this study we have developed and validated a triplex real time reverse transcription PCR assay using hydrolysis probes targeting the non-structural 5 (NS5) region of ZIKV, non-structural protein 4 (nsP4) from CHIKV and 3' untranslated region (3'UTR) of DENV 1-4. The 95% LOD by the triplex assay was 15 copies/reaction for DENV-1 and less than 10 copies/reaction for all other viruses. The triplex assay was 100% specific and did not amplify any of the other viruses tested. The assay was reproducible and adaptable to testing different specimen types including serum, plasma, urine, placental tissue, brain tissue and amniotic fluid. This assay can be easily implemented for diagnostic testing of patient samples, even in a high throughput laboratory. Copyright © 2016 Elsevier B.V. All rights reserved.

Real-time multiple-look synthetic aperture radar processor for spacecraft applications

NASA Technical Reports Server (NTRS)

Wu, C.; Tyree, V. C. (Inventor)

1981-01-01

A spaceborne synthetic aperture radar (SAR) having pipeline multiple-look data processing is described which makes use of excessive azimuth bandwidth in radar echo signals to produce multiple-looking images. Time multiplexed single-look image lines from an azimuth correlator go through an energy analyzer which analyzes the mean energy in each separate look to determine the radar antenna electric boresight for use in generating the correct reference functions for the production of high quality SAR images. The multiplexed single look image lines also go through a registration delay to produce multi-look images.

Development of a Real-Time Pulse Processing Algorithm for TES-Based X-Ray Microcalorimeters

NASA Technical Reports Server (NTRS)

Tan, Hui; Hennig, Wolfgang; Warburton, William K.; Doriese, W. Bertrand; Kilbourne, Caroline A.

2011-01-01

We report here a real-time pulse processing algorithm for superconducting transition-edge sensor (TES) based x-ray microcalorimeters. TES-based. microca1orimeters offer ultra-high energy resolutions, but the small volume of each pixel requires that large arrays of identical microcalorimeter pixe1s be built to achieve sufficient detection efficiency. That in turn requires as much pulse processing as possible must be performed at the front end of readout electronics to avoid transferring large amounts of data to a host computer for post-processing. Therefore, a real-time pulse processing algorithm that not only can be implemented in the readout electronics but also achieve satisfactory energy resolutions is desired. We have developed an algorithm that can be easily implemented. in hardware. We then tested the algorithm offline using several data sets acquired with an 8 x 8 Goddard TES x-ray calorimeter array and 2x16 NIST time-division SQUID multiplexer. We obtained an average energy resolution of close to 3.0 eV at 6 keV for the multiplexed pixels while preserving over 99% of the events in the data sets.

Real-Time Fourier Transformed Holographic Associative Memory With Photorefractive Material

NASA Astrophysics Data System (ADS)

Changsuk, Oh; Hankyu, Park

1989-02-01

We describe a volume holographic associative memory using photorefractive material and conventional planar mirror. Multiple hologram is generated with two angular multiplexed writing beams and Fourier transformed object beam in BaTiO3 crystal at 0.6328 μm. Complete image can be recalled successfully by partial input of original stored image. It is proved that our system is useful for optical implementation of real-time associative memory and location addressable memory.

Real time interrogation technique for fiber Bragg grating enhanced fiber loop ringdown sensors array.

PubMed

Zhang, Yunlong; Li, Ruoming; Shi, Yuechun; Zhang, Jintao; Chen, Xiangfei; Liu, Shengchun

2015-06-01

A novel fiber Bragg grating aided fiber loop ringdown (FLRD) sensor array and the wavelength-time multiplexing based interrogation technique for the FLRD sensors array are proposed. The interrogation frequency of the system is formulated and the interrelationships among the parameters of the system are analyzed. To validate the performance of the proposed system, a five elements array is experimentally demonstrated, and the system shows the capability of real time monitoring every FLRD element with interrogation frequency of 125.5 Hz.

Awareness of Emerging Wireless Technologies: Ad-hoc and Personal Area Networks Standards and Emerging Technologies (Sensibilisation a l’emergence des technologies sans fil: technologies emergeantes et normes de reseaux personnels et ad-hoc)

DTIC Science & Technology

2007-04-01

for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data...Control Organization NRL Navy Research Laboratory nrtPS Non-real- time Polling Services OFDM Orthogonal frequency division multiplex OFDMA...Routeur IDentifier RTG RTO Task Group RTO Research & Technology Organization rtPS Real- time Polling Services SC Single-carrier modulation

Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms.

PubMed

Cottenet, Geoffrey; Blancpain, Carine; Sonnard, Véronique; Chuah, Poh Fong

2013-08-01

Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers' perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories.

Real-time multiplex PCR assay for detection of Yersinia pestis and Yersinia pseudotuberculosis.

PubMed

Matero, Pirjo; Pasanen, Tanja; Laukkanen, Riikka; Tissari, Päivi; Tarkka, Eveliina; Vaara, Martti; Skurnik, Mikael

2009-01-01

A multiplex real-time polymerase chain reaction (PCR) assay was developed for the detection of Yersinia pestis and Yersinia pseudotuberculosis. The assay includes four primer pairs, two of which are specific for Y. pestis, one for Y. pestis and Y. pseudotuberculosis and one for bacteriophage lambda; the latter was used as an internal amplification control. The Y. pestis-specific target genes in the assay were ypo2088, a gene coding for a putative methyltransferase, and the pla gene coding for the plasminogen activator. In addition, the wzz gene was used as a target to specifically identify both Y. pestis and the closely related Y. pseudotuberculosis group. The primer and probe sets described for the different genes can be used either in single or in multiplex PCR assays because the individual probes were designed with different fluorochromes. The assays were found to be both sensitive and specific; the lower limit of the detection was 10-100 fg of extracted Y. pestis or Y. pseudotuberculosis total DNA. The sensitivity of the tetraplex assay was determined to be 1 cfu for the ypo2088 and pla probe labelled with FAM and JOE fluorescent dyes, respectively.

Simultaneous detection of the three ilarviruses affecting stone fruit trees by nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction.

PubMed

Saade, M; Aparicio, F; Sánchez-Navarro, J A; Herranz, M C; Myrta, A; Di Terlizzi, B; Pallás, V

2000-12-01

ABSTRACT The three most economically damaging ilarviruses affecting stone fruit trees on a worldwide scale are the related Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Apple mosaic virus (ApMV). Nonisotopic molecular hybridization and multiplex reverse-transcription polymerase chain reaction (RT-PCR) methodologies were developed that could detect all these viruses simultaneously. The latter technique was advantageous because it was discriminatory. For RT-PCR, a degenerate antisense primer was designed which was used in conjunction with three virus-specific sense primers. The amplification efficiencies for the detection of the three viruses in the multiplex RT-PCR reaction were identical to those obtained in the single RT-PCR reactions for individual viruses. This cocktail of primers was able to amplify sequences from all of the PNRSV, ApMV, and PDV isolates tested in five Prunus spp. hosts (almond, apricot, cherry, peach, and plum) occurring naturally in single or multiple infections. For ApMV isolates, differences in the electrophoretic mobilities of the PCR products were observed. The nucleotide sequence of the amplified products of two representative ApMV isolates was determined, and comparative analysis revealed the existence of a 28-nucleotide deletion in the sequence of isolates showing the faster electrophoretic mobility. To our knowledge, this is the first report on the simultaneous detection of three plant viruses by multiplex RT-PCR in woody hosts. This multiplex RT-PCR could be a useful time and cost saving method for indexing these three ilarviruses, which damage stone fruit tree yields, and for the analysis of mother plants in certification programs.

Direct multiplexed measurement of gene expression with color-coded probe pairs.

PubMed

Geiss, Gary K; Bumgarner, Roger E; Birditt, Brian; Dahl, Timothy; Dowidar, Naeem; Dunaway, Dwayne L; Fell, H Perry; Ferree, Sean; George, Renee D; Grogan, Tammy; James, Jeffrey J; Maysuria, Malini; Mitton, Jeffrey D; Oliveri, Paola; Osborn, Jennifer L; Peng, Tao; Ratcliffe, Amber L; Webster, Philippa J; Davidson, Eric H; Hood, Leroy; Dimitrov, Krassen

2008-03-01

We describe a technology, the NanoString nCounter gene expression system, which captures and counts individual mRNA transcripts. Advantages over existing platforms include direct measurement of mRNA expression levels without enzymatic reactions or bias, sensitivity coupled with high multiplex capability, and digital readout. Experiments performed on 509 human genes yielded a replicate correlation coefficient of 0.999, a detection limit between 0.1 fM and 0.5 fM, and a linear dynamic range of over 500-fold. Comparison of the NanoString nCounter gene expression system with microarrays and TaqMan PCR demonstrated that the nCounter system is more sensitive than microarrays and similar in sensitivity to real-time PCR. Finally, a comparison of transcript levels for 21 genes across seven samples measured by the nCounter system and SYBR Green real-time PCR demonstrated similar patterns of gene expression at all transcript levels.

All Inkjet-Printed Amperometric Multiplexed Biosensors Based on Nanostructured Conductive Hydrogel Electrodes.

PubMed

Li, Lanlan; Pan, Lijia; Ma, Zhong; Yan, Ke; Cheng, Wen; Shi, Yi; Yu, Guihua

2018-06-13

Multiplexing, one of the main trends in biosensors, aims to detect several analytes simultaneously by integrating miniature sensors on a chip. However, precisely depositing electrode materials and selective enzymes on distinct microelectrode arrays remains an obstacle to massively produced multiplexed sensors. Here, we report on a "drop-on-demand" inkjet printing process to fabricate multiplexed biosensors based on nanostructured conductive hydrogels in which the electrode material and several kinds of enzymes were printed on the electrode arrays one by one by employing a multinozzle inkjet system. The whole inkjet printing process can be finished within three rounds of printing and only one round of alignment. For a page of sensor arrays containing 96 working electrodes, the printing process took merely ∼5 min. The multiplexed assays can detect glucose, lactate, and triglycerides in real time with good selectivity and high sensitivity, and the results in phosphate buffer solutions and calibration serum samples are comparable. The inkjet printing process exhibited advantages of high efficiency and accuracy, which opens substantial possibilities for massive fabrication of integrated multiplexed biosensors for human health monitoring.

Evaluation of 793/B-like and Mass-like vaccine strain kinetics in experimental and field conditions by real-time RT-PCR quantification.

PubMed

Tucciarone, C M; Franzo, G; Berto, G; Drigo, M; Ramon, G; Koutoulis, K C; Catelli, E; Cecchinato, M

2018-01-01

Infectious bronchitis virus (IBV) is a great economic burden both for productive losses and costs of the control strategies. Many different vaccination protocols are applied in the same region and even in consecutive cycles on the same farm in order to find the perfect balance between costs and benefits. In Northern Italy, the usual second vaccination is more and more often moved up to the chick's first d of life. The second strain administration together with the common Mass priming by spray at the hatchery allows saving money and time and reducing animal stress. The present work compared the different vaccine strains (Mass-like or B48, and 1/96) kinetics both in field conditions and in a 21-day-long experimental trial in broilers, monitoring the viral replication by upper respiratory tract swabbing and vaccine specific real time reverse transcription PCR (RT-PCR) quantification. In both field and experimental conditions, titers for all the vaccines showed an increasing trend in the first 2 wk and then a decrease, though still remaining detectable during the whole monitored period. IBV field strain and avian Metapneumovirus (aMPV) presence also was also investigated by RT-PCR and sequencing, and by multiplex real-time RT-PCR, respectively, revealing a consistency in the pathogen introduction timing at around 30 d, in correspondence with the vaccine titer's main decrease. These findings suggest the need for an accurate knowledge of live vaccine kinetics, whose replication can compete with the other pathogen one, providing additional protection to be added to what is conferred by the adaptive immune response. © 2017 Poultry Science Association Inc.

Presence of the vancomycin resistance gene cluster vanC1, vanXYc, and vanT in Enterococcus casseliflavus.

PubMed

Hölzel, Christina; Bauer, Johann; Stegherr, Eva-Maria; Schwaiger, Karin

2014-04-01

The three chromosomally located clustered genes vanC1, vanXYc, and vanT confer intrinsic resistance to vancomycin and are used for species identification of Enterococcus gallinarum. In this study, 28 strains belonging to the E. gallinarum/casseliflavus group isolated from cloacal swabs from laying hens were screened for the presence of vanC1. As confirmed by species-specific multiplex PCR, 11 vanC1-positive strains were identified as E. gallinarum. Surprisingly, one yellow pigmented strain, verified as E. casseliflavus by species-specific multiplex PCR, was also vanC1 positive; vanXYc and vanT were additionally detectable in this strain. To our knowledge, this is the first report of vanC1, vanXYc, and vanT in E. casseliflavus. The minimum inhibitory concentration of vancomycin was 4 mg/L. Real-time reverse transcription-PCR revealed that none of the clustered genes was expressed in this strain. Even if the genes seem not to be active, there is a certain risk that they will be transferred to other bacteria where they might be functionally expressed. Therefore, it may be advisable to expand the search for vanC1, vanXYc, and vanT from E. gallinarum to other (enterococcal) species. This study confirms that enterococci live up to their name as being reservoir bacteria and should therefore always be closely monitored.

Investigation of avian influenza infection in wild birds in Ismailia and Damietta cities, Egypt

PubMed Central

Fadel, Hanaa Mohamed; Afifi, Rabab

2017-01-01

Aim: This study was carried out to monitor avian influenza (AI) infection in wild birds in Egypt. Materials and Methods: A total of 135 wild birds were examined for the presence of H5, H7, and H9 hemagglutination inhibition antibodies. Organs and swab samples of 75 birds were screened by multiplex real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) to detect AI subtypes H5, H7, and H9 matrix genes. Results: The highest seropositive result was recorded in cattle egrets (90.9%) followed by crows (88.6%), semi-captive pigeons (44.8%), and moorhens (39.1%). In cattle egrets, semi-captive pigeons and moorhens, H5 antibodies predominated. In crows, H9 antibodies predominated. Multiple infections with two or three virus subtypes were highest in crows (6/39, 15.4%) followed by cattle egrets (3/30, 10%) and moorhens’ (1/9, 11.1%) positive samples. Multiplex RRT-PCR results revealed two positive samples in cattle egrets and moorhens. Conclusion: The results indicated high seropositive rates against AI virus subtypes H5 and H9 in the examined wild birds. Multiple infections with more than one AI virus (AIV) subtypes were detected in some birds. This requires a collaboration of efforts to monitor AIV infection in wild birds and implement suitable early intervention measures. PMID:28717324

Diagnostic accuracy of two multiplex real-time polymerase chain reaction assays for the diagnosis of meningitis in children in a resource-limited setting.

PubMed

Khumalo, Jermaine; Nicol, Mark; Hardie, Diana; Muloiwa, Rudzani; Mteshana, Phindile; Bamford, Colleen

2017-01-01

Accurate etiological diagnosis of meningitis is important, but difficult in resource-limited settings due to prior administration of antibiotics and lack of viral diagnostics. We aimed to develop and validate 2 real-time multiplex PCR (RT-PCR) assays for the detection of common causes of community-acquired bacterial and viral meningitis in South African children. We developed 2 multiplex RT- PCRs for detection of S. pneumoniae, N. meningitidis, H. influenzae, enteroviruses, mumps virus and herpes simplex virus. We tested residual CSF samples from children presenting to a local paediatric hospital over a one-year period, whose CSF showed an abnormal cell count. Results were compared with routine diagnostic tests and the final discharge diagnosis. We calculated accuracy of the bacterial RT-PCR assay compared to CSF culture and using World Health Organisation definitions of laboratory-confirmed bacterial meningitis. From 292 samples, bacterial DNA was detected in 12 (4.1%) and viral nucleic acids in 94 (32%). Compared to CSF culture, the sensitivity and specificity of the bacterial RT-PCR was 100% and 97.2% with complete agreement in organism identification. None of the cases positive by viral RT-PCR had a bacterial cause confirmed on CSF culture. Only 9/90 (10%) of patients diagnosed clinically as bacterial meningitis or partially treated bacterial meningitis tested positive with the bacterial RT-PCR. In this population the use of 2 multiplex RT-PCRs targeting 6 common pathogens gave promising results. If introduced into routine diagnostic testing, these multiplex RT-PCR assays would supplement other diagnostic tests, and have the potential to limit unnecessary antibiotic therapy and hospitalisation.

Diagnostic accuracy of two multiplex real-time polymerase chain reaction assays for the diagnosis of meningitis in children in a resource-limited setting

PubMed Central

Khumalo, Jermaine; Nicol, Mark; Hardie, Diana; Muloiwa, Rudzani; Mteshana, Phindile

2017-01-01

Introduction Accurate etiological diagnosis of meningitis is important, but difficult in resource-limited settings due to prior administration of antibiotics and lack of viral diagnostics. We aimed to develop and validate 2 real-time multiplex PCR (RT-PCR) assays for the detection of common causes of community-acquired bacterial and viral meningitis in South African children. Methods We developed 2 multiplex RT- PCRs for detection of S. pneumoniae, N. meningitidis, H. influenzae, enteroviruses, mumps virus and herpes simplex virus. We tested residual CSF samples from children presenting to a local paediatric hospital over a one-year period, whose CSF showed an abnormal cell count. Results were compared with routine diagnostic tests and the final discharge diagnosis. We calculated accuracy of the bacterial RT-PCR assay compared to CSF culture and using World Health Organisation definitions of laboratory-confirmed bacterial meningitis. Results From 292 samples, bacterial DNA was detected in 12 (4.1%) and viral nucleic acids in 94 (32%). Compared to CSF culture, the sensitivity and specificity of the bacterial RT-PCR was 100% and 97.2% with complete agreement in organism identification. None of the cases positive by viral RT-PCR had a bacterial cause confirmed on CSF culture. Only 9/90 (10%) of patients diagnosed clinically as bacterial meningitis or partially treated bacterial meningitis tested positive with the bacterial RT-PCR. Discussion In this population the use of 2 multiplex RT-PCRs targeting 6 common pathogens gave promising results. If introduced into routine diagnostic testing, these multiplex RT-PCR assays would supplement other diagnostic tests, and have the potential to limit unnecessary antibiotic therapy and hospitalisation. PMID:28346504

Preliminary study of visual effect of multiplex hologram

NASA Astrophysics Data System (ADS)

Fu, Huaiping; Xiong, Bingheng; Yang, Hong; Zhang, Xueguo

2004-06-01

The process of any movement of real object can be recorded and displayed by a multiplex holographic stereogram. An embossing multiplex holographic stereogram and a multiplex rainbow holographic stereogram have been made by us, the multiplex rainbow holographic stereogram reconstructs the dynamic 2D line drawing of speech organs, the embossing multiplex holographic stereogram reconstructs the process of an old man drinking water. In this paper, we studied the visual result of an embossing multiplex holographic stereogram made with 80 films of 2-D pictures. Forty-eight persons of aged from 13 to 67 were asked to see the hologram and then to answer some questions about the feeling of viewing. The results indicate that this kind of holograms could be accepted by human visual sense organ without any problem. This paper also discusses visual effect of the multiplex holography stereograms base on visual perceptual psychology. It is open out that the planar multiplex holograms can be recorded and present the movement of real animal and object. Not only have the human visual perceptual constancy for shape, just as that size, color, etc... but also have visual perceptual constancy for binocular parallax.

Evaluation of a shortened QIAsymphony DNA extraction protocol for stool samples using a multiplex real-time PCR for the detection of enteric pathogens.

PubMed

van Zanten, E; Wisselink, G J; Stoll, S; Alvarez, R; Kooistra-Smid, A M D

2011-02-01

A shortened DNA extraction protocol for the QIAsymphony SP was evaluated by quantitative and qualitative comparison of real-time PCR results of 150 co-extracted stool samples. The average ∆Cycle threshold value for positive pathogenic targets was 0.28 Ct. A consensus of 96.91%, with a correlation coefficient of 0.9880 was recorded. Copyright © 2010 Elsevier B.V. All rights reserved.

Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection.

PubMed

Hernández, Marta; Rodríguez-Lázaro, David; Esteve, Teresa; Prat, Salomé; Pla, Maria

2003-12-15

Commercialization of several genetically modified crops has been approved worldwide to date. Uniplex polymerase chain reaction (PCR)-based methods to identify these different insertion events have been developed, but their use in the analysis of all commercially available genetically modified organisms (GMOs) is becoming progressively insufficient. These methods require a large number of assays to detect all possible GMOs present in the sample and thereby the development of multiplex PCR systems using combined probes and primers targeted to sequences specific to various GMOs is needed for detection of this increasing number of GMOs. Here we report on the development of a multiplex real-time PCR suitable for multiple GMO identification, based on the intercalating dye SYBR Green I and the analysis of the melting curves of the amplified products. Using this method, different amplification products specific for Maximizer 176, Bt11, MON810, and GA21 maize and for GTS 40-3-2 soybean were obtained and identified by their specific Tm. We have combined amplification of these products in a number of multiplex reactions and show the suitability of the methods for identification of GMOs with a sensitivity of 0.1% in duplex reactions. The described methods offer an economic and simple alternative to real-time PCR systems based on sequence-specific probes (i.e., TaqMan chemistry). These methods can be used as selection tests and further optimized for uniplex GMO quantification.

Multiplex real-time PCR for the detection and quantification of latent and persistent viral genomes in cellular or plasma blood fractions.

PubMed

Compston, Lara Isobel; Sarkobie, Francis; Li, Chengyao; Candotti, Daniel; Opare-Sem, Ohene; Allain, Jean-Pierre

2008-07-01

In common with latent viruses such as herpesviruses, parvovirus B19, HBV and GBV-C are contained successfully by the immune response and persist in the host. When immune control breaks down, reactivation of both latent and persistent viruses occurs. Two multiplex assays were developed (B19, HBV, HHV-8), (EBV, CMV, VZV) for blood screening, and tested on blood donor samples from Ghana to determine baseline prevalence of viraemia in immunocompetent persons. Single-virus real-time quantitative PCR (qPCR) assays were optimised for viral load determination of positive initial screening. The qPCR method utilised was absolute quantification with external standards. Multiplex and single-virus qPCR assays had similar sensitivity, except for the B19 assay in which sensitivity was 100-fold lower. Assays were optimised for reproducibility and repeatability, with R(2) of 0.9 being obtained for most assays. With the exception of B19 and CMV, assays had 100% detection limit ranging between 10(1) and 10(2) copies, IU or arbitrary units under single-virus and multiplex assay conditions. The prevalence of viraemia was 1.6% HBV (0.8% DNA+/HBsAg-, 0.8% DNA+/HBsAg+), 0.8% parvovirus B19, and 3.3% GBV-C viraemia in the plasma fraction. The prevalence of four herpesviruses was 1.0% HHV-8, 0.85% CMV, and 8.3% EBV, and no detectable VZV viraemia.

Real-time ESI-MS of enzymatic conversion: impact of organic solvents and multiplexing.

PubMed

Scheerle, Romy K; Grassmann, Johanna; Letzel, Thomas

2012-01-01

Different enzymatic assays were characterized systematically by real-time electrospray ionization mass spectrometry (ESI-MS) in the presence of organic solvents as well as in multiplex approaches and in a combination of both. Typically, biological enzymatic reactions are studied in aqueous solutions, since most enzymes show their full activity solely in aqueous solutions. However, in recent years, the use of organic solvents in combination with enzymatic reactions has gained increasing interest due to biotechnological advantages in chemical synthesis, development of online coupled setups screening for enzyme regulatory compounds, advantages regarding mass spectrometric detection and others. In the current study, the influence of several common organic solvents (methanol, ethanol, isopropanol, acetone, acetonitrile) on enzymatic activity (hen egg white lysozyme, chitinase, α-chymotrypsin, elastase from human neutrophils and porcine pancreas, acetylcholinesterase) was tested. Moreover, multiplexing is a promising approach enabling fast and cost-efficient screening methods, e.g. for determination of inhibitors in complex mixtures or in the field of biomedical research. Although in multiplexed setups the enzymatic activity may be affected by the presence of other substrates and/or enzymes, the expected advantages possibly will predominate. To investigate those effects, we measured multiple enzymatic assays simultaneously. For all conducted measurements, the conversion rate of the substrate(s) was calculated, which reflects the enzymatic activity. The results provide an overview about the susceptibility of the selected enzymes towards diverse factors and a reference point for many applications in analytical chemistry and biotechnology.

Simultaneous detection of eight avian influenza A virus subtypes by multiplex reverse transcription-PCR using a GeXP analyser.

PubMed

Li, Meng; Xie, Zhixun; Xie, Zhiqin; Liu, Jiabo; Xie, Liji; Deng, Xianwen; Luo, Sisi; Fan, Qing; Huang, Li; Huang, Jiaoling; Zhang, Yanfang; Zeng, Tingting; Wang, Sheng

2018-04-18

Recent studies have demonstrated that at least eight subtypes of avian influenza virus (AIV) can infect humans, including H1, H2, H3, H5, H6, H7, H9 and H10. A GeXP analyser-based multiplex reverse transcription (RT)-PCR (GeXP-multiplex RT-PCR) assay was developed in our recent studies to simultaneously detect these eight AIV subtypes using the haemagglutinin (HA) gene. The assay consists of chimeric primer-based PCR amplification with fluorescent labelling and capillary electrophoresis separation. RNA was extracted from chick embryo allantoic fluid or liquid cultures of viral isolates. In addition, RNA synthesised via in vitro transcription was used to determine the specificity and sensitivity of the assay. After selecting the primer pairs, their concentrations and GeXP-multiplex RT-PCR conditions were optimised. The established GeXP-multiplex RT-PCR assay can detect as few as 100 copies of premixed RNA templates. In the present study, 120 clinical specimens collected from domestic poultry at live bird markets and from wild birds were used to evaluate the performance of the assay. The GeXP-multiplex RT-PCR assay specificity was the same as that of conventional RT-PCR. Thus, the GeXP-multiplex RT-PCR assay is a rapid and relatively high-throughput method for detecting and identifying eight AIV subtypes that may infect humans.

A new biosensor for noninvasive determination of fetal RHD status in maternal blood of RhD negative pregnant women.

PubMed

Dündar Yenilmez, Ebru; Kökbaş, Umut; Kartlaşmış, Kezban; Kayrın, Levent; Tuli, Abdullah

2018-01-01

Prenatal detection of the fetal RHD status can be useful in the management of RhD incompatibility to identify fetuses at risk of hemolytic disease. Hemolytic disease causes morbidity and mortality of the fetus in the neonatal period. The routine use of antenatal and postnatal anti-D prophylaxis has reduced the incidence of hemolytic disease of the fetus and newborn. This study describe the detection of fetal RhD antigens in blood of RhD negative pregnant women using a nanopolymer coated electrochemical biosensor for medical diagnosis. Cell free fetal DNA in maternal plasma was also used to genotyping fetal RHD status using multiplex real-time PCR. Twenty-six RhD negative pregnant women in different gestational ages were included in the study. RhD positive fetal antibodies detected with a developed biosensor in maternal blood of RhD negative mothers. The electrochemical measurements were performed on a PalmSens potentiostat, and corundum ceramic based screen printed gold electrode combined with the reference Ag/AgCl electrode, and the auxiliary Au/Pd (98/2%) electrode. Fetal RHD genotyping performed using fluorescence-based multiplex real-time PCR exons 5 and 7 of the RHD gene. The fetal RHD status of 26 RhD negative cases were detected 21 as RhD positive and 5 as RhD negative with electrochemical biosensor. Fetal RHD status confirmed with extracted fetal DNA in maternal plasma using multiplex real-time PCR RHD genotyping and by serological test after delivery. The new method for fetal RhD detection in early pregnancy is useful and can be carry out rapidly in clinical diagnosis. Using automated biosensors are reproducible, quick and results can be generated within a few minutes compared to noninvasive fetal RHD genotyping from maternal plasma with real-time PCR-based techniques. We suggest the biosensor techniques could become an alternative part of fetal RHD genotyping from maternal plasma as a prenatal screening in the management of RhD incompatibility.

Development of a multiplexing fingerprint and high wavenumber Raman spectroscopy technique for real-time in vivo tissue Raman measurements at endoscopy

NASA Astrophysics Data System (ADS)

Bergholt, Mads Sylvest; Zheng, Wei; Huang, Zhiwei

2013-03-01

We report on the development of a novel multiplexing Raman spectroscopy technique using a single laser light together with a volume phase holographic (VPH) grating that simultaneously acquires both fingerprint (FP) and high wavenumber (HW) tissue Raman spectra at endoscopy. We utilize a customized VPH dual-transmission grating, which disperses the incident Raman scattered light vertically onto two separate segments (i.e., -150 to 1950 cm-1 1750 to 3600 cm-1) of a charge-coupled device camera. We demonstrate that the multiplexing Raman technique can acquire high quality in vivo tissue Raman spectra ranging from 800 to 3600 cm-1 within 1.0 s with a spectral resolution of 3 to 6 cm-1 during clinical endoscopy. The rapid multiplexing Raman spectroscopy technique covering both FP and HW ranges developed in this work has potential for improving in vivo tissue diagnosis and characterization at endoscopy.

Multiplex nucleic acid sequence-based amplification for simultaneous detection of several enteric viruses in model ready-to-eat foods.

PubMed

Jean, Julie; D'Souza, Doris H; Jaykus, Lee-Ann

2004-11-01

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10(-1) reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 10(0) to 10(2) reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods.

Optimization of the segmented method for optical compression and multiplexing system

NASA Astrophysics Data System (ADS)

Al Falou, Ayman

2002-05-01

Because of the constant increasing demands of images exchange, and despite the ever increasing bandwidth of the networks, compression and multiplexing of images is becoming inseparable from their generation and display. For high resolution real time motion pictures, electronic performing of compression requires complex and time-consuming processing units. On the contrary, by its inherent bi-dimensional character, coherent optics is well fitted to perform such processes that are basically bi-dimensional data handling in the Fourier domain. Additionally, the main limiting factor that was the maximum frame rate is vanishing because of the recent improvement of spatial light modulator technology. The purpose of this communication is to benefit from recent optical correlation algorithms. The segmented filtering used to store multi-references in a given space bandwidth product optical filter can be applied to networks to compress and multiplex images in a given bandwidth channel.

Multiplexed plasmonic sensing based on small-dimension nanohole arrays and intensity interrogation

PubMed Central

Yang, Jiun-Chan; Ji, Jin; Hogle, James M.; Larson, Dale N.

2009-01-01

We performed multiplexed sensing on nanohole array devices to simultaneously obtain information on molecular absorption, scattering, and refractive-index change, which were distinguished by using different array structures with distinct optical behavior. Up to 25 arrays were fabricated within a 65 μm × 50 μm area to provide real-time information of the local surface environment. The performance of multiplexed sensing was examined by flowing NaCl, coomassie blue, bovine serum albumin, and liposome solutions that exhibit different visible light absorption / scattering properties and different refractive indices. Experimental artifacts from light source fluctuation, sample injections, and light scattering induced by aggregates in solutions were detected by monitoring superwavelength holes or nanohole arrays with different periodicity and hole diameters. PMID:19157848

The increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections.

PubMed

Gray, J; Coupland, L J

2014-01-01

On 14 January 2013, the US Food and Drug Administration (FDA) announced permission for a multiplex nucleic acid test, the xTAG® Gastrointestinal Pathogen Panel (GPP) (Luminex Corporation, USA), which simultaneously detects 11 common viral, bacterial and parasitic causes of infectious gastroenteritis, to be marketed in the USA. This announcement reflects the current move towards the development and commercialization of detection technologies based on nucleic acid amplification techniques for diagnosis of syndromic infections. We discuss the limitations and advantages of nucleic acid amplification techniques and the recent advances in Conformité Européene - in-vitro diagnostic (CE-IVD)-approved multiplex real-time PCR kits for the simultaneous detection of multiple targets within the clinical diagnostics market.

Development of VIS/NIR spectroscopic system for real-time prediction of fresh pork quality

NASA Astrophysics Data System (ADS)

Zhang, Haiyun; Peng, Yankun; Zhao, Songwei; Sasao, Akira

2013-05-01

Quality attributes of fresh meat will influence nutritional value and consumers' purchasing power. The aim of the research was to develop a prototype for real-time detection of quality in meat. It consisted of hardware system and software system. A VIS/NIR spectrograph in the range of 350 to 1100 nm was used to collect the spectral data. In order to acquire more potential information of the sample, optical fiber multiplexer was used. A conveyable and cylindrical device was designed and fabricated to hold optical fibers from multiplexer. High power halogen tungsten lamp was collected as the light source. The spectral data were obtained with the exposure time of 2.17ms from the surface of the sample by press down the trigger switch on the self-developed system. The system could automatically acquire, process, display and save the data. Moreover the quality could be predicted on-line. A total of 55 fresh pork samples were used to develop prediction model for real time detection. The spectral data were pretreated with standard normalized variant (SNV) and partial least squares regression (PLSR) was used to develop prediction model. The correlation coefficient and root mean square error of the validation set for water content and pH were 0.810, 0.653, and 0.803, 0.098 respectively. The research shows that the real-time non-destructive detection system based on VIS/NIR spectroscopy can be efficient to predict the quality of fresh meat.

Divergent Synthesis and Real-Time Biological Annotation of Optically Active Tetrahydrocyclopenta[c]pyranone Derivatives

PubMed Central

2016-01-01

Sp3-rich compounds are underrepresented in libraries for probe- and drug-discovery, despite their promise of extending the range of accessible molecular shapes beyond planar geometries. With this in mind, a collection of single-enantiomer bicyclic, fused cyclopentenones underpinned by a complexity-generating Pauson–Khand cyclization was synthesized. A fingerprint of biological actions of these compounds was determined immediately after synthesis using real-time annotation−a process relying on multiplexed measurements of alterations in cell morphological features. PMID:27978655

Simultaneous Detection of Four Foodborne Viruses in Food Samples Using a One-Step Multiplex Reverse Transcription PCR.

PubMed

Lee, Shin-Young; Kim, Mi-Ju; Kim, Hyun-Joong; Jeong, KwangCheol Casey; Kim, Hae-Yeong

2018-02-28

A one-step multiplex reverse transcription PCR (RT-PCR) method comprising six primer sets (for the detection of norovirus GI and GII, hepatitis A virus, rotavirus, and astrovirus) was developed to simultaneously detect four kinds of pathogenic viruses. The size of the PCR products for norovirus GI and GII, hepatitis A virus (VP3/VP1 and P2A regions), rotavirus, and astrovirus were 330, 164, 244, 198, 629, and 449 bp, respectively. The RT-PCR with the six primer sets showed specificity for the pathogenic viruses. The detection limit of the developed multiplex RT-PCR, as evaluated using serially diluted viral RNAs, was comparable to that of one-step single RT-PCR. Moreover, this multiplex RT-PCR was evaluated using food samples such as water, oysters, lettuce, and vegetable product. These food samples were artificially spiked with the four kinds of viruses in diverse combinations, and the spiked viruses in all food samples were detected successfully.

A robust and scalable neuromorphic communication system by combining synaptic time multiplexing and MIMO-OFDM.

PubMed

Srinivasa, Narayan; Zhang, Deying; Grigorian, Beayna

2014-03-01

This paper describes a novel architecture for enabling robust and efficient neuromorphic communication. The architecture combines two concepts: 1) synaptic time multiplexing (STM) that trades space for speed of processing to create an intragroup communication approach that is firing rate independent and offers more flexibility in connectivity than cross-bar architectures and 2) a wired multiple input multiple output (MIMO) communication with orthogonal frequency division multiplexing (OFDM) techniques to enable a robust and efficient intergroup communication for neuromorphic systems. The MIMO-OFDM concept for the proposed architecture was analyzed by simulating large-scale spiking neural network architecture. Analysis shows that the neuromorphic system with MIMO-OFDM exhibits robust and efficient communication while operating in real time with a high bit rate. Through combining STM with MIMO-OFDM techniques, the resulting system offers a flexible and scalable connectivity as well as a power and area efficient solution for the implementation of very large-scale spiking neural architectures in hardware.

Effect of Shot Noise on Simultaneous Sensing in Frequency Division Multiplexed Diffuse Optical Tomographic Imaging Process.

PubMed

Jang, Hansol; Lim, Gukbin; Hong, Keum-Shik; Cho, Jaedu; Gulsen, Gultekin; Kim, Chang-Seok

2017-11-28

Diffuse optical tomography (DOT) has been studied for use in the detection of breast cancer, cerebral oxygenation, and cognitive brain signals. As optical imaging studies have increased significantly, acquiring imaging data in real time has become increasingly important. We have developed frequency-division multiplexing (FDM) DOT systems to analyze their performance with respect to acquisition time and imaging quality, in comparison with the conventional time-division multiplexing (TDM) DOT. A large tomographic area of a cylindrical phantom 60 mm in diameter could be successfully reconstructed using both TDM DOT and FDM DOT systems. In our experiment with 6 source-detector (S-D) pairs, the TDM DOT and FDM DOT systems required 6.18 and 1 s, respectively, to obtain a single tomographic data set. While the absorption coefficient of the reconstruction image was underestimated in the case of the FDM DOT, we experimentally confirmed that the abnormal region can be clearly distinguished from the background phantom using both methods.

Simultaneous detection of four garlic viruses by multiplex reverse transcription PCR and their distribution in Indian garlic accessions.

PubMed

Majumder, S; Baranwal, V K

2014-06-01

Indian garlic is infected with Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and allexiviruses. Identity and distribution of garlic viruses in various garlic accessions from different geographical regions of India were investigated. OYDV and allexiviruses were observed in all the garlic accessions, while SLV and GarCLV were observed only in a few accessions. A multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection and identification of OYDV, SLV, GarCLV and Allexivirus infecting garlic accessions in India. This multiplex protocol standardized in this study will be useful in indexing of garlic viruses and production of virus free seed material. Copyright © 2014 Elsevier B.V. All rights reserved.

Measuring and modeling correlations in multiplex networks.

PubMed

Nicosia, Vincenzo; Latora, Vito

2015-09-01

The interactions among the elementary components of many complex systems can be qualitatively different. Such systems are therefore naturally described in terms of multiplex or multilayer networks, i.e., networks where each layer stands for a different type of interaction between the same set of nodes. There is today a growing interest in understanding when and why a description in terms of a multiplex network is necessary and more informative than a single-layer projection. Here we contribute to this debate by presenting a comprehensive study of correlations in multiplex networks. Correlations in node properties, especially degree-degree correlations, have been thoroughly studied in single-layer networks. Here we extend this idea to investigate and characterize correlations between the different layers of a multiplex network. Such correlations are intrinsically multiplex, and we first study them empirically by constructing and analyzing several multiplex networks from the real world. In particular, we introduce various measures to characterize correlations in the activity of the nodes and in their degree at the different layers and between activities and degrees. We show that real-world networks exhibit indeed nontrivial multiplex correlations. For instance, we find cases where two layers of the same multiplex network are positively correlated in terms of node degrees, while other two layers are negatively correlated. We then focus on constructing synthetic multiplex networks, proposing a series of models to reproduce the correlations observed empirically and/or to assess their relevance.

Toward Precision Medicine: A Cancer Molecular Subtyping Nano-Strategy for RNA Biomarkers in Tumor and Urine.

PubMed

Koo, Kevin M; Wee, Eugene J H; Mainwaring, Paul N; Wang, Yuling; Trau, Matt

2016-12-01

Cancer is a heterogeneous disease which manifests as different molecular subtypes due to the complex nature of tumor initiation, progression, and metastasis. The concept of precision medicine aims to exploit this cancer heterogeneity by incorporating diagnostic technology to characterize each cancer patient's molecular subtype for tailored treatments. To characterize cancer molecular subtypes accurately, a suite of multiplexed bioassays have currently been developed to detect multiple oncogenic biomarkers. Despite the reliability of current multiplexed detection techniques, novel strategies are still needed to resolve limitations such as long assay time, complex protocols, and difficulty in interpreting broad overlapping spectral peaks of conventional fluorescence readouts. Herein a rapid (80 min) multiplexed platform strategy for subtyping prostate cancer tumor and urine samples based on their RNA biomarker profiles is presented. This is achieved by combining rapid multiplexed isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) of target RNA biomarkers with surface-enhanced Raman spectroscopy (SERS) nanotags for "one-pot" readout. This is the first translational application of a RT-RPA/SERS-based platform for multiplexed cancer biomarker detection to address a clinical need. With excellent sensitivity of 200 zmol (100 copies) and specificity, we believed that this platform methodology could be a useful tool for rapid multiplexed subtyping of cancers. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Paradigm for diagnosing mycobacterial disease: direct detection and differentiation of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in clinical specimens using multiplex real-time PCR.

PubMed

Kim, Jeong-Uk; Ryu, Dae-Shick; Cha, Choong-Hwan; Park, Seon-Hee

2018-03-20

Mycobacterium tuberculosis and non-tuberculous mycobacteria (NTM) are clinically different, and the rapid detection and differentiation of M. tuberculosis complex (MTBC) and NTM is crucial for patient management and infection control. Given the slow growth of most pathogenic mycobacteria, nucleic acid amplification assays are excellent tools for direct identification of mycobacteria in clinical specimens. Recently, a multiplex real-time PCR assay was developed that can directly detect 20 mycobacterial species in clinical specimens. Here, we evaluated the diagnostic performance of the assay for diagnosing mycobacterial disease under routine laboratory conditions. A total of 3334 specimens collected from 1437 patients suspected of tuberculosis infection were subjected to acid-fast bacilli staining, conventional culture and the multiplex real-time PCR assay. To evaluate the sensitivity and specificity of the assay, the overall diagnosis of tuberculosis was defined by positive culture plus medical history, and the 2007 American Thoracic Society and Infectious Disease Society of America diagnostic criteria for NTM disease were applied. The sensitivity, specificity, positive predictive value and negative predictive value were 87.5%, 99.6%, 96.1% and 98.5%, respectively, for the detection of MTBC isolates and 53.3%, 99.9%, 95.2%, and 98.9%, respectively, for detecting NTM isolates. Thus, the assay can correctly differentiate between MTBC and NTM isolates in clinical specimens and would be a useful tool for the rapid differentiation of tuberculosis and NTM disease, despite its limited sensitivity for the diagnosis of NTM disease. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

A novel multiplex real-time PCR assay for the concurrent detection of hepatitis A, B and C viruses in patients with acute hepatitis.

PubMed

Park, Yongjung; Kim, Beom Seok; Choi, Kyu Hun; Shin, Dong Ho; Lee, Mi Jung; Cho, Yonggeun; Kim, Hyon-Suk

2012-01-01

A novel multiplex real-time PCR assay for concurrent detection of hepatitis viruses was evaluated for its clinical performance in screening patients with acute hepatitis. A total of 648 serum samples were collected from patients with acute symptoms of hepatitis. Concurrent detection of nucleic acids of HAV, HBV and HCV was performed using the Magicplex™ HepaTrio Real-time Detection test. Serum nucleic acid levels of HBV and HCV were also quantified by the Cobas® AmpliPrep/Cobas® TaqMan® (CAP/CTM) HBV and HCV tests. Patients' medical records were also reviewed. Concordance rates between the results from the HepaTrio and the CAP/CTM tests for the detection of HBV and HCV were 94.9% (k = 0.88) and 99.2% (k = 0.98), respectively. The cycle threshold values with the HepaTrio test were also correlated well with the levels of HBV DNA (r = -0.9230) and HCV RNA (r = -0.8458). The sensitivity and specificity of the HepaTrio test were 93.8% and 98.2%, respectively, for detecting HBV infection, and 99.1% and 100.0%, respectively, for HCV infection. For the HepaTrio test, 21 (3.2%) cases were positive for both HBV and HCV. Among the positive cases, 6 (0.9%) were true coinfections. This test also detected 18 (2.8%) HAV positives. The HepaTrio test demonstrated good clinical performance and produced results that agreed well with those of the CAP/CTM assays, especially for the detection of HCV. This assay was also able to detect HAV RNA from anti-HAV IgM-positive individuals. Therefore, this new multiplex PCR assay could be useful for the concurrent detection of the three hepatitis viruses.

Evaluation of a multiplex real-time PCR for detection of four bacterial agents commonly associated with bovine respiratory disease in bronchoalveolar lavage fluid.

PubMed

Wisselink, Henk J; Cornelissen, Jan B W J; van der Wal, Fimme J; Kooi, Engbert A; Koene, Miriam G; Bossers, Alex; Smid, Bregtje; de Bree, Freddy M; Antonis, Adriaan F G

2017-07-13

Pasteurella multocida, Mannheimia haemolytica, Histophilus somni and Trueperella pyogenes are four bacterial agents commonly associated with bovine respiratory disease (BRD). In this study a bacterial multiplex real-time PCR (the RespoCheck PCR) was evaluated for the detection in bronchoalveolar lavage fluid (BALF) of these four bacterial agents. The analytical sensitivity of the multiplex real-time PCR assay determined on purified DNA and on bacterial cells of the four target pathogens was one to ten fg DNA/assay and 4 × 10 -1 to 2 × 10 0  CFU/assay. The analytical specificity of the test was, as evaluated on a collection of 118 bacterial isolates, 98.3% for M. haemolytica and 100% for the other three target bacteria. A set of 160 BALF samples of calves originating from ten different herds with health problems related to BRD was examined with bacteriological methods and with the RespoCheck PCR. Using bacteriological examination as the gold standard, the diagnostic sensitivities and specificities of the four bacterial agents were respectively between 0.72 and 1.00 and between 0.70 and 0.99. Kappa values for agreement between results of bacteriological examination and PCRs were low for H. somni (0.17), moderate for P. multocida (0.52) and M. haemolytica (0.57), and good for T. pyogenes (0.79). The low and moderate kappa values seemed to be related to limitations of the bacteriological examination, this was especially the case for H. somni. It was concluded that the RespoCheck PCR assay is a valuable diagnostic tool for the simultaneous detection of the four bacterial agents in BALF of calves.

Evaluation of the new AmpliSens multiplex real-time PCR assay for simultaneous detection of Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, and Trichomonas vaginalis.

PubMed

Rumyantseva, Tatiana; Golparian, Daniel; Nilsson, Christian S; Johansson, Emma; Falk, My; Fredlund, Hans; Van Dam, Alje; Guschin, Alexander; Unemo, Magnus

2015-10-01

In this study, we performed an evaluation of the new CE-marked multiplex real-time AmpliSens N.gonorrhoeae/C.trachomatis/M.genitalium/T.vaginalis-MULTIPRIME-FRT PCR assay compared to APTIMA tests, i.e., APTIMA COMBO 2 assay, APTIMA Trichomonas vaginalis assay (FDA-approved), and two different APTIMA Mycoplasma genitalium assays (research use only; one of them only used for discrepancy analysis). Vaginal swabs (n = 209) and first-void urine (FVU) specimens from females (n = 498) and males (n = 554), consecutive attendees (n = 1261) at a dermatovenerological clinic in Sweden, were examined. The sensitivity of the AmpliSens PCR assay for detection of C. trachomatis (6.3% prevalence), M. genitalium (5.7% prevalence), N. gonorrhoeae (0.3% prevalence), and T. vaginalis (0.08% prevalence) was 97.5% (95% confidence interval (CI): 91.2-99.6%), 81.9% (95% CI: 70.7-89.7%), 100% (95% CI: 40.2-100%) and 100% (95% CI: 16.5-100%), respectively. The specificity of the AmpliSens PCR assay was 100% (95% CI: 99.6-100%) for all agents. The analytical sensitivity and specificity for N. gonorrhoeae detection was excellent, i.e., 55 international gonococcal strains detected and 135 isolates of 13 non-gonococcal Neisseria species were negative. In conclusion, the multiplex real-time AmpliSens N.gonorrhoeae/C.trachomatis/M.genitalium/T.vaginalis-MULTIPRIME-FRT PCR assay demonstrated high sensitivity and excellent specificity for the detection of C. trachomatis, N. gonorrhoeae, and T. vaginalis, and excellent specificity but suboptimal sensitivity for M. genitalium detection. © 2015 APMIS. Published by John Wiley & Sons Ltd.

Implementation and Evaluation of a Fully Automated Multiplex Real-Time PCR Assay on the BD Max Platform to Detect and Differentiate Herpesviridae from Cerebrospinal Fluids

PubMed Central

Köller, Thomas; Kurze, Daniel; Lange, Mirjam; Scherdin, Martin; Podbielski, Andreas; Warnke, Philipp

2016-01-01

A fully automated multiplex real-time PCR assay—including a sample process control and a plasmid based positive control—for the detection and differentiation of herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2) and varicella-zoster virus (VZV) from cerebrospinal fluids (CSF) was developed on the BD Max platform. Performance was compared to an established accredited multiplex real time PCR protocol utilizing the easyMAG and the LightCycler 480/II, both very common devices in viral molecular diagnostics. For clinical validation, 123 CSF specimens and 40 reference samples from national interlaboratory comparisons were examined with both methods, resulting in 97.6% and 100% concordance for CSF and reference samples, respectively. Utilizing the BD Max platform revealed sensitivities of 173 (CI 95%, 88–258) copies/ml for HSV1, 171 (CI 95%, 148–194) copies/ml for HSV2 and 84 (CI 95%, 5–163) copies/ml for VZV. Cross reactivity could be excluded by checking 25 common viral, bacterial and fungal human pathogens. Workflow analyses displayed shorter test duration as well as remarkable fewer and easier preparation steps with the potential to reduce error rates occurring when manually assessing patient samples. This protocol allows for a fully automated PCR assay on the BD Max platform for the simultaneously detection of herpesviridae from CSF specimens. Singular or multiple infections due to HSV1, HSV2 and VZV can reliably be differentiated with good sensitivities. Control parameters are included within the assay, thereby rendering its suitability for current quality management requirements. PMID:27092772

High-Resolution Melting Analysis as a Powerful Tool to Discriminate and Genotype Pseudomonas savastanoi Pathovars and Strains

PubMed Central

Gori, Andrea; Cerboneschi, Matteo; Tegli, Stefania

2012-01-01

Pseudomonas savastanoi is a serious pathogen of Olive, Oleander, Ash, and several other Oleaceae. Its epiphytic or endophytic presence in asymptomatic plants is crucial for the spread of Olive and Oleander knot disease, as already ascertained for P. savastanoi pv. savastanoi (Psv) on Olive and for pv. nerii (Psn) on Oleander, while no information is available for pv. fraxini (Psf) on Ash. Nothing is known yet about the distribution on the different host plants and the real host range of these pathovars in nature, although cross-infections were observed following artificial inoculations. A multiplex Real-Time PCR assay was recently developed to simultaneously and quantitatively discriminate in vitro and in planta these P. savastanoi pathovars, for routine culture confirmation and for epidemiological and diagnostical studies. Here an innovative High-Resolution Melting Analysis (HRMA)-based assay was set up to unequivocally discriminate Psv, Psn and Psf, according to several single nucleotide polymorphisms found in their Type Three Secretion System clusters. The genetic distances among 56 P. savastanoi strains belonging to these pathovars were also evaluated, confirming and refining data previously obtained by fAFLP. To our knowledge, this is the first time that HRMA is applied to a bacterial plant pathogen, and one of the few multiplex HRMA-based assays developed so far. This protocol provides a rapid, sensitive, specific tool to differentiate and detect Psv, Psn and Psf strains, also in vivo and against other related bacteria, with lower costs than conventional multiplex Real-Time PCR. Its application is particularly suitable for sanitary certification programs for P. savastanoi, aimed at avoiding the spreading of this phytopathogen through asymptomatic plants. PMID:22295075

Development of a qualitative, multiplex real-time PCR kit for screening of genetically modified organisms (GMOs).

PubMed

Dörries, Hans-Henno; Remus, Ivonne; Grönewald, Astrid; Grönewald, Cordt; Berghof-Jäger, Kornelia

2010-03-01

The number of commercially available genetically modified organisms (GMOs) and therefore the diversity of possible target sequences for molecular detection techniques are constantly increasing. As a result, GMO laboratories and the food production industry currently are forced to apply many different methods to reliably test raw material and complex processed food products. Screening methods have become more and more relevant to minimize the analytical effort and to make a preselection for further analysis (e.g., specific identification or quantification of the GMO). A multiplex real-time PCR kit was developed to detect the 35S promoter of the cauliflower mosaic virus, the terminator of the nopaline synthase gene of Agrobacterium tumefaciens, the 35S promoter from the figwort mosaic virus, and the bar gene of the soil bacterium Streptomyces hygroscopicus as the most widely used sequences in GMOs. The kit contains a second assay for the detection of plant-derived DNA to control the quality of the often processed and refined sample material. Additionally, the plant-specific assay comprises a homologous internal amplification control for inhibition control. The determined limits of detection for the five assays were 10 target copies/reaction. No amplification products were observed with DNAs of 26 bacterial species, 25 yeasts, 13 molds, and 41 not genetically modified plants. The specificity of the assays was further demonstrated to be 100% by the specific amplification of DNA derived from reference material from 22 genetically modified crops. The applicability of the kit in routine laboratory use was verified by testing of 50 spiked and unspiked food products. The herein described kit represents a simple and sensitive GMO screening method for the reliable detection of multiple GMO-specific target sequences in a multiplex real-time PCR reaction.

Rapid Detection of Chlamydia trachomatis and Typing of the Lymphogranuloma venereum associated L-Serovars by TaqMan PCR

PubMed Central

Schaeffer, Anke; Henrich, Birgit

2008-01-01

Background Infection due to Chlamydia trachomatis is the most common sexually transmitted bacterial disease of global health significance, and especially the L-serovars causing lymphogranuloma venereum are increasingly being found in Europe in men who have sex with men. Results The design and evaluation of a rapid, multiplex, real-time PCR targeting the major outer membrane protein (omp-1) -gene and a L-serovar-specific region of the polymorphic protein H (pmp-H) -gene for the detection of Chlamydia trachomatis is reported here. The PCR takes place as a single reaction with an internal control. For L1-, L2- and L3-serovar differentiation a second set of real-time PCRs was evaluated based on the amplification of serovar-specific omp-1-regions. The detection limit of each real-time PCR, multiplexed or not, was 50 genome copies per reaction with an efficiency ranging from 90,5–95,2%. In a retrospective analysis of 50 ocular, rectal and urogenital specimens formerly tested to be positive for C. trachomatis we identified six L2-serovars in rectal specimens of HIV-positive men, one in a double-infection with L3, and one L2 in a urethral specimen of an HIV-negative male. Conclusion This unique real-time PCR is specific and convenient for the rapid routine-diagnostic detection of lymphogranuloma venereum-associated L-serovars and enables the subsequent differentiation of L1, L2 and L3 for epidemiologic studies. PMID:18447917

Multiplex, Rapid, and Sensitive Isothermal Detection of Nucleic-Acid Sequence by Endonuclease Restriction-Mediated Real-Time Multiple Cross Displacement Amplification.

PubMed

Wang, Yi; Wang, Yan; Zhang, Lu; Liu, Dongxin; Luo, Lijuan; Li, Hua; Cao, Xiaolong; Liu, Kai; Xu, Jianguo; Ye, Changyun

2016-01-01

We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA), which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5' end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labeled at the 5' end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 min, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here, we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism.

Pilot-multiplexed continuous-variable quantum key distribution with a real local oscillator

NASA Astrophysics Data System (ADS)

Wang, Tao; Huang, Peng; Zhou, Yingming; Liu, Weiqi; Zeng, Guihua

2018-01-01

We propose a pilot-multiplexed continuous-variable quantum key distribution (CVQKD) scheme based on a local local oscillator (LLO). Our scheme utilizes time-multiplexing and polarization-multiplexing techniques to dramatically isolate the quantum signal from the pilot, employs two heterodyne detectors to separately detect the signal and the pilot, and adopts a phase compensation method to almost eliminate the multifrequency phase jitter. In order to analyze the performance of our scheme, a general LLO noise model is constructed. Besides the phase noise and the modulation noise, the photon-leakage noise from the reference path and the quantization noise due to the analog-to-digital converter (ADC) are also considered, which are first analyzed in the LLO regime. Under such general noise model, our scheme has a higher key rate and longer secure distance compared with the preexisting LLO schemes. Moreover, we also conduct an experiment to verify our pilot-multiplexed scheme. Results show that it maintains a low level of the phase noise and is expected to obtain a 554-Kbps secure key rate within a 15-km distance under the finite-size effect.

Multiplex Nucleic Acid Sequence-Based Amplification for Simultaneous Detection of Several Enteric Viruses in Model Ready-To-Eat Foods†

PubMed Central

Jean, Julie; D'Souza, Doris H.; Jaykus, Lee-Ann

2004-01-01

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10−1 reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 100 to 102 reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods. PMID:15528524

High-throughput multiplexed T-cell-receptor excision circle quantitative PCR assay with internal controls for detection of severe combined immunodeficiency in population-based newborn screening.

PubMed

Gerstel-Thompson, Jacalyn L; Wilkey, Jonathan F; Baptiste, Jennifer C; Navas, Jennifer S; Pai, Sung-Yun; Pass, Kenneth A; Eaton, Roger B; Comeau, Anne Marie

2010-09-01

Real-time quantitative PCR (qPCR) targeting a specific marker of functional T cells, the T-cell-receptor excision circle (TREC), detects the absence of functional T cells and has a demonstrated clinical validity for detecting severe combined immunodeficiency (SCID) in infants. There is need for a qPCR TREC assay with an internal control to monitor DNA quality and the relative cellular content of the particular dried blood spot punch sampled in each reaction. The utility of the qPCR TREC assay would also be far improved if more tests could be performed on the same newborn screening sample. We approached the multiplexing of qPCR for TREC by attenuating the reaction for the reference gene, with focus on maintaining tight quality assurance for reproducible slopes and for prevention of sample-to-sample cross contamination. Statewide newborn screening for SCID using the multiplexed assay was implemented, and quality-assurance data were recorded. The multiplex qPCR TREC assay showed nearly 100% amplification efficiency for each of the TREC and reference sequences, clinical validity for multiple forms of SCID, and an analytic limit of detection consistent with prevention of contamination. The eluate and residual ghost from a 3.2-mm dried blood spot could be used as source material for multiplexed immunoassays and multiplexed DNA tests (Multiplex Plus), with no disruption to the multiplex TREC qPCR. Population-based SCID newborn screening programs should consider multiplexing for quality assurance purposes. Potential benefits of using Multiplex Plus include the ability to perform multianalyte profiling.

Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus.

PubMed

Mari, Viviana; Losurdo, Michele; Lucente, Maria Stella; Lorusso, Eleonora; Elia, Gabriella; Martella, Vito; Patruno, Giovanni; Buonavoglia, Domenico; Decaro, Nicola

2016-03-01

HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 10(0)-10(1) viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Development and evaluation of a Quadruplex Taq Man real-time PCR assay for simultaneous detection of clinical isolates of Enterococcus faecalis, Enterococcus faecium and their vanA and vanB genotypes.

PubMed

Naserpour Farivar, Taghi; Najafipour, Reza; Johari, Pouran; Aslanimehr, Masoumeh; Peymani, Amir; Jahani Hashemi, Hoasan; Mirzaui, Baman

2014-10-01

We developed and evaluated the utility of a quadruplex Taqman real-time PCR assay that allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci. The specificity of the assay was tested using reference strains of vancomycin-resistant and susceptible enterococci. In total, 193 clinical isolates were identified and subsequently genotyped using a Quadruplex Taqman real-time PCR assay and melting curve analysis. Representative Quadruplex Taqman real-time PCR amplification curve were obtained for Enterococcus faecium, Enterococcus faecalis, vanA-containing E. faecium, vanB-containing E. faecalis. Phenotypic and genotypic analysis of the isolates gave same results for 82 enterococcal isolates, while in 5 isolates, they were inconsistent. We had three mixed strains, which were detected by the TaqMan real-time PCR assay and could not be identified correctly using phenotypic methods. Vancomycin resistant enterococci (VRE) genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using TaqMan real-time multiplex real-time PCR assay.

QUANTIFICATION OF ENTEROVIRUS AND HEPATITIS A VIRUSES IN WELLS AND SPRINGS IN EAST TENNESSEE USING REAL-TIME REVERSE TRANSCIPTION PCR

EPA Science Inventory

This project involves development, validation testing and application of a fast, efficient method of quantitatively measuring occurrence and concentration of common human viral pathogens, enterovirus and hepatitis A virus, in ground water samples using real-time reverse transcrip...

A multiplex PCR method for the identification of commercially important salmon and trout species (Oncorhynchus and Salmo) in North America.

PubMed

Rasmussen Hellberg, Rosalee S; Morrissey, Michael T; Hanner, Robert H

2010-09-01

The purpose of this study was to develop a species-specific multiplex polymerase chain reaction (PCR) method that allows for the detection of salmon species substitution on the commercial market. Species-specific primers and TaqMan® probes were developed based on a comprehensive collection of mitochondrial 5' cytochrome c oxidase subunit I (COI) deoxyribonucleic acid (DNA) "barcode" sequences. Primers and probes were combined into multiplex assays and tested for specificity against 112 reference samples representing 25 species. Sensitivity and linearity tests were conducted using 10-fold serial dilutions of target DNA (single-species samples) and DNA admixtures containing the target species at levels of 10%, 1.0%, and 0.1% mixed with a secondary species. The specificity tests showed positive signals for the target DNA in both real-time and conventional PCR systems. Nonspecific amplification in both systems was minimal; however, false positives were detected at low levels (1.2% to 8.3%) in conventional PCR. Detection levels were similar for admixtures and single-species samples based on a 30 PCR cycle cut-off, with limits of 0.25 to 2.5 ng (1% to 10%) in conventional PCR and 0.05 to 5.0 ng (0.1% to 10%) in real-time PCR. A small-scale test with food samples showed promising results, with species identification possible even in heavily processed food items. Overall, this study presents a rapid, specific, and sensitive method for salmon species identification that can be applied to mixed-species and heavily processed samples in either conventional or real-time PCR formats. This study provides a newly developed method for salmon and trout species identification that will assist both industry and regulatory agencies in the detection and prevention of species substitution. This multiplex PCR method allows for rapid, high-throughput species identification even in heavily processed and mixed-species samples. An inter-laboratory study is currently being carried out to assess the ability of this method to identify species in a variety of commercial salmon and trout products.

Surface plasmon resonance biosensors for highly sensitive detection in real samples

NASA Astrophysics Data System (ADS)

Sepúlveda, B.; Carrascosa, L. G.; Regatos, D.; Otte, M. A.; Fariña, D.; Lechuga, L. M.

2009-08-01

In this work we summarize the main results obtained with the portable surface plasmon resonance (SPR) device developed in our group (commercialised by SENSIA, SL, Spain), highlighting its applicability for the real-time detection of extremely low concentrations of toxic pesticides in environmental water samples. In addition, we show applications in clinical diagnosis as, on the one hand, the real-time and label-free detection of DNA hybridization and single point mutations at the gene BRCA-1, related to the predisposition in women to develop an inherited breast cancer and, on the other hand, the analysis of protein biomarkers in biological samples (urine, serum) for early detection of diseases. Despite the large number of applications already proven, the SPR technology has two main drawbacks: (i) not enough sensitivity for some specific applications (where pM-fM or single-molecule detection are needed) (ii) low multiplexing capabilities. In order solve such drawbacks, we work in several alternative configurations as the Magneto-optical Surface Plasmon Resonance sensor (MOSPR) based on a combination of magnetooptical and ferromagnetic materials, to improve the SPR sensitivity, or the Localized Surface Plasmon Resonance (LSPR) based on nanostructures (nanoparticles, nanoholes,...), for higher multiplexing capabilities.

Multiplexed Holographic Data Storage in Bacteriorhodopsin

NASA Technical Reports Server (NTRS)

Mehrl, David J.; Krile, Thomas F.

1997-01-01

High density optical data storage, driven by the information revolution, remains at the forefront of current research areas. Much of the current research has focused on photorefractive materials (SBN and LiNbO3) and polymers, despite various problems with expense, durability, response time and retention periods. Photon echo techniques, though promising, are questionable due to the need for cryogenic conditions. Bacteriorhodopsin (BR) films are an attractive alternative recording medium. Great strides have been made in refining BR, and materials with storage lifetimes as long as 100 days have recently become available. The ability to deposit this robust polycrystalline material as high quality optical films suggests the use of BR as a recording medium for commercial optical disks. Our own recent research has demonstrated the suitability of BR films for real time spatial filtering and holography. We propose to fully investigate the feasibility of performing holographic mass data storage in BR. Important aspects of the problem to be investigated include various data multiplexing techniques (e.g. angle- amplitude- and phase-encoded multiplexing, and in particular shift-multiplexing), multilayer recording techniques, SLM selection and data readout using crossed polarizers for noise rejection. Systems evaluations of storage parameters, including access times, memory refresh constraints, erasure, signal-to-noise ratios and bit error rates, will be included in our investigations.

QoS for Real Time Applications over Next Generation Data Networks

NASA Technical Reports Server (NTRS)

Ivancic, William; Atiquzzaman, Mohammed; Bai, Haowei; Su, Hongjun; Chitri, Jyotsna; Ahamed, Faruque

2001-01-01

Viewgraphs on Qualtity of Service (QOS) for real time applications over next generation data networks are presented. The progress to date include: Task 1: QoS in Integrated Services over DiffServ networks (UD); Task 2: Interconnecting ATN with the next generation Internet (UD); Task 3: QoS in DiffServ over ATM (UD); Task 4: Improving Explicit Congestion Notification with the Mark-Front Strategy (OSU); Task 5: Multiplexing VBR over VBR (OSU); and Task 6: Achieving QoS for TCP traffic in Satellite Networks with Differentiated Services (OSU).

Multiplex real-time PCR detection and differentiation of Colletotrichum species infecting soybean

USDA-ARS?s Scientific Manuscript database

Colletotrichum species are fungal plant pathogens of worldwide significance. We isolated Colletotrichum species from soybean [Glycine max (L.) Merr.] with anthracnose symptoms in the U.S. states of Alabama, Arkansas, Illinois, Mississippi, and North Dakota from 2009 to 2013. Thirty-five strains from...

Uplink Packet-Data Scheduling in DS-CDMA Systems

NASA Astrophysics Data System (ADS)

Choi, Young Woo; Kim, Seong-Lyun

In this letter, we consider the uplink packet scheduling for non-real-time data users in a DS-CDMA system. As an effort to jointly optimize throughput and fairness, we formulate a time-span minimization problem incorporating the time-multiplexing of different simultaneous transmission schemes. Based on simple rules, we propose efficient scheduling algorithms and compare them with the optimal solution obtained by linear programming.

Development of a multiplexing fingerprint and high wavenumber Raman spectroscopy technique for real-time in vivo tissue Raman measurements at endoscopy.

PubMed

Bergholt, Mads Sylvest; Zheng, Wei; Huang, Zhiwei

2013-03-01

We report on the development of a novel multiplexing Raman spectroscopy technique using a single laser light together with a volume phase holographic (VPH) grating that simultaneously acquires both fingerprint (FP) and high wavenumber (HW) tissue Raman spectra at endoscopy. We utilize a customized VPH dual-transmission grating, which disperses the incident Raman scattered light vertically onto two separate segments (i.e., -150 to 1950  cm⁻¹; 1750 to 3600  cm⁻¹) of a charge-coupled device camera. We demonstrate that the multiplexing Raman technique can acquire high quality in vivo tissue Raman spectra ranging from 800 to 3600  cm⁻¹ within 1.0 s with a spectral resolution of 3 to 6  cm⁻¹ during clinical endoscopy. The rapid multiplexing Raman spectroscopy technique covering both FP and HW ranges developed in this work has potential for improving in vivo tissue diagnosis and characterization at endoscopy.

SensiScreen® KRAS exon 2-sensitive simplex and multiplex real-time PCR-based assays for detection of KRAS exon 2 mutations

PubMed Central

Guldmann-Christensen, Mariann; Hauge Kyneb, Majbritt; Voogd, Kirsten; Andersen, Christina; Epistolio, Samantha; Merlo, Elisabetta; Yding Wolff, Tine; Hamilton-Dutoit, Stephen; Lorenzen, Jan; Christensen, Ulf Bech

2017-01-01

Activating mutations in codon 12 and codon 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) gene are implicated in the development of several human cancer types and influence their clinical evaluation, treatment and prognosis. Numerous different methods for KRAS genotyping are currently available displaying a wide range of sensitivities, time to answer and requirements for laboratory equipment and user skills. Here we present SensiScreen® KRAS exon 2 simplex and multiplex CE IVD assays, that use a novel real-time PCR-based method for KRAS mutation detection based on PentaBase’s proprietary DNA analogue technology and designed to work on standard real-time PCR instruments. By means of the included BaseBlocker™ technology, we show that SensiScreen® specifically amplifies the mutated alleles of interest with no or highly subdued amplification of the wild type allele. Furthermore, serial dilutions of mutant DNA in a wild type background demonstrate that all SensiScreen® assays display a limit of detection that falls within the range of 0.25–1%. Finally, in three different colorectal cancer patient populations, SensiScreen® assays confirmed the KRAS genotype previously determined by commonly used methods for KRAS mutation testing, and notably, in two of the populations, SensiScreen® identified additional mutant positive cases not detected by common methods. PMID:28636636

Nano metal-organic framework (NMOF)-based strategies for multiplexed microRNA detection in solution and living cancer cells

NASA Astrophysics Data System (ADS)

Wu, Yafeng; Han, Jianyu; Xue, Peng; Xu, Rong; Kang, Yuejun

2015-01-01

MiRNAs are an emerging type of biomarker for diagnostics and prognostics. A reliable sensing strategy that can monitor miRNA expression in living cancer cells would be critical in view of its extensive advantages for fundamental research related to miRNA-associated bioprocesses and biomedical applications. Conventional miRNA sensing methods include northern blot, microarrays and real-time quantitative PCR. However, none of them is able to monitor miRNA levels expressed in living cancer cells in a real-time fashion. Some fluorescennt biosensors developed recently from carbon nanomaterials, such as single-walled carbon nanotubes (SWNTs), graphene oxide (GO), and carbon nanoparticles, have been successfully used for assaying miRNA in vitro; however the preparation processes are often expensive, complicated and time-consuming, which have motivated the research on other substitute and novel materials. Herein we present a novel sensing strategy based on peptide nucleic acid (PNA) probes labeled with fluorophores and conjugated with an NMOF vehicle to monitor multiplexed miRNAs in living cancer cells. The NMOF works as a fluorescence quencher of the labelled PNA that is firmly bound with the metal center. In the presence of a target miRNA, PNA is hybridized and released from the NMOF leading to the recovery of fluorescence. This miRNA sensor not only enables the quantitative and highly specific detection of multiplexed miRNAs in living cancer cells, but it also allows the precise and in situ monitoring of the spatiotemporal changes of miRNA expression.MiRNAs are an emerging type of biomarker for diagnostics and prognostics. A reliable sensing strategy that can monitor miRNA expression in living cancer cells would be critical in view of its extensive advantages for fundamental research related to miRNA-associated bioprocesses and biomedical applications. Conventional miRNA sensing methods include northern blot, microarrays and real-time quantitative PCR. However, none of them is able to monitor miRNA levels expressed in living cancer cells in a real-time fashion. Some fluorescennt biosensors developed recently from carbon nanomaterials, such as single-walled carbon nanotubes (SWNTs), graphene oxide (GO), and carbon nanoparticles, have been successfully used for assaying miRNA in vitro; however the preparation processes are often expensive, complicated and time-consuming, which have motivated the research on other substitute and novel materials. Herein we present a novel sensing strategy based on peptide nucleic acid (PNA) probes labeled with fluorophores and conjugated with an NMOF vehicle to monitor multiplexed miRNAs in living cancer cells. The NMOF works as a fluorescence quencher of the labelled PNA that is firmly bound with the metal center. In the presence of a target miRNA, PNA is hybridized and released from the NMOF leading to the recovery of fluorescence. This miRNA sensor not only enables the quantitative and highly specific detection of multiplexed miRNAs in living cancer cells, but it also allows the precise and in situ monitoring of the spatiotemporal changes of miRNA expression. Electronic supplementary information (ESI) available: Extra figures and tables. See DOI: 10.1039/c4nr05447d

Real-time PCR Demonstrates Ancylostoma duodenale Is a Key Factor in the Etiology of Severe Anemia and Iron Deficiency in Malawian Pre-school Children

PubMed Central

Jonker, Femkje A. M.; Calis, Job C. J.; Phiri, Kamija; Brienen, Eric A. T.; Khoffi, Harriet; Brabin, Bernard J.; Verweij, Jaco J.; van Hensbroek, Michael Boele; van Lieshout, Lisette

2012-01-01

Background Hookworm infections are an important cause of (severe) anemia and iron deficiency in children in the tropics. Type of hookworm species (Ancylostoma duodenale or Necator americanus) and infection load are considered associated with disease burden, although these parameters are rarely assessed due to limitations of currently used diagnostic methods. Using multiplex real-time PCR, we evaluated hookworm species-specific prevalence, infection load and their contribution towards severe anemia and iron deficiency in pre-school children in Malawi. Methodology and Findings A. duodenale and N. americanus DNA loads were determined in 830 fecal samples of pre-school children participating in a case control study investigating severe anemia. Using multiplex real-time PCR, hookworm infections were found in 34.1% of the severely anemic cases and in 27.0% of the non-severely anemic controls (p<0.05) whereas a 5.6% hookworm prevalence was detected by microscopy. Prevalence of A. duodenale and N. americanus was 26.1% and 4.9% respectively. Moderate and high load A. duodenale infections were positively associated with severe anemia (adjusted odds ratio: 2.49 (95%CI 1.16–5.33) and 9.04 (95%CI 2.52–32.47) respectively). Iron deficiency (assessed through bone marrow examination) was positively associated with intensity of A. duodenale infection (adjusted odds ratio: 3.63 (95%CI 1.18–11.20); 16.98 (95%CI 3.88–74.35) and 44.91 (95%CI 5.23–385.77) for low, moderate and high load respectively). Conclusions/Significance This is the first report assessing the association of hookworm load and species differentiation with severe anemia and bone marrow iron deficiency. By revealing a much higher than expected prevalence of A. duodenale and its significant and load-dependent association with severe anemia and iron deficiency in pre-school children in Malawi, we demonstrated the need for quantitative and species-specific screening of hookworm infections. Multiplex real-time PCR is a powerful diagnostic tool for public health research to combat (severe) anemia and iron deficiency in children living in resource poor settings. PMID:22514750

A screening method for the detection of the 35S promoter and the nopaline synthase terminator in genetically modified organisms in a real-time multiplex polymerase chain reaction using high-resolution melting-curve analysis.

PubMed

Akiyama, Hiroshi; Nakamura, Fumi; Yamada, Chihiro; Nakamura, Kosuke; Nakajima, Osamu; Kawakami, Hiroshi; Harikai, Naoki; Furui, Satoshi; Kitta, Kazumi; Teshima, Reiko

2009-11-01

To screen for unauthorized genetically modified organisms (GMO) in the various crops, we developed a multiplex real-time polymerase chain reaction high-resolution melting-curve analysis method for the simultaneous qualitative detection of 35S promoter sequence of cauliflower mosaic virus (35SP) and the nopaline synthase terminator (NOST) in several crops. We selected suitable primer sets for the simultaneous detection of 35SP and NOST and designed the primer set for the detection of spiked ColE1 plasmid to evaluate the validity of the polymerase chain reaction (PCR) analyses. In addition, we optimized the multiplex PCR conditions using the designed primer sets and EvaGreen as an intercalating dye. The contamination of unauthorized GMO with single copy similar to NK603 maize can be detected as low as 0.1% in a maize sample. Furthermore, we showed that the present method would be applicable in identifying GMO in various crops and foods like authorized GM soybean, authorized GM potato, the biscuit which is contaminated with GM soybeans and the rice which is contaminated with unauthorized GM rice. We consider this method to be a simple and reliable assay for screening for unauthorized GMO in crops and the processing food products.

Dynamic Sensor Interrogation Using Wavelength-Swept Laser with a Polygon-Scanner-Based Wavelength Filter

PubMed Central

Kwon, Yong Seok; Ko, Myeong Ock; Jung, Mi Sun; Park, Ik Gon; Kim, Namje; Han, Sang-Pil; Ryu, Han-Cheol; Park, Kyung Hyun; Jeon, Min Yong

2013-01-01

We report a high-speed (∼2 kHz) dynamic multiplexed fiber Bragg grating (FBG) sensor interrogation using a wavelength-swept laser (WSL) with a polygon-scanner-based wavelength filter. The scanning frequency of the WSL is 18 kHz, and the 10 dB scanning bandwidth is more than 90 nm around a center wavelength of 1,540 nm. The output from the WSL is coupled into the multiplexed FBG array, which consists of five FBGs. The reflected Bragg wavelengths of the FBGs are 1,532.02 nm, 1,537.84 nm, 1,543.48 nm, 1,547.98 nm, and 1,553.06 nm, respectively. A dynamic periodic strain ranging from 500 Hz to 2 kHz is applied to one of the multiplexed FBGs, which is fixed on the stage of the piezoelectric transducer stack. Good dynamic performance of the FBGs and recording of their fast Fourier transform spectra have been successfully achieved with a measuring speed of 18 kHz. The signal-to-noise ratio and the bandwidth over the whole frequency span are determined to be more than 30 dB and around 10 Hz, respectively. We successfully obtained a real-time measurement of the abrupt change of the periodic strain. The dynamic FBG sensor interrogation system can be read out with a WSL for high-speed and high-sensitivity real-time measurement. PMID:23899934

Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

PubMed

Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L

2016-07-01

Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR.

Real-time, multiplexed electrochemical DNA detection using an active complementary metal-oxide-semiconductor biosensor array with integrated sensor electronics.

PubMed

Levine, Peter M; Gong, Ping; Levicky, Rastislav; Shepard, Kenneth L

2009-03-15

Optical biosensing based on fluorescence detection has arguably become the standard technique for quantifying extents of hybridization between surface-immobilized probes and fluorophore-labeled analyte targets in DNA microarrays. However, electrochemical detection techniques are emerging which could eliminate the need for physically bulky optical instrumentation, enabling the design of portable devices for point-of-care applications. Unlike fluorescence detection, which can function well using a passive substrate (one without integrated electronics), multiplexed electrochemical detection requires an electronically active substrate to analyze each array site and benefits from the addition of integrated electronic instrumentation to further reduce platform size and eliminate the electromagnetic interference that can result from bringing non-amplified signals off chip. We report on an active electrochemical biosensor array, constructed with a standard complementary metal-oxide-semiconductor (CMOS) technology, to perform quantitative DNA hybridization detection on chip using targets conjugated with ferrocene redox labels. A 4 x 4 array of gold working electrodes and integrated potentiostat electronics, consisting of control amplifiers and current-input analog-to-digital converters, on a custom-designed 5 mm x 3 mm CMOS chip drive redox reactions using cyclic voltammetry, sense DNA binding, and transmit digital data off chip for analysis. We demonstrate multiplexed and specific detection of DNA targets as well as real-time monitoring of hybridization, a task that is difficult, if not impossible, with traditional fluorescence-based microarrays.

Development of a two-step, non-probed multiplex real-time PCR for surveilling Vibrio anguillarum in seawater

USDA-ARS?s Scientific Manuscript database

Vibrio anguillarum is an aggressive and halophilic bacterial pathogen commonly found in seawater. Its presence in aquaculture facilities causes significant morbidity and mortality among aquaculture species primarily from hemorrhaging of the body and skin of the infected fish that eventually leads t...

Multiplex qPCR for serodetection and serotyping of hepatitis viruses: A brief review.

PubMed

Irshad, Mohammad; Gupta, Priyanka; Mankotia, Dhananjay Singh; Ansari, Mohammad Ahmad

2016-05-28

The present review describes the current status of multiplex quantitative real time polymerase chain reaction (qPCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex qPCR for the detection of hepatitis viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). In addition, multiplex qPCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex qPCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex qPCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process.

Multiplex qPCR for serodetection and serotyping of hepatitis viruses: A brief review

PubMed Central

Irshad, Mohammad; Gupta, Priyanka; Mankotia, Dhananjay Singh; Ansari, Mohammad Ahmad

2016-01-01

The present review describes the current status of multiplex quantitative real time polymerase chain reaction (qPCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex qPCR for the detection of hepatitis viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). In addition, multiplex qPCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex qPCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex qPCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process. PMID:27239109

A novel real-time duplex PCR assay for detecting penA and ponA genotypes in Neisseria gonorrhoeae: Comparison with phenotypes determined by the E-test.

PubMed

Vernel-Pauillac, Frédérique; Merien, Fabrice

2006-12-01

For many years, the pathogenic bacterium Neisseria gonorrhoeae, the etiologic agent of gonorrhea, was generally susceptible to penicillin, until the emergence of resistant strains. Well-characterized genetic variations in the penicillin resistance-determining region correlate with decreased susceptibility to penicillin. At least 5 genes (penA, penB, mtrR, ponA, and penC) are involved in the chromosomally mediated resistance to this antibiotic. To date, no development of multiplex PCR assays targeting a range of gonococcal genes and variations as a means of predicting antibiotic resistance has been reported. The aim of this study was to develop a duplex assay using DNA from isolated strains. We describe the development and evaluation on the LightCycler platform of a real-time duplex PCR assay (hybridization probe format) for rapid and specific detection of ponA and penA variations, predicting penicillin susceptibilities. The real-time duplex PCR assay successfully detected variations in ponA and penA genes by use of distinct melting temperatures from a total of 120 Neisseria gonorrhoeae isolates. Moreover, the variation profiles obtained with the real-time PCR and the melting analysis showed good correlation with the pattern of penicillin susceptibility generated with classical antibiograms. Nucleotide sequencing data were in complete agreement with multiplex assay results. The presented assay is suitable for the detection of chromosomally mediated resistant strains of Neisseria gonorrhoeae in genotyping studies and could be valuable in the effective antimicrobial strategy to gonococci.

Simultaneous detection of four causal agents of tobacco bushy top disease by a multiplex one-step RT-PCR

USDA-ARS?s Scientific Manuscript database

Tobacco bushy top disease is a complex disease caused by mixed infection of Tobacco bushy top virus (TBTV), Tobacco vein distorting virus (TVDV), satellite RNA of TBTV (Sat-TBTV) and Tobacco vein distorting virus associate RNA (TVDVaRNA). A one-tube multiplex reverse transcription-PCR (RT-PCR) assay...

Mini-midi-mito: adapting the amplification and sequencing strategy of mtDNA to the degradation state of crime scene samples.

PubMed

Berger, Cordula; Parson, Walther

2009-06-01

The degradation state of some biological traces recovered from the crime scene requires the amplification of very short fragments to attain a useful mitochondrial (mt)DNA sequence. We have previously introduced two mini-multiplex assays that amplify 10 overlapping control region (CR) fragments in two separate multiplex PCRs, which brought successful CR consensus sequences from even highly degraded DNA extracts. This procedure requires a total of 20 sequencing reactions per sample, which is laborious and cost intensive. For only moderately degraded samples that we encounter more frequently with typical mtDNA casework material, we developed two new multiplex assays that use a subset of the mini-amplicon primers but embrace larger fragments (midis) and require only 10 sequencing reactions to build a double-stranded CR consensus sequence. We used a preceding mtDNA quantitation step by real-time PCR with two different target fragments (143 and 283 bp) that roughly correspond to the average fragment sizes of the different multiplex approaches to estimate size-dependent mtDNA quantities and to aid the choice of the appropriate PCR multiplexes with respect to quality of the results and required costs.

Design and Assessment of a Real Time Reverse Transcription-PCR Method to Genotype Single-Stranded RNA Male-Specific Coliphages (Family Leviviridae).

EPA Science Inventory

A real-time, reverse transcription-PCR (RT-qPCR) assay was developed to differentiate the four genogroups of male-specific ssRNA coliphages (FRNA) (family Leviviridae). As FRNA display a trend of source-specificity (human sewage or animal waste) at the genogroup level, this assa...

A rocket-borne pulse-height analyzer for energetic particle measurements

NASA Technical Reports Server (NTRS)

Leung, W.; Smith, L. G.; Voss, H. D.

1979-01-01

The pulse-height analyzer basically resembles a time-sharing multiplexing data-acquisition system which acquires analog data (from energetic particle spectrometers) and converts them into digital code. The PHA simultaneously acquires pulse-height information from the analog signals of the four input channels and sequentially multiplexes the digitized data to a microprocessor. The PHA together with the microprocessor form an on-board real-time data-manipulation system. The system processes data obtained during the rocket flight and reduces the amount of data to be sent back to the ground station. Consequently the data-reduction process for the rocket experiments is speeded up. By using a time-sharing technique, the throughput rate of the microprocessor is increased. Moreover, data from several particle spectrometers are manipulated to share one information channel; consequently, the TM capacity is increased.

Deffuant model of opinion formation in one-dimensional multiplex networks

NASA Astrophysics Data System (ADS)

Shang, Yilun

2015-10-01

Complex systems in the real world often operate through multiple kinds of links connecting their constituents. In this paper we propose an opinion formation model under bounded confidence over multiplex networks, consisting of edges at different topological and temporal scales. We determine rigorously the critical confidence threshold by exploiting probability theory and network science when the nodes are arranged on the integers, {{Z}}, evolving in continuous time. It is found that the existence of ‘multiplexity’ impedes the convergence, and that working with the aggregated or summarized simplex network is inaccurate since it misses vital information. Analytical calculations are confirmed by extensive numerical simulations.

Soft fruit traceability in food matrices using real-time PCR.

PubMed

Palmieri, Luisa; Bozza, Elisa; Giongo, Lara

2009-02-01

Food product authentication provides a means of monitoring and identifying products for consumer protection and regulatory compliance. There is a scarcity of analytical methods for confirming the identity of fruit pulp in products containing Soft Fruit. In the present work we have developed a very sensible qualitative and quantitative method to determine the presence of berry DNAs in different food matrices. To our knowledge, this is the first study that shows the applicability, to Soft Fruit traceability, of melting curve analysis and multiplexed fluorescent probes, in a Real-Time PCR platform. This methodology aims to protect the consumer from label misrepresentation.

Design and laboratory validation of a structural element instrumented with multiplexed interferometric fiber optic sensors

NASA Astrophysics Data System (ADS)

Zonta, Daniele; Pozzi, Matteo; Wu, Huayong; Inaudi, Daniele

2008-03-01

This paper introduces a concept of smart structural elements for the real-time condition monitoring of bridges. These are prefabricated reinforced concrete elements embedding a permanent sensing system and capable of self-diagnosis when in operation. The real-time assessment is automatically controlled by a numerical algorithm founded on Bayesian logic: the method assigns a probability to each possible damage scenario, and estimates the statistical distribution of the damage parameters involved (such as location and extent). To verify the effectiveness of the technology, we produced and tested in the laboratory a reduced-scale smart beam prototype. The specimen is 3.8 m long and has cross-section 0.3 by 0.5m, and has been prestressed using a Dywidag bar, in such a way as to control the preload level. The sensor system includes a multiplexed version of SOFO interferometric sensors mounted on a composite bar, along with a number of traditional metal-foil strain gauges. The method allowed clear recognition of increasing fault states, simulated on the beam by gradually reducing the prestress level.

Two alternative multiplex PCRs for the identification of the seven species of anglerfish (Lophius spp.) using an end-point or a melting curve analysis real-time protocol.

PubMed

Castigliego, Lorenzo; Armani, Andrea; Tinacci, Lara; Gianfaldoni, Daniela; Guidi, Alessandra

2015-01-01

Anglerfish (Lophius spp.) is consumed worldwide and is an important economic resource though its seven species are often fraudulently interchanged due to their different commercial value, especially when sold in the form of fillets or pieces. Molecular analysis is the only possible mean to verify traceability and counteract fraud. We developed two multiplex PCRs, one end-point and one real-time with melting curve post-amplification analysis, which can even be run with the simplest two-channel thermocyclers. The two methods were tested on seventy-five reference samples. Their specificity was checked in twenty more species of those most commonly available on the market and in other species of the Lophiidae family. Both methods, the choice of which depends on the equipment and budget of the lab, provide a rapid and easy-to-read response, improving both the simplicity and cost-effectiveness of existing methods for identifying Lophius species. Copyright © 2014 Elsevier Ltd. All rights reserved.

Metallic Nanohole Arrays on Fluoropolymer Substrates as Small Label-Free Real-Time Bioprobes

PubMed Central

Yang, Jiun-Chan; Ji, Jin; Hogle, James M.; Larson, Dale N.

2009-01-01

We describe a nanoplasmonic probing platform that exploits small-dimension (≤ 20 μm2) ordered arrays of subwavelength holes for multiplexed, high spatial resolution, and real-time analysis on biorecognition events. Nanohole arrays are perforated on a super smooth gold surface (roughness RMS < 2.7 Å) attached on a fluoropolymer (FEP) substrate fabricated by a replica technique. The smooth surface of gold provides a superb environment for fabricating nanometer features and uniform immobilization of biomolecules. The refractive index matching between FEP and biological solutions contributes to ∼ 20% improvement on the sensing performance. Spectral studies on a series of small-dimension nanohole arrays from 1 μm2 to 20 μm2 indicate that the plasmonic sensing sensitivity improves as the gold-solution contact area increases. Our results also demonstrate that nanohole arrays with dimension as small as 1 μm2 can be used to effectively detect biomolecular binding events and analyze the binding kinetics. The future scientific opportunities opened by this nanohole platform include highly multiplexed analysis of ligand interactions with membrane proteins on high quality supported lipid bilayers. PMID:18710296

Evaluation of a multiplex real-time PCR assay for detecting pathogens in cardiac valve tissue in patients with endocarditis.

PubMed

Fernández, Angel L; Varela, Eduardo; Martínez, Lucía; Martínez, Amparo; Sierra, Juan; González-Juanatey, José R; Regueiro, Benito

2010-10-01

With a novel real-time multiplex polymerase chain reaction test, the LightCycler SeptiFast® test, 25 bacterial and fungal species can be identified directly in blood. The SeptiFast® test has been used for rapid etiologic diagnosis in infectious endocarditis using blood samples but has not been evaluated directly on cardiac vegetations in patients being treated for infectious endocarditis. We prospectively analyzed 15 samples of heart valve tissue with active infectious endocarditis using the SeptiFast® test and compared the test's sensitivity with that of blood culture, valve tissue culture, and the SeptiFast® test in blood. The sensitivity of the SeptiFast test in heart valve tissue was 100%. The test results confirmed the diagnosis obtained using blood culture in 13 cases and identified the pathogen in 2 cases where blood culture tested negative. The sensitivity of the SeptiFast® test in heart valve tissue was greater than that obtained with conventional culture of vegetations or with the SeptiFast test in blood.

Interlaboratory comparison of real-time pcr protocols for quantification of general fecal indicator bacteria

USGS Publications Warehouse

Shanks, O.C.; Sivaganesan, M.; Peed, L.; Kelty, C.A.; Blackwood, A.D.; Greene, M.R.; Noble, R.T.; Bushon, R.N.; Stelzer, E.A.; Kinzelman, J.; Anan'Eva, T.; Sinigalliano, C.; Wanless, D.; Griffith, J.; Cao, Y.; Weisberg, S.; Harwood, V.J.; Staley, C.; Oshima, K.H.; Varma, M.; Haugland, R.A.

2012-01-01

The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized protocol requires information on the reproducibility and sources of variation associated with qPCR methodology across laboratories. This study examines interlaboratory variability in the measurement of enterococci and Bacteroidales concentrations from standardized, spiked, and environmental sources of DNA using the Entero1a and GenBac3 qPCR methods, respectively. Comparisons are based on data generated from eight different research facilities. Special attention was placed on the influence of the DNA isolation step and effect of simplex and multiplex amplification approaches on interlaboratory variability. Results suggest that a crude lysate is sufficient for DNA isolation unless environmental samples contain substances that can inhibit qPCR amplification. No appreciable difference was observed between simplex and multiplex amplification approaches. Overall, interlaboratory variability levels remained low (<10% coefficient of variation) regardless of qPCR protocol. ?? 2011 American Chemical Society.

A multiplexed fluorescent assay for independent second-messenger systems: decoding GPCR activation in living cells.

PubMed

Tewson, Paul H; Quinn, Anne Marie; Hughes, Thomas E

2013-08-01

There is a growing need in drug discovery and basic research to measure multiple second-messenger components of cell signaling pathways in real time and in relevant tissues and cell types. Many G-protein-coupled receptors activate the heterotrimeric protein, Gq, which in turn activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce two second messengers: diacylglycerol (DAG), which remains in the plasma membrane, and inositol triphosphate (IP3), which diffuses through the cytosol to release stores of intracellular calcium ions (Ca(2+)). Our goal was to create a series of multiplex sensors that would make it possible to simultaneously measure two different components of the Gq pathway in living cells. Here we describe new fluorescent sensors for DAG and PIP2 that produce robust changes in green or red fluorescence and can be combined with one another, or with existing Ca(2+) sensors, in a live-cell assay. These assays can detect multiple components of Gq signaling, simultaneously in real time, on standard fluorescent plate readers or live-cell imaging systems.

Specific detection of rinderpest virus by real-time reverse transcription-PCR in preclincal and clinical samples of experimentally infected cattle

USDA-ARS?s Scientific Manuscript database

A highly sensitive detection test for Rinderpest virus (RPV), based on a real-time reverse transcription-PCR (RT-PR) system, was developed. Five different RPV genomic targets were examined, and one was selected and optimized to detect viral RNA in infected tissue culture fluid with a level of detec...

Evaluation of different embryonating bird eggs and cell cultures for isolation efficiency of avian influenza A virus and avian paramyxovirus serotype 1 from real-time reverse transcription polymerase chain reaction--positive

USDA-ARS?s Scientific Manuscript database

Two hundred samples collected from Anseriformes, Charadriiformes, Gruiformes, and Galliformes were assayed using real-time reverse transcriptase polymerase chain reaction (RRT-PCR) for presence of avian influenza virus and avian paramyxovirus-1. Virus isolation using embryonating chicken eggs, embr...

Development of duplex SYBR Green I-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses

USDA-ARS?s Scientific Manuscript database

A SYBR® Green-based real-time quantitative reverse transcription PCR (qRT-PCR) assay in combination with melt curve analysis (MCA) was developed for the detection of nine grapevine viruses. The detection limits for singleplex qRT-PCR for all nine grapevine viruses were determined to be in the range ...

Strand-specific real-time RT-PCR quantitation of Maize fine streak virus genomic and positive-sense RNAs using high temperature reverse transcription

USDA-ARS?s Scientific Manuscript database

Efforts to analyze the replicative RNA produced by Maize fine streak virus (MVSF) within maize tissue was complicated by the lack of specificity during cDNA generation using standard reverse transcriptase protocols. Real-time qRT-PCR using cDNA generated by priming with random hexamers does not dist...

Development and Interlaboratory Validation of a Simple Screening Method for Genetically Modified Maize Using a ΔΔC(q)-Based Multiplex Real-Time PCR Assay.

PubMed

Noguchi, Akio; Nakamura, Kosuke; Sakata, Kozue; Sato-Fukuda, Nozomi; Ishigaki, Takumi; Mano, Junichi; Takabatake, Reona; Kitta, Kazumi; Teshima, Reiko; Kondo, Kazunari; Nishimaki-Mogami, Tomoko

2016-04-19

A number of genetically modified (GM) maize events have been developed and approved worldwide for commercial cultivation. A screening method is needed to monitor GM maize approved for commercialization in countries that mandate the labeling of foods containing a specified threshold level of GM crops. In Japan, a screening method has been implemented to monitor approved GM maize since 2001. However, the screening method currently used in Japan is time-consuming and requires generation of a calibration curve and experimental conversion factor (C(f)) value. We developed a simple screening method that avoids the need for a calibration curve and C(f) value. In this method, ΔC(q) values between the target sequences and the endogenous gene are calculated using multiplex real-time PCR, and the ΔΔC(q) value between the analytical and control samples is used as the criterion for determining analytical samples in which the GM organism content is below the threshold level for labeling of GM crops. An interlaboratory study indicated that the method is applicable independently with at least two models of PCR instruments used in this study.

Development of Multiplex Reverse Transcription-Polymerase Chain Reaction for Simultaneous Detection of Influenza A, B and Adenoviruses

PubMed Central

Nakhaie, Mohsen; Soleimanjahi, Hoorieh; Mollaie, Hamid Reza; Arabzadeh, Seyed Mohamad Ali

2018-01-01

Background and objective: Millions of people in developing countries lose their lives due to acute respiratory infections, such as Influenza A & B and Adeno viruses. Given the importance of rapid identification of the virus, in this study the researchers attempted to design a method that enables detection of influenza A, B, and adenoviruses, quickly and simultaneously. The Multiplex RT PCR method was the preferred method for the detection of influenza A, B, and adenoviruses in clinical specimens because it is rapid, sensitive, specific, and more cost-effective than alternative methods Methods: After collecting samples from patients with respiratory disease, virus genome was extracted, then Monoplex PCR was used on positive samples and Multiplex RT-PCR on clinical specimens. Finally, by comparing the bands of these samples, the type of virus in the clinical samples was determined. Results: Performing Multiplex RT-PCR on 50 samples of respiratory tract led to following results; flu A: 12.5%, fluB: 50%, adeno: 27.5%, negative: 7.5%, and 2.5% contamination. Conclusion: Reverse transcription-multiplex Polymerase Chain Reaction (PCR) technique, a rapid diagnostic tool, has potential for high-throughput testing. This method has a significant advantage, which provides simultaneous amplification of numerous viruses in a single reaction. This study concentrates on multiplex molecular technologies and their clinical application for the detection and quantification of respiratory pathogens. The improvement in diagnostic testing for viral respiratory pathogens effects patient management, and leads to more cost-effective delivery of care. It limits unnecessary antibiotic use and improves clinical management by use of suitable treatment. PMID:29731796

Vancomycin-resistance phenotypes, vancomycin-resistance genes, and resistance to antibiotics of enterococci isolated from food of animal origin.

PubMed

Gousia, Panagiota; Economou, Vangelis; Bozidis, Petros; Papadopoulou, Chrissanthy

2015-03-01

In the present study, 500 raw beef, pork, and chicken meat samples and 100 pooled egg samples were analyzed for the presence of vancomycin-resistant enterococci, vancomycin-resistance phenotypes, and resistance genes. Of 141 isolates of enterococci, 88 strains of Enterococcus faecium and 53 strains of E. faecalis were identified. The most prevalent species was E. faecium. Resistance to ampicillin (n = 93, 66%), ciprofloxacin (n = 74, 52.5%), erythromycin (n = 73, 51.8%), penicillin (n = 59, 41.8%) and tetracycline (n = 52, 36.9%) was observed, while 53.2% (n = 75) of the isolates were multiresistant and 15.6% (n = 22) were susceptible to all antibiotics. Resistance to vancomycin was exhibited in 34.1% (n = 30) of the E. faecium isolates (n = 88) and 1.9% (n = 1) of the E. faecalis isolates (n = 53) using the disc-diffusion test and the E-test. All isolates were tested for vanA and vanB using real-time polymerase chain reaction (PCR) and multiplex PCR, and for vanC, vanD, vanE, vanG genes using multiplex PCR only. Among E. faecalis isolates, no resistance genes were identified. Among the E. faecium isolates, 28 carried the vanA gene when tested by multiplex PCR and 29 when tested with real-time PCR. No isolate carrying the vanC, vanD, vanE, or vanG genes was identified. Melting-curve analysis of the positive real-time PCR E. faecium isolates showed that 22 isolates carried the vanA gene only, 2 isolates the vanB2,3 genes only, and seven isolates carried both the vanA and vanB2,3 genes. Enterococci should be considered a significant zoonotic pathogen and a possible reservoir of genes encoding resistance potentially transferred to other bacterial species.

A Carbon Nanotube Reporter of miRNA Hybridization Events In Vivo

PubMed Central

Harvey, Jackson D.; Jena, Prakrit V.; Baker, Hanan A.; Zerze, Gül H.; Williams, Ryan M.; Galassi, Thomas V.; Roxbury, Daniel; Mittal, Jeetain

2017-01-01

MicroRNAs and other small oligonucleotides in biofluids are promising disease biomarkers, yet conventional assays require complex processing steps that are unsuitable for point-of-care testing or for implantable or wearable sensors. Single-walled carbon nanotubes are an ideal material for implantable sensors, owing to their emission in the near-infrared spectral region, photostability and exquisite sensitivity. Here, we report an engineered carbon-nanotube-based sensor capable of real-time optical quantification of hybridization events of microRNA and other oligonucleotides. The mechanism of the sensor arises from competitive effects between displacement of both oligonucleotide charge groups and water from the nanotube surface, which result in a solvatochromism-like response. The sensor, which allows for detection via single-molecule sensor elements and for multiplexing by using multiple nanotube chiralities, can monitor toehold-based strand-displacement events, which reverse the sensor response and regenerate the sensor complex. We also show that the sensor functions in whole urine and serum, and can non-invasively measure DNA and microRNA after implantation in live mice. PMID:28845337

A Carbon Nanotube Reporter of miRNA Hybridization Events In Vivo.

PubMed

Harvey, Jackson D; Jena, Prakrit V; Baker, Hanan A; Zerze, Gül H; Williams, Ryan M; Galassi, Thomas V; Roxbury, Daniel; Mittal, Jeetain; Heller, Daniel A

2017-01-01

MicroRNAs and other small oligonucleotides in biofluids are promising disease biomarkers, yet conventional assays require complex processing steps that are unsuitable for point-of-care testing or for implantable or wearable sensors. Single-walled carbon nanotubes are an ideal material for implantable sensors, owing to their emission in the near-infrared spectral region, photostability and exquisite sensitivity. Here, we report an engineered carbon-nanotube-based sensor capable of real-time optical quantification of hybridization events of microRNA and other oligonucleotides. The mechanism of the sensor arises from competitive effects between displacement of both oligonucleotide charge groups and water from the nanotube surface, which result in a solvatochromism-like response. The sensor, which allows for detection via single-molecule sensor elements and for multiplexing by using multiple nanotube chiralities, can monitor toehold-based strand-displacement events, which reverse the sensor response and regenerate the sensor complex. We also show that the sensor functions in whole urine and serum, and can non-invasively measure DNA and microRNA after implantation in live mice.

Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes.

PubMed

Treder, Krzysztof; Chołuj, Joanna; Zacharzewska, Bogumiła; Babujee, Lavanya; Mielczarek, Mateusz; Burzyński, Adam; Rakotondrafara, Aurélie M

2018-02-01

Potato virus Y (PVY) infection has been a global challenge for potato production and the leading cause of downgrading and rejection of seed crops for certification. Accurate and timely diagnosis is a key for effective disease control. Here, we have optimized a reverse transcription loop-mediated amplification (RT-LAMP) assay to differentiate the PVY O and N serotypes. The RT-LAMP assay is based on isothermal autocyclic strand displacement during DNA synthesis. The high specificity of this method relies heavily on the primer sets designed for the amplification of the targeted regions. We designed specific primer sets targeting a region within the coat protein gene that contains nucleotide signatures typical for O and N coat protein types, and these primers differ in their annealing temperature. Combining this assay with total RNA extraction by magnetic capture, we have established a highly sensitive, simplified and shortened RT-LAMP procedure as an alternative to conventional nucleic acid assays for diagnosis. This optimized procedure for virus detection may be used as a preliminary test for identifying the viral serotype prior to investing time and effort in multiplex RT-PCR tests when a specific strain is needed.

Multiplex polymerase chain reaction-capillary gel electrophoresis: a promising tool for GMO screening--assay for simultaneous detection of five genetically modified cotton events and species.

PubMed

Nadal, Anna; Esteve, Teresa; Pla, Maria

2009-01-01

A multiplex polymerase chain reaction assay coupled to capillary gel electrophoresis for amplicon identification by size and color (multiplex PCR-CGE-SC) was developed for simultaneous detection of cotton species and 5 events of genetically modified (GM) cotton. Validated real-time-PCR reactions targeting Bollgard, Bollgard II, Roundup Ready, 3006-210-23, and 281-24-236 junction sequences, and the cotton reference gene acp1 were adapted to detect more than half of the European Union-approved individual or stacked GM cotton events in one reaction. The assay was fully specific (<1.7% of false classification rate), with limit of detection values of 0.1% for each event, which were also achieved with simulated mixtures at different relative percentages of targets. The assay was further combined with a second multiplex PCR-CGE-SC assay to allow simultaneous detection of 6 cotton and 5 maize targets (two endogenous genes and 9 GM events) in two multiplex PCRs and a single CGE, making the approach more economic. Besides allowing simultaneous detection of many targets with adequate specificity and sensitivity, the multiplex PCR-CGE-SC approach has high throughput and automation capabilities, while keeping a very simple protocol, e.g., amplification and labeling in one step. Thus, it is an easy and inexpensive tool for initial screening, to be complemented with quantitative assays if necessary.

A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses

PubMed Central

Wadhwa, Ashutosh; Wilkins, Kimberly; Gao, Jinxin; Condori Condori, Rene Edgar; Gigante, Crystal M.; Zhao, Hui; Ma, Xiaoyue; Ellison, James A.; Greenberg, Lauren; Velasco-Villa, Andres; Orciari, Lillian

2017-01-01

Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabies molecular diagnostics. The LN34 assay provides an alternative approach for rabies diagnostics, especially in rural areas and rabies endemic regions that lack the conditions and broad experience required to run the standard DFA assay. PMID:28081126

A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.

PubMed

Wadhwa, Ashutosh; Wilkins, Kimberly; Gao, Jinxin; Condori Condori, Rene Edgar; Gigante, Crystal M; Zhao, Hui; Ma, Xiaoyue; Ellison, James A; Greenberg, Lauren; Velasco-Villa, Andres; Orciari, Lillian; Li, Yu

2017-01-01

Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabies molecular diagnostics. The LN34 assay provides an alternative approach for rabies diagnostics, especially in rural areas and rabies endemic regions that lack the conditions and broad experience required to run the standard DFA assay.

Consistency of influenza A virus detection test results across respiratory specimen collection methods using real-time reverse transcription-PCR.

PubMed

Spencer, Sarah; Gaglani, Manjusha; Naleway, Allison; Reynolds, Sue; Ball, Sarah; Bozeman, Sam; Henkle, Emily; Meece, Jennifer; Vandermause, Mary; Clipper, Lydia; Thompson, Mark

2013-11-01

In our prospective cohort study, we compared the performance of nasopharyngeal, oropharyngeal, and nasal swabs for the detection of influenza virus using real-time reverse transcription-PCR assay. Joint consideration of results from oropharyngeal and nasal swabs was as effective as consideration of results from nasopharyngeal swabs alone, as measured by sensitivity and noninferiority analysis.

Outbreak of hepatitis E virus infection in Darfur, Sudan: effectiveness of real-time reverse transcription-PCR analysis of dried blood spots.

PubMed

Mérens, Audrey; Guérin, Philippe Jean; Guthmann, Jean-Paul; Nicand, Elisabeth

2009-06-01

Biological samples collected in refugee camps during an outbreak of hepatitis E were used to compare the accuracy of hepatitis E virus RNA amplification by real-time reverse transcription-PCR (RT-PCR) for sera and dried blood spots (concordance of 90.6%). Biological profiles (RT-PCR and serology) of asymptomatic individuals were also analyzed.

Rapid and Quantitative Detection of Hepatitis A Virus from Green Onion and Strawberry Rinses by Use of Real-Time Reverse Transcription-PCR

PubMed Central

Shan, X. C.; Wolffs, P.; Griffiths, M. W.

2005-01-01

In this study, an immunomagnetic capture method and a real-time reverse transcription-PCR assay were used to quantify hepatitis A virus (HAV) in green onion and strawberry rinses. This combined protocol detected as low as 0.5 PFU HAV in produce rinses and concentrated HAV levels up to 20-fold. PMID:16151164

Solid-phase proximity ligation assays for individual or parallel protein analyses with readout via real-time PCR or sequencing.

PubMed

Nong, Rachel Yuan; Wu, Di; Yan, Junhong; Hammond, Maria; Gu, Gucci Jijuan; Kamali-Moghaddam, Masood; Landegren, Ulf; Darmanis, Spyros

2013-06-01

Solid-phase proximity ligation assays share properties with the classical sandwich immunoassays for protein detection. The proteins captured via antibodies on solid supports are, however, detected not by single antibodies with detectable functions, but by pairs of antibodies with attached DNA strands. Upon recognition by these sets of three antibodies, pairs of DNA strands brought in proximity are joined by ligation. The ligated reporter DNA strands are then detected via methods such as real-time PCR or next-generation sequencing (NGS). We describe how to construct assays that can offer improved detection specificity by virtue of recognition by three antibodies, as well as enhanced sensitivity owing to reduced background and amplified detection. Finally, we also illustrate how the assays can be applied for parallel detection of proteins, taking advantage of the oligonucleotide ligation step to avoid background problems that might arise with multiplexing. The protocol for the singleplex solid-phase proximity ligation assay takes ~5 h. The multiplex version of the assay takes 7-8 h depending on whether quantitative PCR (qPCR) or sequencing is used as the readout. The time for the sequencing-based protocol includes the library preparation but not the actual sequencing, as times may vary based on the choice of sequencing platform.

On-Orbit Quantitative Real-Time Gene Expression Analysis Using the Wetlab-2 System

NASA Technical Reports Server (NTRS)

Parra, Macarena; Jung, Jimmy; Almeida, Eduardo; Boone, Travis; Tran, Luan; Schonfeld, Julie

2015-01-01

NASA Ames Research Center's WetLab-2 Project enables on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The resulting RNA is dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The project selected the Cepheid SmartCycler (TradeMark), a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, because of its advantages including rugged modular design, low power consumption, rapid thermal ramp times and four-color multiplex detection. Single tube multiplex assays can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to conduct qRT-PCR and generate results on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also eliminate the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples and provide on-orbit gene expression benchmarking prior to sample return. Finally, the system can also be used for analysis of air, surface, water, and clinical samples to monitor environmental pathogens and crew health. The validation flight of the WetLab-2 system using E. coli bacteria and mouse liver launched on SpaceX-7 in June 2015 and will remain on the ISS National Laboratory.

Optimization of the elution buffer and concentration method for detecting hepatitis E virus in swine liver using a nested reverse transcription-polymerase chain reaction and real-time reverse transcription-polymerase chain reaction.

PubMed

Son, Na Ry; Seo, Dong Joo; Lee, Min Hwa; Seo, Sheungwoo; Wang, Xiaoyu; Lee, Bog-Hieu; Lee, Jeong-Su; Joo, In-Sun; Hwang, In-Gyun; Choi, Changsun

2014-09-01

The aim of this study was to develop an optimal technique for detecting hepatitis E virus (HEV) in swine livers. Here, three elution buffers and two concentration methods were compared with respect to enhancing recovery of HEV from swine liver samples. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and nested RT-PCR were performed to detect HEV RNA. When phosphate-buffered saline (PBS, pH 7.4) was used to concentrate HEV in swine liver samples using ultrafiltration, real-time RT-PCR detected HEV in 6 of the 26 samples. When threonine buffer was used to concentrate HEV using polyethylene glycol (PEG) precipitation and ultrafiltration, real-time RT-PCR detected HEV in 1 and 3 of the 26 samples, respectively. When glycine buffer was used to concentrate HEV using ultrafiltration and PEG precipitation, real-time RT-PCR detected HEV in 1 and 3 samples of the 26 samples, respectively. When nested RT-PCR was used to detect HEV, all samples tested negative regardless of the type of elution buffer or concentration method used. Therefore, the combination of real-time RT-PCR and ultrafiltration with PBS buffer was the most sensitive and reliable method for detecting HEV in swine livers. Copyright © 2014 Elsevier B.V. All rights reserved.

FPGA-based multi-channel fluorescence lifetime analysis of Fourier multiplexed frequency-sweeping lifetime imaging

PubMed Central

Zhao, Ming; Li, Yu; Peng, Leilei

2014-01-01

We report a fast non-iterative lifetime data analysis method for the Fourier multiplexed frequency-sweeping confocal FLIM (Fm-FLIM) system [ Opt. Express22, 10221 ( 2014)24921725]. The new method, named R-method, allows fast multi-channel lifetime image analysis in the system’s FPGA data processing board. Experimental tests proved that the performance of the R-method is equivalent to that of single-exponential iterative fitting, and its sensitivity is well suited for time-lapse FLIM-FRET imaging of live cells, for example cyclic adenosine monophosphate (cAMP) level imaging with GFP-Epac-mCherry sensors. With the R-method and its FPGA implementation, multi-channel lifetime images can now be generated in real time on the multi-channel frequency-sweeping FLIM system, and live readout of FRET sensors can be performed during time-lapse imaging. PMID:25321778

Molecular detection of Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, and Burkholderia glumae in infected rice seeds and leaves

USDA-ARS?s Scientific Manuscript database

Polymerase chain reaction (PCR) is particularly useful for plant pathogen detection. In the present study, multiplex PCR and SYBR green real-time PCR were developed to facilitate simultaneous detection of three important rice pathogens, Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, and Bur...

Quantitative real-time imaging of glutathione

USDA-ARS?s Scientific Manuscript database

Glutathione plays many important roles in biological processes; however, the dynamic changes of glutathione concentrations in living cells remain largely unknown. Here, we report a reversible reaction-based fluorescent probe—designated as RealThiol (RT)—that can quantitatively monitor the real-time ...

High-speed wavelength-division multiplexing quantum key distribution system.

PubMed

Yoshino, Ken-ichiro; Fujiwara, Mikio; Tanaka, Akihiro; Takahashi, Seigo; Nambu, Yoshihiro; Tomita, Akihisa; Miki, Shigehito; Yamashita, Taro; Wang, Zhen; Sasaki, Masahide; Tajima, Akio

2012-01-15

A high-speed quantum key distribution system was developed with the wavelength-division multiplexing (WDM) technique and dedicated key distillation hardware engines. Two interferometers for encoding and decoding are shared over eight wavelengths to reduce the system's size, cost, and control complexity. The key distillation engines can process a huge amount of data from the WDM channels by using a 1 Mbit block in real time. We demonstrated a three-channel WDM system that simultaneously uses avalanche photodiodes and superconducting single-photon detectors. We achieved 12 h continuous key generation with a secure key rate of 208 kilobits per second through a 45 km field fiber with 14.5 dB loss.

Flow cytometric detection method for DNA samples

DOEpatents

Nasarabadi, Shanavaz [Livermore, CA; Langlois, Richard G [Livermore, CA; Venkateswaran, Kodumudi S [Round Rock, TX

2011-07-05

Disclosed herein are two methods for rapid multiplex analysis to determine the presence and identity of target DNA sequences within a DNA sample. Both methods use reporting DNA sequences, e.g., modified conventional Taqman.RTM. probes, to combine multiplex PCR amplification with microsphere-based hybridization using flow cytometry means of detection. Real-time PCR detection can also be incorporated. The first method uses a cyanine dye, such as, Cy3.TM., as the reporter linked to the 5' end of a reporting DNA sequence. The second method positions a reporter dye, e.g., FAM.TM. on the 3' end of the reporting DNA sequence and a quencher dye, e.g., TAMRA.TM., on the 5' end.

Flow cytometric detection method for DNA samples

DOEpatents

Nasarabadi, Shanavaz [Livermore, CA; Langlois, Richard G [Livermore, CA; Venkateswaran, Kodumudi S [Livermore, CA

2006-08-01

Disclosed herein are two methods for rapid multiplex analysis to determine the presence and identity of target DNA sequences within a DNA sample. Both methods use reporting DNA sequences, e.g., modified conventional Taqman.RTM. probes, to combine multiplex PCR amplification with microsphere-based hybridization using flow cytometry means of detection. Real-time PCR detection can also be incorporated. The first method uses a cyanine dye, such as, Cy3.TM., as the reporter linked to the 5' end of a reporting DNA sequence. The second method positions a reporter dye, e.g., FAM, on the 3' end of the reporting DNA sequence and a quencher dye, e.g., TAMRA, on the 5' end.

Consistency of Influenza A Virus Detection Test Results across Respiratory Specimen Collection Methods Using Real-Time Reverse Transcription-PCR

PubMed Central

Gaglani, Manjusha; Naleway, Allison; Reynolds, Sue; Ball, Sarah; Bozeman, Sam; Henkle, Emily; Meece, Jennifer; Vandermause, Mary; Clipper, Lydia; Thompson, Mark

2013-01-01

In our prospective cohort study, we compared the performance of nasopharyngeal, oropharyngeal, and nasal swabs for the detection of influenza virus using real-time reverse transcription-PCR assay. Joint consideration of results from oropharyngeal and nasal swabs was as effective as consideration of results from nasopharyngeal swabs alone, as measured by sensitivity and noninferiority analysis. PMID:24108606

Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues.

PubMed

Kilpatrick, David R; Yang, Chen-Fu; Ching, Karen; Vincent, Annelet; Iber, Jane; Campagnoli, Ray; Mandelbaum, Mark; De, Lina; Yang, Su-Ju; Nix, Allan; Kew, Olen M

2009-06-01

We have adapted our previously described poliovirus diagnostic reverse transcription-PCR (RT-PCR) assays to a real-time RT-PCR (rRT-PCR) format. Our highly specific assays and rRT-PCR reagents are designed for use in the WHO Global Polio Laboratory Network for rapid and large-scale identification of poliovirus field isolates.

Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR.

PubMed

Kamau, Everlyn; Agoti, Charles N; Lewa, Clement S; Oketch, John; Owor, Betty E; Otieno, Grieven P; Bett, Anne; Cane, Patricia A; Nokes, D James

2017-03-01

Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses. Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity. Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples. N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses. An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Soft Fruit Traceability in Food Matrices using Real-Time PCR

PubMed Central

Palmieri, Luisa; Bozza, Elisa; Giongo, Lara

2009-01-01

Food product authentication provides a means of monitoring and identifying products for consumer protection and regulatory compliance. There is a scarcity of analytical methods for confirming the identity of fruit pulp in products containing Soft Fruit. In the present work we have developed a very sensible qualitative and quantitative method to determine the presence of berry DNAs in different food matrices. To our knowledge, this is the first study that shows the applicability, to Soft Fruit traceability, of melting curve analysis and multiplexed fluorescent probes, in a Real-Time PCR platform. This methodology aims to protect the consumer from label misrepresentation. PMID:22253987

Use of Multiplex Real-Time PCR To Diagnose Scrub Typhus.

PubMed

Tantibhedhyangkul, Wiwit; Wongsawat, Ekkarat; Silpasakorn, Saowaluk; Waywa, Duangdao; Saenyasiri, Nuttawut; Suesuay, Jintapa; Thipmontree, Wilawan; Suputtamongkol, Yupin

2017-05-01

Scrub typhus, caused by Orientia tsutsugamushi , is a common cause of acute undifferentiated febrile illness in the Asia-Pacific region. However, its nonspecific clinical manifestation often prevents early diagnosis. We propose the use of PCR and serologic tests as diagnostic tools. Here, we developed a multiplex real-time PCR assay using hydrolysis (TaqMan) probes targeting O. tsutsugamushi 47-kDa, groEL , and human interferon beta (IFN-β gene) genes to improve early diagnosis of scrub typhus. The amplification efficiency was higher than 94%, and the lower detection limit was 10 copies per reaction. We used a human gene as an internal DNA quality and quantity control. To determine the sensitivity of this PCR assay, we selected patients with confirmed scrub typhus who exhibited a clear 4-fold increase in the level of IgG and/or IgM. The PCR assay result was positive in 45 of 52 patients, indicating a sensitivity of 86.5% (95% confidence interval [CI]: 74.2 to 94.4). The PCR assessment was negative for all 136 non-scrub typhus patients, indicating a specificity of 100% (95% CI: 97.3 to 100). In addition, this test helped diagnose patients with inconclusive immunofluorescence assay (IFA) results and using single blood samples. In conclusion, the real-time PCR assay proposed here is sensitive and specific in diagnosing scrub typhus. Combining PCR and serologic tests will improve the diagnosis of scrub typhus among patients presenting with acute febrile illness. Copyright © 2017 American Society for Microbiology.

Suppressing disease spreading by using information diffusion on multiplex networks.

PubMed

Wang, Wei; Liu, Quan-Hui; Cai, Shi-Min; Tang, Ming; Braunstein, Lidia A; Stanley, H Eugene

2016-07-06

Although there is always an interplay between the dynamics of information diffusion and disease spreading, the empirical research on the systemic coevolution mechanisms connecting these two spreading dynamics is still lacking. Here we investigate the coevolution mechanisms and dynamics between information and disease spreading by utilizing real data and a proposed spreading model on multiplex network. Our empirical analysis finds asymmetrical interactions between the information and disease spreading dynamics. Our results obtained from both the theoretical framework and extensive stochastic numerical simulations suggest that an information outbreak can be triggered in a communication network by its own spreading dynamics or by a disease outbreak on a contact network, but that the disease threshold is not affected by information spreading. Our key finding is that there is an optimal information transmission rate that markedly suppresses the disease spreading. We find that the time evolution of the dynamics in the proposed model qualitatively agrees with the real-world spreading processes at the optimal information transmission rate.

Development of a versatile tool for the simultaneous differential detection of Pseudomonas savastanoi pathovars by End Point and Real-Time PCR

PubMed Central

2010-01-01

Background Pseudomonas savastanoi pv. savastanoi is the causal agent of olive knot disease. The strains isolated from oleander and ash belong to the pathovars nerii and fraxini, respectively. When artificially inoculated, pv. savastanoi causes disease also on ash, and pv. nerii attacks also olive and ash. Surprisingly nothing is known yet about their distribution in nature on these hosts and if spontaneous cross-infections occur. On the other hand sanitary certification programs for olive plants, also including P. savastanoi, were launched in many countries. The aim of this work was to develop several PCR-based tools for the rapid, simultaneous, differential and quantitative detection of these P. savastanoi pathovars, in multiplex and in planta. Results Specific PCR primers and probes for the pathovars savastanoi, nerii and fraxini of P. savastanoi were designed to be used in End Point and Real-Time PCR, both with SYBR® Green or TaqMan® chemistries. The specificity of all these assays was 100%, as assessed by testing forty-four P. savastanoi strains, belonging to the three pathovars and having different geographical origins. For comparison strains from the pathovars phaseolicola and glycinea of P. savastanoi and bacterial epiphytes from P. savastanoi host plants were also assayed, and all of them tested always negative. The analytical detection limits were about 5 - 0.5 pg of pure genomic DNA and about 102 genome equivalents per reaction. Similar analytical thresholds were achieved in Multiplex Real-Time PCR experiments, even on artificially inoculated olive plants. Conclusions Here for the first time a complex of PCR-based assays were developed for the simultaneous discrimination and detection of P. savastanoi pv. savastanoi, pv. nerii and pv. fraxini. These tests were shown to be highly reliable, pathovar-specific, sensitive, rapid and able to quantify these pathogens, both in multiplex reactions and in vivo. Compared with the other methods already available for P. savastanoi, the identification procedures here reported provide a versatile tool both for epidemiological and ecological studies on these pathovars, and for diagnostic procedures monitoring the asymptomatic presence of P. savastanoi on olive and oleander propagation materials. PMID:20509893

High throughput, multiplexed pathogen detection authenticates plague waves in medieval Venice, Italy.

PubMed

Tran, Thi-Nguyen-Ny; Signoli, Michel; Fozzati, Luigi; Aboudharam, Gérard; Raoult, Didier; Drancourt, Michel

2011-03-10

Historical records suggest that multiple burial sites from the 14th-16th centuries in Venice, Italy, were used during the Black Death and subsequent plague epidemics. High throughput, multiplexed real-time PCR detected DNA of seven highly transmissible pathogens in 173 dental pulp specimens collected from 46 graves. Bartonella quintana DNA was identified in five (2.9%) samples, including three from the 16th century and two from the 15th century, and Yersinia pestis DNA was detected in three (1.7%) samples, including two from the 14th century and one from the 16th century. Partial glpD gene sequencing indicated that the detected Y. pestis was the Orientalis biotype. These data document for the first time successive plague epidemics in the medieval European city where quarantine was first instituted in the 14th century.

Real-time optical laboratory solution of parabolic differential equations

NASA Technical Reports Server (NTRS)

Casasent, David; Jackson, James

1988-01-01

An optical laboratory matrix-vector processor is used to solve parabolic differential equations (the transient diffusion equation with two space variables and time) by an explicit algorithm. This includes optical matrix-vector nonbase-2 encoded laboratory data, the combination of nonbase-2 and frequency-multiplexed data on such processors, a high-accuracy optical laboratory solution of a partial differential equation, new data partitioning techniques, and a discussion of a multiprocessor optical matrix-vector architecture.

Frequency and bandwidth conversion of single photons in a room-temperature diamond quantum memory

PubMed Central

Fisher, Kent A. G.; England, Duncan G.; MacLean, Jean-Philippe W.; Bustard, Philip J.; Resch, Kevin J.; Sussman, Benjamin J.

2016-01-01

The spectral manipulation of photons is essential for linking components in a quantum network. Large frequency shifts are needed for conversion between optical and telecommunication frequencies, while smaller shifts are useful for frequency-multiplexing quantum systems, in the same way that wavelength division multiplexing is used in classical communications. Here we demonstrate frequency and bandwidth conversion of single photons in a room-temperature diamond quantum memory. Heralded 723.5 nm photons, with 4.1 nm bandwidth, are stored as optical phonons in the diamond via a Raman transition. Upon retrieval from the diamond memory, the spectral shape of the photons is determined by a tunable read pulse through the reverse Raman transition. We report central frequency tunability over 4.2 times the input bandwidth, and bandwidth modulation between 0.5 and 1.9 times the input bandwidth. Our results demonstrate the potential for diamond, and Raman memories in general, as an integrated platform for photon storage and spectral conversion. PMID:27045988

Frequency and bandwidth conversion of single photons in a room-temperature diamond quantum memory.

PubMed

Fisher, Kent A G; England, Duncan G; MacLean, Jean-Philippe W; Bustard, Philip J; Resch, Kevin J; Sussman, Benjamin J

2016-04-05

The spectral manipulation of photons is essential for linking components in a quantum network. Large frequency shifts are needed for conversion between optical and telecommunication frequencies, while smaller shifts are useful for frequency-multiplexing quantum systems, in the same way that wavelength division multiplexing is used in classical communications. Here we demonstrate frequency and bandwidth conversion of single photons in a room-temperature diamond quantum memory. Heralded 723.5 nm photons, with 4.1 nm bandwidth, are stored as optical phonons in the diamond via a Raman transition. Upon retrieval from the diamond memory, the spectral shape of the photons is determined by a tunable read pulse through the reverse Raman transition. We report central frequency tunability over 4.2 times the input bandwidth, and bandwidth modulation between 0.5 and 1.9 times the input bandwidth. Our results demonstrate the potential for diamond, and Raman memories in general, as an integrated platform for photon storage and spectral conversion.

A fully sealed plastic chip for multiplex PCR and its application in bacteria identification.

PubMed

Xu, Youchun; Yan, He; Zhang, Yan; Jiang, Kewei; Lu, Ying; Ren, Yonghong; Wang, Hui; Wang, Shan; Xing, Wanli

2015-07-07

Multiplex PCR is an effective tool for simultaneous multiple target detection but is limited by the intrinsic interference and competition among primer pairs when it is performed in one reaction tube. Dividing a multiplex PCR into many single PCRs is a simple strategy to overcome this issue. Here, we constructed a plastic, easy-to-use, fully sealed multiplex PCR chip based on reversible centrifugation for the simultaneous detection of 63 target DNA sequences. The structure of the chip is quite simple, which contains sine-shaped infusing channels and a number of reaction chambers connecting to one side of these channels. Primer pairs for multiplex PCR were sequentially preloaded in the different reaction chambers, and the chip was enclosed with PCR-compatible adhesive tape. For usage, the PCR master mix containing a DNA template is pipetted into the infusing channels and centrifuged into the reaction chambers, leaving the infusing channels filled with air to avoid cross-contamination of the different chambers. Then, the chip is sealed and placed on a flat thermal cycler for PCR. Finally, amplification products can be detected in situ using a fluorescence scanner or recovered by reverse centrifugation for further analyses. Therefore, our chip possesses two functions: 1) it can be used for multi-target detection based on end-point in situ fluorescence detection; and 2) it can work as a sample preparation unit for analyses that need multiplex PCR such as hybridization and target sequencing. The performance of this chip was carefully examined and further illustrated in the identification of 8 pathogenic bacterial genomic DNA samples and 13 drug-resistance genes. Due to simplicity of its structure and operation, accuracy and generality, high-throughput capacity, and versatile functions (i.e., for in situ detection and sample preparation), our multiplex PCR chip has great potential in clinical diagnostics and nucleic acid-based point-of-care testing.

Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species.

PubMed

Gopaul, Krishna K; Sells, Jessica; Lee, Robin; Beckstrom-Sternberg, Stephen M; Foster, Jeffrey T; Whatmore, Adrian M

2014-12-11

The zoonosis brucellosis causes economically significant reproductive problems in livestock and potentially debilitating disease of humans. Although the causative agent, organisms from the genus Brucella, can be differentiated into a number of species based on phenotypic characteristics, there are also significant differences in genotype that are concordant with individual species. This paper describes the development of a five target multiplex assay to identify five terrestrial Brucella species using real-time polymerase chain reaction (PCR) and subsequent high resolution melt curve analysis. This technology offers a robust and cost effective alternative to previously described hydrolysis-probe Single Nucleotide Polymorphism (SNP)-based species defining assays. Through the use of Brucella whole genome sequencing five species defining SNPs were identified. Individual HRM assays were developed to these target these changes and, following optimisation of primer concentrations, it was possible to multiplex all five assays in a single tube. In a validation exercise using a panel of 135 Brucella strains of terrestrial and marine origin, it was possible to distinguish the five target species from the other species within this panel. The HRM multiplex offers a number of diagnostic advantages over previously described SNP-based typing approaches. Further, and uniquely for HRM, the successful multiplexing of five assays in a single tube allowing differentiation of five Brucella species in the diagnostic laboratory in a cost-effective and timely manner is described. However there are possible limitations to using this platform on DNA extractions direct from clinical material.

Polarization-multiplexed plasmonic phase generation with distributed nanoslits.

PubMed

Lee, Seung-Yeol; Kim, Kyuho; Lee, Gun-Yeal; Lee, Byoungho

2015-06-15

Methods for multiplexing surface plasmon polaritons (SPPs) have been attracting much attention due to their potentials for plasmonic integrated systems, plasmonic holography, and optical tweezing. Here, using closely-distanced distributed nanoslits, we propose a method for generating polarization-multiplexed SPP phase profiles which can be applied for implementing general SPP phase distributions. Two independent types of SPP phase generation mechanisms - polarization-independent and polarization-reversible ones - are combined to generate fully arbitrary phase profiles for each optical handedness. As a simple verification of the proposed scheme, we experimentally demonstrate that the location of plasmonic focus can be arbitrary designed, and switched by the change of optical handedness.

Field Experiments on Real-Time 1-Gbps High-Speed Packet Transmission in MIMO-OFDM Broadband Packet Radio Access

NASA Astrophysics Data System (ADS)

Taoka, Hidekazu; Higuchi, Kenichi; Sawahashi, Mamoru

This paper presents experimental results in real propagation channel environments of real-time 1-Gbps packet transmission using antenna-dependent adaptive modulation and channel coding (AMC) with 4-by-4 MIMO multiplexing in the downlink Orthogonal Frequency Division Multiplexing (OFDM) radio access. In the experiment, Maximum Likelihood Detection employing QR decomposition and the M-algorithm (QRM-MLD) with adaptive selection of the surviving symbol replica candidates (ASESS) is employed to achieve such a high data rate at a lower received signal-to-interference plus background noise power ratio (SINR). The field experiments, which are conducted at the average moving speed of 30km/h, show that real-time packet transmission of greater than 1Gbps in a 100-MHz channel bandwidth (i.e., 10bits/second/Hz) is achieved at the average received SINR of approximately 13.5dB using 16QAM modulation and turbo coding with the coding rate of 8/9. Furthermore, we show that the measured throughput of greater than 1Gbps is achieved at the probability of approximately 98% in a measurement course, where the maximum distance from the cell site was approximately 300m with the respective transmitter and receiver antenna separation of 1.5m and 40cm with the total transmission power of 10W. The results also clarify that the minimum required receiver antenna spacing is approximately 10cm (1.5 carrier wave length) to suppress the loss in the required received SINR at 1-Gbps throughput to within 1dB compared to that assuming the fading correlation between antennas of zero both under non-line-of-sight (NLOS) and line-of-sight (LOS) conditions.

An improved method to simultaneously detect Salmonella enteritidis, Escherichia coli O157 and Listeria monocytogenes in ground black pepper using multiplex real-time PCR

USDA-ARS?s Scientific Manuscript database

Introduction: The three common foodborne pathogens implicated in foodborne outbreaks are Salmonella spp., Escherichia coli O157:H7 and Listeria monocytogenes. Hence, it is important to identify these pathogens in contaminated foods so that they can be eliminated from the marketplace. At present, the...

Development of Multiplex Real-time PCR with Internal Amplification Control for Simultaneous Detection of Salmonella and Cronobacter sakazakii in Powdered Infant Formula.

USDA-ARS?s Scientific Manuscript database

Contamination of powdered infant formula (PIF) by the bacteria Cronobacter sakazakii and Salmonella enterica was deemed a matter of great concern by the World Health Organization and the Food and Agriculture Organization of the United Nations in 2004. Therefore, we developed a rapid and sensitive m...

Modification of two capripoxvirus quantitative real-time PCR assays to improve diagnostic sensitivity and include beta-actin as an internal positive control.

PubMed

Das, Amaresh; Deng, Ming Y; Babiuk, Shawn; McIntosh, Michael T

2017-05-01

Capripoxviruses (CaPVs), consisting of Sheeppox virus (SPV), Goatpox virus (GPV), and Lumpy skin disease virus (LSDV) species, cause economically significant diseases in sheep, goats, and cattle, respectively. Quantitative real-time polymerase chain reaction (qPCR) assays are routinely used for rapid detection of CaPVs in surveillance and outbreak management programs. We further modified and optimized 2 previously published CaPV qPCR assays, referred to as the Balinsky and Bowden assays, by changing commercial PCR reagents used in the tests. The modified assays displayed 100% analytical specificity and showed no apparent changes in analytical sensitivities for detection of CaPVs compared with the original assays. Diagnostic sensitivities, assessed using 50 clinical reference samples from experimentally infected sheep, goats, and cattle, improved from 82% to 92% for the modified Balinsky assay and from 58% to 82% for the modified Bowden assay. The modified qPCR assays were multiplexed for detection of beta-actin as an indicator for potential false-negative results. The multiplex modified qPCR assays exhibited the same diagnostic sensitivities as the singleplex assays suggesting their utility in the detection of CaPVs.

Development and Application of a Multiplex Real-Time PCR Assay as an Indicator of Potential Allergenicity in Citrus Fruits.

PubMed

Wu, Jinlong; Chen, Lin; Lin, Dingbo; Ma, Zhaocheng; Deng, Xiuxin

2016-11-30

The effects of tissue type, harvest maturity, and genetic factors on the expression of genes that related to citrus fruit allergies remain poorly understood. In the present study, a multiplex real-time PCR assay was developed to monitor the expression of citrus allergen genes individually with the advantages of much fewer sample requirements and simultaneously multiple target genes detection. Gene specific primer pairs and Taqman probes of three citrus allergen genes Cit s 1.01, Cit s 2.01, and Cit s 3.01 and the house-keeping gene β-actin were designed based on gene sequence differences. The PCR results showed that differential expression patterns were found during the ripening process. The expression levels of Cit s 3.01 were much higher than those of Cit s 1.01 and Cit s 2.01 in both peel and pulp tissues among 10 citrus cultivars. Data suggested that Kao Phuang Pummelo could be safely consumed with a potential low risk in allergenicity. Considering that assessing allergenicity is one of the tests in food safety, this assay might also facilitate the breeding and production of "allergy-friendly" citrus fruits.

Quantifying viable Vibrio parahaemolyticus and Listeria monocytogenes simultaneously in raw shrimp.

PubMed

Zhang, Zhaohuan; Liu, Haiquan; Lou, Yang; Xiao, Lili; Liao, Chao; Malakar, Pradeep K; Pan, Yingjie; Zhao, Yong

2015-08-01

A novel TaqMan-based multiplex real-time PCR method combined with propidium monoazide (PMA) treatment was firstly developed for the simultaneous quantification of viable Vibrio parahaemolyticus and Listeria monocytogenes in raw shrimp. The optimization of PMA concentration showed that 100 μM was considered optimal to effectively inhibit 10(8) CFU/mL dead cells of both bacteria. The high specificity of this method was confirmed on tests using 96 target and non-target strains. The optimized assay could detect as low as 10(1)-10(2) CFU/g of each strain on the artificially contaminated shrimp, and its amplification efficiencies were up to 100 and 106 % for V. parahaemolyticus and L. monocytogenes, respectively. Furthermore, this assay has been successfully applied to describe the behavior of these two pathogens in raw shrimps stored at 4 °C. In conclusion, this PMA TaqMan-based multiplex real-time PCR technique, where the whole procedure takes less than 5 h, provides an effective and rapid tool for monitoring contamination of viable V. parahaemolyticus and L. monocytogenes in seafood, improving seafood safety and protecting public health.

Multiplex Real-Time PCR for Detecting and Typing Clostridium botulinum Group III Organisms and Their Mosaic Variants

PubMed Central

Auricchio, Bruna; Woudstra, Cédric; Fach, Patrick; Fiore, Alfonsina; Skarin, Hanna; Bano, Luca; Segerman, Bo; Knutsson, Rickard; De Medici, Dario

2013-01-01

Botulism is a neuroparalytic disease that can occur in all warm-blooded animals, birds, and fishes. The disease in animals is mainly caused by toxins produced by Clostridium botulinum strains belonging to group III, although outbreaks due to toxins produced by group I and II organisms have been recognized. Group III strains are capable of producing botulinum toxins of type C, D, and C/D and D/C mosaic variants. Definitive diagnosis of animal botulism is made by combining clinical findings with laboratory investigations. Detection of toxins in clinical specimens and feed is the gold standard for laboratory diagnosis. Since toxins may be degraded by organisms contained in the gastrointestinal tract or may be present at levels below the detection limit, the recovery of C. botulinum from sick animal specimens is consistent for laboratory confirmation. In this article we report the development and in-house validation of a new multiplex real-time PCR for detecting and typing the neurotoxin genes found in C. botulinum group III organisms. Validation procedures have been carried out according to ISO 16140, using strains and samples recovered from cases of animal botulism in Italy and France. PMID:23971808

Development of a Multiplex Real-Time PCR for Determination of Apricot in Marzipan Using the Plexor System.

PubMed

Schelm, Stefanie; Haase, Ilka; Fischer, Christin; Fischer, Markus

2017-01-18

Marzipan is a confectionary which is mostly offered in form of filled chocolate, pralines, or pure. According to the German guidelines for oil seeds only almonds, sugar and water are admitted ingredients of marzipan. A product very similar in taste is persipan which is used in the confectionary industry because of its stronger flavor. For persipan production almonds are replaced by debittered apricot or peach kernels. To guarantee high quality products for consumers, German raw paste producers have agreed a limit of apricot kernels in marzipan raw paste of 0.5%. Different DNA-based methods for quantitation of persipan contaminations in marzipan are already published. To increase the detection specificity compared to published intercalation dye-based assays, the present work demonstrate the utilization of a multiplex real-time PCR based on the Plexor technology. Thus, the present work enables the detection of at least 0.1% apricot DNA in almond DNA or less. By analyzing DNA mixtures, the theoretical limit of quantification of the duplex PCR for the quantitation of persipan raw paste DNA in marzipan raw paste DNA was determined as 0.05%.

Optimal percolation on multiplex networks.

PubMed

Osat, Saeed; Faqeeh, Ali; Radicchi, Filippo

2017-11-16

Optimal percolation is the problem of finding the minimal set of nodes whose removal from a network fragments the system into non-extensive disconnected clusters. The solution to this problem is important for strategies of immunization in disease spreading, and influence maximization in opinion dynamics. Optimal percolation has received considerable attention in the context of isolated networks. However, its generalization to multiplex networks has not yet been considered. Here we show that approximating the solution of the optimal percolation problem on a multiplex network with solutions valid for single-layer networks extracted from the multiplex may have serious consequences in the characterization of the true robustness of the system. We reach this conclusion by extending many of the methods for finding approximate solutions of the optimal percolation problem from single-layer to multiplex networks, and performing a systematic analysis on synthetic and real-world multiplex networks.

A multiplexed reverse transcriptase PCR assay for identification of viral respiratory pathogens at point-of-care

DOE Office of Scientific and Technical Information (OSTI.GOV)

Letant, S E; .Ortiz, J I; Tammero, L

2007-04-11

We have developed a nucleic acid-based assay that is rapid, sensitive, specific, and can be used for the simultaneous detection of 5 common human respiratory pathogens including influenza A, influenza B, parainfluenza type 1 and 3, respiratory syncytial virus, and adenovirus group B, C, and E. Typically, diagnosis on an un-extracted clinical sample can be provided in less than 3 hours, including sample collection, preparation, and processing, as well as data analysis. Such a multiplexed panel would enable rapid broad-spectrum pathogen testing on nasal swabs, and therefore allow implementation of infection control measures, and timely administration of antiviral therapies. Thismore » article presents a summary of the assay performance in terms of sensitivity and specificity. Limits of detection are provided for each targeted respiratory pathogen, and result comparisons are performed on clinical samples, our goal being to compare the sensitivity and specificity of the multiplexed assay to the combination of immunofluorescence and shell vial culture currently implemented at the UCDMC hospital. Overall, the use of the multiplexed RT-PCR assay reduced the rate of false negatives by 4% and reduced the rate of false positives by up to 10%. The assay correctly identified 99.3% of the clinical negatives, 97% of adenovirus, 95% of RSV, 92% of influenza B, and 77% of influenza A without any extraction performed on the clinical samples. The data also showed that extraction will be needed for parainfluenza virus, which was only identified correctly 24% of the time on un-extracted samples.« less

Multiplex PCR detection of Cryptosporidium sp, Giardia lamblia and Entamoeba histolytica directly from dried stool samples from Guinea-Bissauan children with diarrhoea.

PubMed

Mero, Sointu; Kirveskari, Juha; Antikainen, Jenni; Ursing, Johan; Rombo, Lars; Kofoed, Poul-Erik; Kantele, Anu

2017-09-01

In developing countries, diarrhoea is the most common cause of death for children under five years of age, with Giardia lamblia, Cryptosporidium and Entamoeba histolytica as the most frequent pathogenic parasites. Traditional microscopy for stool parasites has poor sensitivity and specificity, while new molecular methods may provide more accurate diagnostics. In poor regions with sample storage hampered by uncertain electricity supply, research would benefit from a method capable of analysing dried stools. A real-time multiplex PCR method with internal inhibition control was developed for detecting Giardia lamblia, Cryptosporidium hominis/parvum and Entamoeba histolytica directly from stool specimens. Applicability to dried samples was checked by comparing with fresh ones in a small test material. Finally, the assay was applied to dried specimens collected from Guinea-Bissauan children with diarrhoea. The PCR's analytical sensitivity limit was 0.1 ng/ml for G. lamblia DNA, 0.01 ng/ml for E. histolytica DNA and 0.1 ng/ml for Cryptosporidium sp. In the test material, the assay performed similarly with fresh and dried stools. Of the 52 Guinea-Bissauan samples, local microscopy revealed a parasite in 15%, while PCR detected 62% positive for at least one parasite: 44% of the dried samples had Giardia, 23% Cryptosporidium and 0% E. histolytica. Our new multiplex real-time PCR for protozoa presents a sensitive method applicable to dried samples. As proof of concept, it worked well on stools collected from Guinea-Bissauan children with diarrhoea. It provides an epidemiological tool for analysing dried specimens from regions poor in resources.

Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors

PubMed Central

Ikten, Cengiz; Ustun, Rustem; Catal, Mursel; Yol, Engin; Uzun, Bulent

2016-01-01

Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease. PMID:27195795

Multiplex Molecular Panels for Diagnosis of Gastrointestinal Infection: Performance, Result Interpretation, and Cost-Effectiveness.

PubMed

Binnicker, Matthew J

2015-12-01

Gastrointestinal disease is a major cause of morbidity and mortality worldwide, especially among young children and immunocompromised patients. Diarrhea may result from infection with a variety of microbial pathogens, including bacteria, viruses, or parasites. Historically, the diagnosis of infectious diarrhea has been made using microscopy, antigen tests, culture, and real-time PCR. A combination of these traditional tests is often required due to the inability to distinguish between infectious etiologies based on the clinical presentation alone. Recently, several multiplex molecular assays have been developed for the detection of gastrointestinal pathogens directly from clinical stool samples. These panels allow for the detection and identification of up to 20 pathogens in as little as 1 h. This review will focus on the multiplex molecular panels that have received clearance from the FDA for the diagnosis of diarrheal disease and will highlight issues related to test performance, result interpretation, and cost-effectiveness of these new molecular diagnostic tools.

Sensitive Molecular Diagnostics for Cutaneous Leishmaniasis.

PubMed

Sagi, Orli; Berkowitz, Anat; Codish, Shlomi; Novack, Victor; Rashti, Aviv; Akad, Fouad; Shemer-Avni, Yonat

2017-01-01

Rapid diagnosis of cutaneous leishmaniasis (CL) and identification of Leishmania species is highly important for the disease management. In Israel, CL is caused mainly by Leishmania major and Leishmania tropica species. We established an easy to handle point of care lesion-swabbing, combined with a highly sensitive multiplex real time PCR (multiplex qPCR) for accurate and rapid diagnosis of Leishmania species. Using three probes: one general for: Leishmania species, and two specific for L major , and L tropica , we screened 1783 clinical samples collected during two years. Leishmania species was found in 1086 individuals, 1008 L major , and 70 L tropica . Eight samples positive for Leishmania species only, were further tested using a second set of multiplex qPCR developed, and were found positive for Leishmania braziliensis and Leishmania infantum/donovani (2 and 6 samples, concomitantly). Taken together, the test enabled diagnostics and better treatment of Leishmania infections from the Old World (1078 samples) and the New World (8 samples), and the subtyping of the dominant strains in the region, as well as in returning travelers'.

Real-Time Two-Dimensional Mapping of Relative Local Surface Temperatures with a Thin-Film Sensor Array

PubMed Central

Li, Gang; Wang, Zhenhai; Mao, Xinyu; Zhang, Yinghuang; Huo, Xiaoye; Liu, Haixiao; Xu, Shengyong

2016-01-01

Dynamic mapping of an object’s local temperature distribution may offer valuable information for failure analysis, system control and improvement. In this letter we present a computerized measurement system which is equipped with a hybrid, low-noise mechanical-electrical multiplexer for real-time two-dimensional (2D) mapping of surface temperatures. We demonstrate the performance of the system on a device embedded with 32 pieces of built-in Cr-Pt thin-film thermocouples arranged in a 4 × 8 matrix. The system can display a continuous 2D mapping movie of relative temperatures with a time interval around 1 s. This technique may find applications in a variety of practical devices and systems. PMID:27347969

Real-Time Reverse Transcription–Polymerase Chain Reaction Assay for SARS-associated Coronavirus

PubMed Central

Emery, Shannon L.; Bowen, Michael D.; Newton, Bruce R.; Winchell, Jonas M.; Meyer, Richard F.; Tong, Suxiang; Cook, Byron T.; Holloway, Brian P.; McCaustland, Karen A.; Rota, Paul A.; Bankamp, Bettina; Lowe, Luis E.; Ksiazek, Tom G.; Bellini, William J.; Anderson, Larry J.

2004-01-01

A real-time reverse transcription–polymerase chain reaction (RT-PCR) assay was developed to rapidly detect the severe acute respiratory syndrome–associated coronavirus (SARS-CoV). The assay, based on multiple primer and probe sets located in different regions of the SARS-CoV genome, could discriminate SARS-CoV from other human and animal coronaviruses with a potential detection limit of <10 genomic copies per reaction. The real-time RT-PCR assay was more sensitive than a conventional RT-PCR assay or culture isolation and proved suitable to detect SARS-CoV in clinical specimens. Application of this assay will aid in diagnosing SARS-CoV infection. PMID:15030703

Ring test evaluation of the detection of influenza A virus in swine oral fluids by real-time, reverse transcription polymerase chain reaction (rRT-PCR) and virus isolation

USDA-ARS?s Scientific Manuscript database

The probability of detecting influenza A virus (IAV) in oral fluid (OF) specimens was calculated for each of 13 real-time, reverse transcription polymerase chain reaction (rRT-PCR) and 7 virus isolation (VI) assays. To conduct the study, OF was inoculated with H1N1 or H3N2 IAV and serially 10-fold d...

Detection of mRNA by reverse transcription PCR as an indicator of viability in Phytophthora ramorum

Treesearch

Antonio Chimento; Santa Olga Cacciola; Matteo Garbelotto

2008-01-01

Real-Time PCR technologies offer increasing opportunities to detect and study phytopathogenic fungi. They combine the sensitivity of conventional PCR with the generation of a specific fluorescent signal providing both real-time analysis of the reaction kinetics and quantification of specific DNA targets. Before the development of Real-Time PCR and...

Performance optimization of PM-16QAM transmission system enabled by real-time self-adaptive coding.

PubMed

Qu, Zhen; Li, Yao; Mo, Weiyang; Yang, Mingwei; Zhu, Shengxiang; Kilper, Daniel C; Djordjevic, Ivan B

2017-10-15

We experimentally demonstrate self-adaptive coded 5×100  Gb/s WDM polarization multiplexed 16 quadrature amplitude modulation transmission over a 100 km fiber link, which is enabled by a real-time control plane. The real-time optical signal-to-noise ratio (OSNR) is measured using an optical performance monitoring device. The OSNR measurement is processed and fed back using control plane logic and messaging to the transmitter side for code adaptation, where the binary data are adaptively encoded with three types of low-density parity-check (LDPC) codes with code rates of 0.8, 0.75, and 0.7 of large girth. The total code-adaptation latency is measured to be 2273 ms. Compared with transmission without adaptation, average net capacity improvements of 102%, 36%, and 7.5% are obtained, respectively, by adaptive LDPC coding.

Application of a molecular beacon based real-time isothermal amplification (MBRTIA) technology for simultaneous detection of Bacillus cereus and Staphylococcus aureus.

PubMed

Mandappa, I M; Joglekar, Prasanna; Manonmani, H K

2015-07-01

A multiplex real-time isothermal amplification assay was developed using molecular beacons for the detection of Bacillus cereus and Staphylococcus aureus by targeting four important virulence genes. A correlation between targeting highly accessible DNA sequences and isothermal amplification based molecular beacon efficiency and sensitivity was demonstrated using phi(Φ)29 DNA polymerase at a constant isothermal temperature of 30 °C. It was very selective and consistently detected down to 10(1) copies of DNA. The specificity and sensitivity of this assay, when tested with pure culture were high, surpassing those of currently used PCR assays for the detection of these organisms. The molecular beacon based real-time isothermal amplification (MBRTIA) assay could be carried out entirely in 96 well plates or well strips, enabling a rapid and high-throughput detection of food borne pathogens.

Automated high-throughput flow-through real-time diagnostic system

DOEpatents

Regan, John Frederick

2012-10-30

An automated real-time flow-through system capable of processing multiple samples in an asynchronous, simultaneous, and parallel fashion for nucleic acid extraction and purification, followed by assay assembly, genetic amplification, multiplex detection, analysis, and decontamination. The system is able to hold and access an unlimited number of fluorescent reagents that may be used to screen samples for the presence of specific sequences. The apparatus works by associating extracted and purified sample with a series of reagent plugs that have been formed in a flow channel and delivered to a flow-through real-time amplification detector that has a multiplicity of optical windows, to which the sample-reagent plugs are placed in an operative position. The diagnostic apparatus includes sample multi-position valves, a master sample multi-position valve, a master reagent multi-position valve, reagent multi-position valves, and an optical amplification/detection system.

Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification.

PubMed

Janse, Ingmar; Hamidjaja, Raditijo A; Bok, Jasper M; van Rotterdam, Bart J

2010-12-08

Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples. Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains. The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum.

Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification

PubMed Central

2010-01-01

Background Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples. Results Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains. Conclusions The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum PMID:21143837

Parallel transmit beamforming using orthogonal frequency division multiplexing applied to harmonic imaging--a feasibility study.

PubMed

Demi, Libertario; Verweij, Martin D; Van Dongen, Koen W A

2012-11-01

Real-time 2-D or 3-D ultrasound imaging systems are currently used for medical diagnosis. To achieve the required data acquisition rate, these systems rely on parallel beamforming, i.e., a single wide-angled beam is used for transmission and several narrow parallel beams are used for reception. When applied to harmonic imaging, the demand for high-amplitude pressure wave fields, necessary to generate the harmonic components, conflicts with the use of a wide-angled beam in transmission because this results in a large spatial decay of the acoustic pressure. To enhance the amplitude of the harmonics, it is preferable to do the reverse: transmit several narrow parallel beams and use a wide-angled beam in reception. Here, this concept is investigated to determine whether it can be used for harmonic imaging. The method proposed in this paper relies on orthogonal frequency division multiplexing (OFDM), which is used to create distinctive parallel beams in transmission. To test the proposed method, a numerical study has been performed, in which the transmit, receive, and combined beam profiles generated by a linear array have been simulated for the second-harmonic component. Compared with standard parallel beamforming, application of the proposed technique results in a gain of 12 dB for the main beam and in a reduction of the side lobes. Experimental verification in water has also been performed. Measurements obtained with a single-element emitting transducer and a hydrophone receiver confirm the possibility of exciting a practical ultrasound transducer with multiple Gaussian modulated pulses, each having a different center frequency, and the capability to generate distinguishable second-harmonic components.

Viremia and Clinical Presentation in Nicaraguan Patients Infected With Zika Virus, Chikungunya Virus, and Dengue Virus.

PubMed

Waggoner, Jesse J; Gresh, Lionel; Vargas, Maria Jose; Ballesteros, Gabriela; Tellez, Yolanda; Soda, K James; Sahoo, Malaya K; Nuñez, Andrea; Balmaseda, Angel; Harris, Eva; Pinsky, Benjamin A

2016-12-15

 Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV) cocirculate in Nicaragua. In this study, we sought to compare the quantified viremia and clinical presentation of patients infected with 1 or more of these viruses.  Acute-phase serum samples from 346 patients with a suspected arboviral illness were tested using a multiplex real-time reverse-transcription polymerase chain reaction for ZIKV, CHIKV, and DENV. Viremia was quantitated for each detected virus, and clinical information from request forms submitted with each sample was recorded.  A total of 263 patients tested positive for 1 or more viruses: 192 patients tested positive for a single virus (monoinfections) and 71 patients tested positive for 2 or all 3 viruses (coinfections). Quantifiable viremia was lower in ZIKV infections compared with CHIKV or DENV (mean 4.70 vs 6.42 and 5.84 log 10 copies/mL serum, respectively; P < .001 for both comparisons), and for each virus, mean viremia was significantly lower in coinfections than in monoinfections. Compared with patients with CHIKV or DENV, ZIKV patients were more likely to have a rash (P < .001) and less likely to be febrile (P < .05) or require hospitalization (P < .001). Among all patients, hospitalized cases had higher viremia than those who did not require hospitalization (7.1 vs 4.1 log10 copies/mL serum, respectively; P < .001).  ZIKV, CHIKV, and DENV result in similar clinical presentations, and coinfections may be relatively common. Our findings illustrate the need for accurate, multiplex diagnostics for patient care and epidemiologic surveillance. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.

Viremia and Clinical Presentation in Nicaraguan Patients Infected With Zika Virus, Chikungunya Virus, and Dengue Virus

PubMed Central

Waggoner, Jesse J.; Gresh, Lionel; Vargas, Maria Jose; Ballesteros, Gabriela; Tellez, Yolanda; Soda, K. James; Sahoo, Malaya K.; Nuñez, Andrea; Balmaseda, Angel; Harris, Eva; Pinsky, Benjamin A.

2016-01-01

Background. Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV) cocirculate in Nicaragua. In this study, we sought to compare the quantified viremia and clinical presentation of patients infected with 1 or more of these viruses. Methods. Acute-phase serum samples from 346 patients with a suspected arboviral illness were tested using a multiplex real-time reverse-transcription polymerase chain reaction for ZIKV, CHIKV, and DENV. Viremia was quantitated for each detected virus, and clinical information from request forms submitted with each sample was recorded. Results. A total of 263 patients tested positive for 1 or more viruses: 192 patients tested positive for a single virus (monoinfections) and 71 patients tested positive for 2 or all 3 viruses (coinfections). Quantifiable viremia was lower in ZIKV infections compared with CHIKV or DENV (mean 4.70 vs 6.42 and 5.84 log10 copies/mL serum, respectively; P < .001 for both comparisons), and for each virus, mean viremia was significantly lower in coinfections than in monoinfections. Compared with patients with CHIKV or DENV, ZIKV patients were more likely to have a rash (P < .001) and less likely to be febrile (P < .05) or require hospitalization (P < .001). Among all patients, hospitalized cases had higher viremia than those who did not require hospitalization (7.1 vs 4.1 log10 copies/mL serum, respectively; P < .001). Conclusions. ZIKV, CHIKV, and DENV result in similar clinical presentations, and coinfections may be relatively common. Our findings illustrate the need for accurate, multiplex diagnostics for patient care and epidemiologic surveillance. PMID:27578819

Method for collecting thermocouple data via secured shell over a wireless local area network in real time

NASA Astrophysics Data System (ADS)

Arnold, F.; DeMallie, I.; Florence, L.; Kashinski, D. O.

2015-03-01

This manuscript addresses the design, hardware details, construction, and programming of an apparatus allowing an experimenter to monitor and record high-temperature thermocouple measurements of dynamic systems in real time. The apparatus uses wireless network technology to bridge the gap between a dynamic (moving) sample frame and the static laboratory frame. Our design is a custom solution applied to samples that rotate through large angular displacements where hard-wired and typical slip-ring solutions are not practical because of noise considerations. The apparatus consists of a Raspberry PI mini-Linux computer, an Arduino micro-controller, an Ocean Controls thermocouple multiplexer shield, and k-type thermocouples.

Method for collecting thermocouple data via secured shell over a wireless local area network in real time.

PubMed

Arnold, F; DeMallie, I; Florence, L; Kashinski, D O

2015-03-01

This manuscript addresses the design, hardware details, construction, and programming of an apparatus allowing an experimenter to monitor and record high-temperature thermocouple measurements of dynamic systems in real time. The apparatus uses wireless network technology to bridge the gap between a dynamic (moving) sample frame and the static laboratory frame. Our design is a custom solution applied to samples that rotate through large angular displacements where hard-wired and typical slip-ring solutions are not practical because of noise considerations. The apparatus consists of a Raspberry PI mini-Linux computer, an Arduino micro-controller, an Ocean Controls thermocouple multiplexer shield, and k-type thermocouples.

Single-shot ultrafast tomographic imaging by spectral multiplexing

NASA Astrophysics Data System (ADS)

Matlis, N. H.; Axley, A.; Leemans, W. P.

2012-10-01

Computed tomography has profoundly impacted science, medicine and technology by using projection measurements scanned over multiple angles to permit cross-sectional imaging of an object. The application of computed tomography to moving or dynamically varying objects, however, has been limited by the temporal resolution of the technique, which is set by the time required to complete the scan. For objects that vary on ultrafast timescales, traditional scanning methods are not an option. Here we present a non-scanning method capable of resolving structure on femtosecond timescales by using spectral multiplexing of a single laser beam to perform tomographic imaging over a continuous range of angles simultaneously. We use this technique to demonstrate the first single-shot ultrafast computed tomography reconstructions and obtain previously inaccessible structure and position information for laser-induced plasma filaments. This development enables real-time tomographic imaging for ultrafast science, and offers a potential solution to the challenging problem of imaging through scattering surfaces.

High Throughput, Multiplexed Pathogen Detection Authenticates Plague Waves in Medieval Venice, Italy

PubMed Central

Tran, Thi-Nguyen-Ny; Signoli, Michel; Fozzati, Luigi; Aboudharam, Gérard; Raoult, Didier; Drancourt, Michel

2011-01-01

Background Historical records suggest that multiple burial sites from the 14th–16th centuries in Venice, Italy, were used during the Black Death and subsequent plague epidemics. Methodology/Principal Findings High throughput, multiplexed real-time PCR detected DNA of seven highly transmissible pathogens in 173 dental pulp specimens collected from 46 graves. Bartonella quintana DNA was identified in five (2.9%) samples, including three from the 16th century and two from the 15th century, and Yersinia pestis DNA was detected in three (1.7%) samples, including two from the 14th century and one from the 16th century. Partial glpD gene sequencing indicated that the detected Y. pestis was the Orientalis biotype. Conclusions These data document for the first time successive plague epidemics in the medieval European city where quarantine was first instituted in the 14th century. PMID:21423736

Fast reversible learning based on neurons functioning as anisotropic multiplex hubs

NASA Astrophysics Data System (ADS)

Vardi, Roni; Goldental, Amir; Sheinin, Anton; Sardi, Shira; Kanter, Ido

2017-05-01

Neural networks are composed of neurons and synapses, which are responsible for learning in a slow adaptive dynamical process. Here we experimentally show that neurons act like independent anisotropic multiplex hubs, which relay and mute incoming signals following their input directions. Theoretically, the observed information routing enriches the computational capabilities of neurons by allowing, for instance, equalization among different information routes in the network, as well as high-frequency transmission of complex time-dependent signals constructed via several parallel routes. In addition, this kind of hubs adaptively eliminate very noisy neurons from the dynamics of the network, preventing masking of information transmission. The timescales for these features are several seconds at most, as opposed to the imprint of information by the synaptic plasticity, a process which exceeds minutes. Results open the horizon to the understanding of fast and adaptive learning realities in higher cognitive brain's functionalities.

Validation and application of a quantitative real-time PCR assay to detect common wheat adulteration of durum wheat for pasta production.

PubMed

Carloni, Elisa; Amagliani, Giulia; Omiccioli, Enrica; Ceppetelli, Veronica; Del Mastro, Michele; Rotundo, Luca; Brandi, Giorgio; Magnani, Mauro

2017-06-01

Pasta is the Italian product par excellence and it is now popular worldwide. Pasta of a superior quality is made with pure durum wheat. In Italy, addition of Triticum aestivum (common wheat) during manufacturing is not allowed and, without adequate labeling, its presence is considered an adulteration. PCR-related techniques can be employed for the detection of common wheat contaminations. In this work, we demonstrated that a previously published method for the detection of T. aestivum, based on the gliadin gene, is inadequate. Moreover, a new molecular method, based on DNA extraction from semolina and real-time PCR determination of T. aestivum in Triticum spp., was validated. This multiplex real-time PCR, based on the dual-labeled probe strategy, guarantees target detection specificity and sensitivity in a short period of time. Moreover, the molecular analysis of common wheat contamination in commercial wheat and flours is described for the first time. Copyright © 2016 Elsevier Ltd. All rights reserved.

Use of the melting curve assay as a means for high-throughput quantification of Illumina sequencing libraries.

PubMed

Shinozuka, Hiroshi; Forster, John W

2016-01-01

Background. Multiplexed sequencing is commonly performed on massively parallel short-read sequencing platforms such as Illumina, and the efficiency of library normalisation can affect the quality of the output dataset. Although several library normalisation approaches have been established, none are ideal for highly multiplexed sequencing due to issues of cost and/or processing time. Methods. An inexpensive and high-throughput library quantification method has been developed, based on an adaptation of the melting curve assay. Sequencing libraries were subjected to the assay using the Bio-Rad Laboratories CFX Connect(TM) Real-Time PCR Detection System. The library quantity was calculated through summation of reduction of relative fluorescence units between 86 and 95 °C. Results.PCR-enriched sequencing libraries are suitable for this quantification without pre-purification of DNA. Short DNA molecules, which ideally should be eliminated from the library for subsequent processing, were differentiated from the target DNA in a mixture on the basis of differences in melting temperature. Quantification results for long sequences targeted using the melting curve assay were correlated with those from existing methods (R (2) > 0.77), and that observed from MiSeq sequencing (R (2) = 0.82). Discussion.The results of multiplexed sequencing suggested that the normalisation performance of the described method is equivalent to that of another recently reported high-throughput bead-based method, BeNUS. However, costs for the melting curve assay are considerably lower and processing times shorter than those of other existing methods, suggesting greater suitability for highly multiplexed sequencing applications.

A robust multiplex real-time PCR method for simultaneous detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes in fresh fruits and vegetables

USDA-ARS?s Scientific Manuscript database

Introduction: On average, about 48 million people per year in the U.S. are affected by food borne diseases. A major portion of these illnesses are caused by Salmonella spp., Escherichia coli O157:H7 and Listeria monocytogenes. Hence, it is important to identify the specific pathogens in contaminate...

Development of a real-time RT-PCR assay for squash mosaic virus useful for broad spectrum detection of various serotypes and its incorporation into a multiplex seed health assay

USDA-ARS?s Scientific Manuscript database

Seed-borne pathogens pose a serious threat to modern agricultural cropping systems as they can be disseminated to many geographical regions around the world. With trends of increasing global seed production and trade, seed-health testing is an important quality control step to prevent the introduct...

Rapid multiplex PCR and Real-Time TaqMan PCR assays for detection of Salmonella enterica and the highly virulent serovars Choleraesuis and Paratyphi C

USDA-ARS?s Scientific Manuscript database

Salmonella enterica is a human pathogen with over 2,500 serovars characterized. S. enterica serovars Choleraesuis (Cs) and Paratyphi C (Pc) are two globally distributed serovars. We have developed a rapid molecular typing method to detect Cs and Pc in food samples by using a comparative genomics ap...

Emergence of Multiplex Communities in Collaboration Networks.

PubMed

Battiston, Federico; Iacovacci, Jacopo; Nicosia, Vincenzo; Bianconi, Ginestra; Latora, Vito

2016-01-01

Community structures in collaboration networks reflect the natural tendency of individuals to organize their work in groups in order to better achieve common goals. In most of the cases, individuals exploit their connections to introduce themselves to new areas of interests, giving rise to multifaceted collaborations which span different fields. In this paper, we analyse collaborations in science and among movie actors as multiplex networks, where the layers represent respectively research topics and movie genres, and we show that communities indeed coexist and overlap at the different layers of such systems. We then propose a model to grow multiplex networks based on two mechanisms of intra and inter-layer triadic closure which mimic the real processes by which collaborations evolve. We show that our model is able to explain the multiplex community structure observed empirically, and we infer the strength of the two underlying social mechanisms from real-world systems. Being also able to correctly reproduce the values of intra-layer and inter-layer assortativity correlations, the model contributes to a better understanding of the principles driving the evolution of social networks.

A novel duplex real time quantitative reverse transcription polymerase chain reaction for rubella virus with armored RNA as a noncompetitive internal positive control.

PubMed

Zhao, Lihong; Li, Ruiying; Liu, Aihua; Zhao, Shuping

2015-07-01

The objective of this study was to build and apply a duplex real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) for rubella virus. Firstly, a 60-bp-long armored RV RNA was constructed in the laboratory. Secondly, a duplex real time RT-PCR assay was established. Thirdly, the 60-bp-long armored RV RNA was used as an internal positive control (IPC) for the duplex real time RT-PCR. And finally the duplex real time RT-PCR assay was applied to detect RV RNA in clinical specimens. The in-house assay has a high amplification efficiency (0.99), a high analytical sensitivity (200 copies/mL), and a good reproducibility. The diagnostic specificity and sensitivity of the in-house assay were both 100%, due to the monitoring of the armored RV RNA IPC. Therefore, the in-house duplex real time quantitative RT-PCR assay is a specific, sensitive, reproducible and accurate assay for quantitation of RV RNA in clinical specimens. And noncompetitive armored RV RNA IPC can monitor RT-PCR inhibition and prevent false-negative and inaccurate results in the real time detection system. Copyright © 2015 Elsevier B.V. All rights reserved.

Real-time PCR in virology.

PubMed

Mackay, Ian M; Arden, Katherine E; Nitsche, Andreas

2002-03-15

The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5' endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease.

Prospective evaluation of the SeptiFAST multiplex real-time PCR assay for surveillance and diagnosis of infections in haematological patients after allogeneic stem cell transplantation compared to routine microbiological assays and an in-house real-time PCR method.

PubMed

Elges, Sandra; Arnold, Renate; Liesenfeld, Oliver; Kofla, Grzegorz; Mikolajewska, Agata; Schwartz, Stefan; Uharek, Lutz; Ruhnke, Markus

2017-12-01

We prospectively evaluated a multiplex real-time PCR assay (SeptiFast, SF) in a cohort of patients undergoing allo-BMT in comparison to an in-house PCR method (IH-PCR). Overall 847 blood samples (mean 8 samples/patient) from 104 patients with haematological malignancies were analysed. The majority of patients had acute leukaemia (62%) with a mean age of 52 years (54% female). Pathogens could be detected in 91 of 847 (11%) samples by SF compared to 38 of 205 (18.5%) samples by BC, and 57 of 847 (6.7%) samples by IH-PCR. Coagulase-negative staphylococci (n=41 in SF, n=29 in BC) were the most frequently detected bacteria followed by Escherichia coli (n=9 in SF, n=6 in BC). Candida albicans (n=17 in SF, n=0 in BC, n=24 in IH-PCR) was the most frequently detected fungal pathogen. SF gave positive results in 5% of samples during surveillance vs in 26% of samples during fever episodes. Overall, the majority of blood samples gave negative results in both PCR methods resulting in 93% overall agreement resulting in a negative predictive value of 0.96 (95% CI: 0.95-0.97), and a positive predictive value of 0.10 (95% CI: -0.01 to 0.21). SeptiFast appeared to be superior over BC and the IH-PCR method. © 2017 Blackwell Verlag GmbH.

The new challenges of multiplex networks: Measures and models

NASA Astrophysics Data System (ADS)

Battiston, Federico; Nicosia, Vincenzo; Latora, Vito

2017-02-01

What do societies, the Internet, and the human brain have in common? They are all examples of complex relational systems, whose emerging behaviours are largely determined by the non-trivial networks of interactions among their constituents, namely individuals, computers, or neurons, rather than only by the properties of the units themselves. In the last two decades, network scientists have proposed models of increasing complexity to better understand real-world systems. Only recently we have realised that multiplexity, i.e. the coexistence of several types of interactions among the constituents of a complex system, is responsible for substantial qualitative and quantitative differences in the type and variety of behaviours that a complex system can exhibit. As a consequence, multilayer and multiplex networks have become a hot topic in complexity science. Here we provide an overview of some of the measures proposed so far to characterise the structure of multiplex networks, and a selection of models aiming at reproducing those structural properties and quantifying their statistical significance. Focusing on a subset of relevant topics, this brief review is a quite comprehensive introduction to the most basic tools for the analysis of multiplex networks observed in the real-world. The wide applicability of multiplex networks as a framework to model complex systems in different fields, from biology to social sciences, and the colloquial tone of the paper will make it an interesting read for researchers working on both theoretical and experimental analysis of networked systems.

[Study on high accuracy detection of multi-component gas in oil-immerse power transformer].

PubMed

Fan, Jie; Chen, Xiao; Huang, Qi-Feng; Zhou, Yu; Chen, Gang

2013-12-01

In order to solve the problem of low accuracy and mutual interference in multi-component gas detection, a kind of multi-component gas detection network with high accuracy was designed. A semiconductor laser with narrow bandwidth was utilized as light source and a novel long-path gas cell was also used in this system. By taking the single sine signal to modulate the spectrum of laser and using space division multiplexing (SDM) and time division multiplexing (TDM) technique, the detection of multi-component gas was achieved. The experiments indicate that the linearity relevance coefficient is 0. 99 and the measurement relative error is less than 4%. The system dynamic response time is less than 15 s, by filling a volume of multi-component gas into the gas cell gradually. The system has advantages of high accuracy and quick response, which can be used in the fault gas on-line monitoring for power transformers in real time.

Avian Metapneumovirus circulation in Italian broiler farms.

PubMed

Tucciarone, Claudia Maria; Franzo, Giovanni; Lupini, Caterina; Alejo, Carolina Torres; Listorti, Valeria; Mescolini, Giulia; Brandão, Paulo Eduardo; Martini, Marco; Catelli, Elena; Cecchinato, Mattia

2018-02-01

With increasing frequency, avian Metapneumovirus (aMPV) is reported to induce respiratory signs in chickens. An adequate knowledge of current aMPV prevalence among Italian broilers is lacking, with little information available on its economical and health impact on the poultry industry. In order to collect preliminary data on the epidemiological context of aMPV in broiler flocks, a survey was performed in areas of Northern Italy with high poultry density from 2014 to 2016. Upper respiratory tract swabs were collected and processed by A and B subtype-specific multiplex real-time reverse transcription PCR (RT-PCR). Samples were also screened for infectious bronchitis virus (IBV) by generic RT-PCR and sequencing. Productive data and respiratory signs were detailed where possible. The high prevalence of aMPV was confirmed in broilers older than 26 d and also attested in IBV-negative farms. All aMPV detections belonged to subtype B. Italian strain genetic variability was evaluated by the partial attachment (G) gene sequencing of selected strains and compared with contemporary turkey strains and previously published aMPV references, revealing no host specificity and the progressive evolution of this virus in Italy. © 2017 Poultry Science Association Inc.

Sequence Optimized Real-Time RT-PCR Assay for Detection of Crimean-Congo Hemorrhagic Fever Virus

DTIC Science & Technology

2017-03-21

19-23]. Real-56 time reverse-transcription PCR remains the gold standard for quantitative , sensitive, and specific 57 detection of CCHFV; however...five-fold in two different series , and samples were run by real- time RT-PCR 116 in triplicate. The preliminary LOD was the lowest RNA dilution where...1 Sequence optimized real- time RT-PCR assay for detection of Crimean-Congo hemorrhagic fever 1 virus 2 3 JW Koehler1, KL Delp1, AT Hall1, SP

Application of a Multiplex Quantitative PCR to Assess Prevalence and Intensity Of Intestinal Parasite Infections in a Controlled Clinical Trial

PubMed Central

Llewellyn, Stacey; Inpankaew, Tawin; Nery, Susana Vaz; Gray, Darren J.; Verweij, Jaco J.; Clements, Archie C. A.; Gomes, Santina J.; Traub, Rebecca; McCarthy, James S.

2016-01-01

Background Accurate quantitative assessment of infection with soil transmitted helminths and protozoa is key to the interpretation of epidemiologic studies of these parasites, as well as for monitoring large scale treatment efficacy and effectiveness studies. As morbidity and transmission of helminth infections are directly related to both the prevalence and intensity of infection, there is particular need for improved techniques for assessment of infection intensity for both purposes. The current study aimed to evaluate two multiplex PCR assays to determine prevalence and intensity of intestinal parasite infections, and compare them to standard microscopy. Methodology/Principal Findings Faecal samples were collected from a total of 680 people, originating from rural communities in Timor-Leste (467 samples) and Cambodia (213 samples). DNA was extracted from stool samples and subject to two multiplex real-time PCR reactions the first targeting: Necator americanus, Ancylostoma spp., Ascaris spp., and Trichuris trichiura; and the second Entamoeba histolytica, Cryptosporidium spp., Giardia. duodenalis, and Strongyloides stercoralis. Samples were also subject to sodium nitrate flotation for identification and quantification of STH eggs, and zinc sulphate centrifugal flotation for detection of protozoan parasites. Higher parasite prevalence was detected by multiplex PCR (hookworms 2.9 times higher, Ascaris 1.2, Giardia 1.6, along with superior polyparasitism detection with this effect magnified as the number of parasites present increased (one: 40.2% vs. 38.1%, two: 30.9% vs. 12.9%, three: 7.6% vs. 0.4%, four: 0.4% vs. 0%). Although, all STH positive samples were low intensity infections by microscopy as defined by WHO guidelines the DNA-load detected by multiplex PCR suggested higher intensity infections. Conclusions/Significance Multiplex PCR, in addition to superior sensitivity, enabled more accurate determination of infection intensity for Ascaris, hookworms and Giardia compared to microscopy, especially in samples exhibiting polyparasitism. The superior performance of multiplex PCR to detect polyparasitism and more accurately determine infection intensity suggests that it is a more appropriate technique for use in epidemiologic studies and for monitoring large-scale intervention trials. PMID:26820626

Experimental demonstration of the real-time online fault monitoring technique for chaos-based passive optical networks

NASA Astrophysics Data System (ADS)

Dou, Xinyu; Yin, Hongxi; Yue, Hehe; Jin, Yu; Shen, Jing; Li, Lin

2015-09-01

In this paper, a real-time online fault monitoring technique for chaos-based passive optical networks (PONs) is proposed and experimentally demonstrated. The fault monitoring is performed by the chaotic communication signal. The proof-of-concept experiments are demonstrated for two PON structures, i.e., wavelength-division-multiplexing (WDM) PON and Ethernet PON (EPON), respectively. For WDM PON, two monitoring approaches are investigated, one deploying a chaotic optical time domain reflectometry (OTDR) for each transmitter, and the other using only one tunable chaotic OTDR. The experimental results show that the faults at beyond 20 km from the OLT can be detected and located. The spatial resolution of the tunable chaotic OTDR is an order of magnitude of centimeter. Meanwhile, the monitoring process can operate in parallel with the chaotic optical secure communications. The proposed technique has benefits of real-time, online, precise fault location, and simple realization, which will significantly reduce the cost of operation, administration and maintenance (OAM) of PON.

Real-Time Spatio-Temporal Twice Whitening for MIMO Energy Detector

DOE Office of Scientific and Technical Information (OSTI.GOV)

Humble, Travis S; Mitra, Pramita; Barhen, Jacob

2010-01-01

While many techniques exist for local spectrum sensing of a primary user, each represents a computationally demanding task to secondary user receivers. In software-defined radio, computational complexity lengthens the time for a cognitive radio to recognize changes in the transmission environment. This complexity is even more significant for spatially multiplexed receivers, e.g., in SIMO and MIMO, where the spatio-temporal data sets grow in size with the number of antennae. Limits on power and space for the processor hardware further constrain SDR performance. In this report, we discuss improvements in spatio-temporal twice whitening (STTW) for real-time local spectrum sensing by demonstratingmore » a form of STTW well suited for MIMO environments. We implement STTW on the Coherent Logix hx3100 processor, a multicore processor intended for low-power, high-throughput software-defined signal processing. These results demonstrate how coupling the novel capabilities of emerging multicore processors with algorithmic advances can enable real-time, software-defined processing of large spatio-temporal data sets.« less

Multiplex detection of protein-protein interactions using a next generation luciferase reporter.

PubMed

Verhoef, Lisette G G C; Mattioli, Michela; Ricci, Fernanda; Li, Yao-Cheng; Wade, Mark

2016-02-01

Cell-based assays of protein-protein interactions (PPIs) using split reporter proteins can be used to identify PPI agonists and antagonists. Generally, such assays measure one PPI at a time, and thus counterscreens for on-target activity must be run in parallel or at a subsequent stage; this increases both the cost and time during screening. Split luciferase systems offer advantages over those that use split fluorescent proteins (FPs). This is since split luciferase offers a greater signal:noise ratio and, unlike split FPs, the PPI can be reversed upon small molecule treatment. While multiplexed PPI assays using luciferase have been reported, they suffer from low signal:noise and require fairly complex spectral deconvolution during analysis. Furthermore, the luciferase enzymes used are large, which limits the range of PPIs that can be interrogated due to steric hindrance from the split luciferase fragments. Here, we report a multiplexed PPI assay based on split luciferases from Photinus pyralis (firefly luciferase, FLUC) and the deep-sea shrimp, Oplophorus gracilirostris (NanoLuc, NLUC). Specifically, we show that the binding of the p53 tumor suppressor to its two major negative regulators, MDM2 and MDM4, can be simultaneously measured within the same sample, without the requirement for complex filters or deconvolution. We provide chemical and genetic validation of this system using MDM2-targeted small molecules and mutagenesis, respectively. Combined with the superior signal:noise and smaller size of split NanoLuc, this multiplexed PPI assay format can be exploited to study the induction or disruption of pairwise interactions that are prominent in many cell signaling pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

Persistence of Penaeus stylirostris densovirus delays mortality caused by white spot syndrome virus infection in black tiger shrimp (Penaeus monodon)

PubMed Central

2013-01-01

Background Persistent infection of Penaeus stylirostris densovirus (PstDNV) (also called IHHNV) and its non-infectious inserts in the black tiger shrimp, Penaeus monodon (P. monodon) genome are commonly found without apparent disease. Here, we introduced the method of multiplex PCR in order to differentiate shrimp with viral inserts from ones with the infectious virus. The method allowed us to study the effect of pre-infection of IHHNV, in comparison to IHHNV inserts, on WSSV resistance in P. monodon. Results A multiplex PCR system was developed to amplify the entire IHHNV genome, ensuring the accurate diagnosis. Field samples containing IHHNV DNA templates as low as 20 pg or equivalent 150 viral copies can be detected by this method. By challenging the two groups of diagnosed shrimp with WSSV, we found that shrimp with IHHNV infection and those with viral inserts responded to WSSV differently. Considering cumulative mortality, average time to death of shrimp in IHHNV-infected group (day 14) was significantly delayed relative to that (day 10) of IHHNV-inserted group. Real-time PCR analysis of WSSV copy number indicated the lower amount of WSSV in the IHHNV-infected group than the virus-inserted group. The ratio of IHHNV: WSSV copy number in all determined IHHNV-infected samples ranged from approximately 4 to 300-fold. Conclusion The multiplex PCR assay developed herein proved optimal for convenient differentiation of shrimp specimens with real IHHNV infection and those with insert types. Diagnosed shrimp were also found to exhibit different WSSV tolerance. After exposed to WSSV, the naturally pre-infected IHHNV P. monodon were less susceptible to WSSV and, consequently, survived longer than the IHHNV-inserted shrimp. PMID:23414329

Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood.

PubMed

Zheng, Zhi; Luo, Yuling; McMaster, Gary K

2006-07-01

Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variations and biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format. We performed single- and multiplex gene expression analysis with multiple hybridization probes to capture mRNA directly from blood lysate and used branched DNA to amplify the signal. The 96-well plate singleplex assay uses chemiluminescence detection, and the multiplex assay combines Luminex-encoded beads with fluorescent detection. The single- and multiplex assays could quantitatively measure as few as 6000 and 24,000 mRNA target molecules (0.01 and 0.04 amoles), respectively, in up to 25 microL of whole blood. Both formats had CVs < 10% and dynamic ranges of 3-4 logs. Assay sensitivities allowed quantitative measurement of gene expression in the minority of cells in whole blood. The signals from whole-blood lysate correlated well with signals from purified RNA of the same sample, and absolute mRNA quantification results from the assay were similar to those obtained by quantitative reverse transcription-PCR. Both single- and multiplex assay formats were compatible with common anticoagulants and PAXgene-treated samples; however, PAXgene preparations induced expression of known antiapoptotic genes in whole blood. Both the singleplex and the multiplex branched DNA assays can quantitatively measure mRNA expression directly from small volumes of whole blood. The assay offers an alternative to current technologies that depend on RNA isolation and is amenable to high-throughput gene expression analysis of whole blood.

Multicentre study of Y chromosome microdeletions in 1,808 Chinese infertile males using multiplex and real-time polymerase chain reaction.

PubMed

Zhu, X-B; Gong, Y-H; He, J; Guo, A-L; Zhi, E-L; Yao, J-E; Zhu, B-S; Zhang, A-J; Li, Z

2017-06-01

Azoospermia factor (AZF) genes on the long arm of the human Y chromosome are involved in spermatogenesis, and microdeletions in the AZF region have been recognised to be the second major genetic cause of spermatogenetic failure resulting in male infertility. While screening for these microdeletions can avoid unnecessary medical and surgical treatments, current methods are generally time-consuming. Therefore, we established a new method to detect and analyse microdeletions in the AZF region quickly, safely and efficiently. In total, 1,808 patients with spermatogenetic failure were recruited from three hospitals in southern China, of which 600 patients were randomly selected for screening for Y chromosome microdeletions in AZF regions employing real-time polymerase chain reaction with a TaqMan probe. In our study, of 1,808 infertile patients, 150 (8.3%) were found to bear microdeletions in the Y chromosome using multiplex PCR, while no deletions were found in the controls. Among the AZF deletions detected, two were in AZFa, three in AZFb, 35 in AZFc, three in AZFb+c and two in AZFa+b+c. Our method is fast-it permits the scanning of DNA from a patient in one and a half hours-and reliable, minimising the risk of cross-contamination and false-positive and false-negative results. © 2016 Blackwell Verlag GmbH.

Analysis of Multiplexed Nanosensor Arrays Based on Near-Infrared Fluorescent Single-Walled Carbon Nanotubes.

PubMed

Dong, Juyao; Salem, Daniel P; Sun, Jessica H; Strano, Michael S

2018-04-24

The high-throughput, label-free detection of biomolecules remains an important challenge in analytical chemistry with the potential of nanosensors to significantly increase the ability to multiplex such assays. In this work, we develop an optical sensor array, printable from a single-walled carbon nanotube/chitosan ink and functionalized to enable a divalent ion-based proximity quenching mechanism for transducing binding between a capture protein or an antibody with the target analyte. Arrays of 5 × 6, 200 μm near-infrared (nIR) spots at a density of ≈300 spots/cm 2 are conjugated with immunoglobulin-binding proteins (proteins A, G, and L) for the detection of human IgG, mouse IgM, rat IgG2a, and human IgD. Binding kinetics are measured in a parallel, multiplexed fashion from each sensor spot using a custom laser scanning imaging configuration with an nIR photomultiplier tube detector. These arrays are used to examine cross-reactivity, competitive and nonspecific binding of analyte mixtures. We find that protein G and protein L functionalized sensors report selective responses to mouse IgM on the latter, as anticipated. Optically addressable platforms such as the one examined in this work have potential to significantly advance the real-time, multiplexed biomolecular detection of complex mixtures.

Molecular beacon probes-base multiplex NASBA Real-time for detection of HIV-1 and HCV.

PubMed

Mohammadi-Yeganeh, S; Paryan, M; Mirab Samiee, S; Kia, V; Rezvan, H

2012-06-01

Developed in 1991, nucleic acid sequence-based amplification (NASBA) has been introduced as a rapid molecular diagnostic technique, where it has been shown to give quicker results than PCR, and it can also be more sensitive. This paper describes the development of a molecular beacon-based multiplex NASBA assay for simultaneous detection of HIV-1 and HCV in plasma samples. A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used for primers and molecular beacon design. The performance features of HCV/HIV-1 multiplex NASBA assay including analytical sensitivity and specificity, clinical sensitivity and clinical specificity were evaluated. The analysis of scalar concentrations of the samples indicated that the limit of quantification of the assay was <1000 copies/ml for HIV-1 and <500 copies/ml for HCV with 95% confidence interval. Multiplex NASBA assay showed a 98% sensitivity and 100% specificity. The analytical specificity study with BLAST software demonstrated that the primers do not attach to any other sequences except for that of HIV-1 or HCV. The primers and molecular beacon probes detected all HCV genotypes and all major variants of HIV-1. This method may represent a relatively inexpensive isothermal method for detection of HIV-1/HCV co-infection in monitoring of patients.

Force-Balance Dynamic Display

NASA Technical Reports Server (NTRS)

Ferris, Alice T.; White, William C.

1988-01-01

Balance dynamic display unit (BDDU) is compact system conditioning six dynamic analog signals so they are monitored simultaneously in real time on single-trace oscilloscope. Typical BDDU oscilloscope display in scan mode shows each channel occupying one-sixth of total trace. System features two display modes usable with conventional, single-channel oscilloscope: multiplexed six-channel "bar-graph" format and single-channel display. Two-stage visual and audible limit alarm provided for each channel.

Holographic three-dimensional telepresence using large-area photorefractive polymer.

PubMed

Blanche, P-A; Bablumian, A; Voorakaranam, R; Christenson, C; Lin, W; Gu, T; Flores, D; Wang, P; Hsieh, W-Y; Kathaperumal, M; Rachwal, B; Siddiqui, O; Thomas, J; Norwood, R A; Yamamoto, M; Peyghambarian, N

2010-11-04

Holography is a technique that is used to display objects or scenes in three dimensions. Such three-dimensional (3D) images, or holograms, can be seen with the unassisted eye and are very similar to how humans see the actual environment surrounding them. The concept of 3D telepresence, a real-time dynamic hologram depicting a scene occurring in a different location, has attracted considerable public interest since it was depicted in the original Star Wars film in 1977. However, the lack of sufficient computational power to produce realistic computer-generated holograms and the absence of large-area and dynamically updatable holographic recording media have prevented realization of the concept. Here we use a holographic stereographic technique and a photorefractive polymer material as the recording medium to demonstrate a holographic display that can refresh images every two seconds. A 50 Hz nanosecond pulsed laser is used to write the holographic pixels. Multicoloured holographic 3D images are produced by using angular multiplexing, and the full parallax display employs spatial multiplexing. 3D telepresence is demonstrated by taking multiple images from one location and transmitting the information via Ethernet to another location where the hologram is printed with the quasi-real-time dynamic 3D display. Further improvements could bring applications in telemedicine, prototyping, advertising, updatable 3D maps and entertainment.

Multiplex real-time PCR using temperature sensitive primer-supplying hydrogel particles and its application for malaria species identification

PubMed Central

Byoun, Mun Sub; Yoo, Changhoon; Sim, Sang Jun; Lim, Chae Seung; Kim, Sung Woo

2018-01-01

Real-time PCR, also called quantitative PCR (qPCR), has been powerful analytical tool for detection of nucleic acids since it developed. Not only for biological research but also for diagnostic needs, qPCR technique requires capacity to detect multiple genes in recent years. Solid phase PCR (SP-PCR) where one or two directional primers are immobilized on solid substrates could analyze multiplex genetic targets. However, conventional SP-PCR was subjected to restriction of application for lack of PCR efficiency and quantitative resolution. Here we introduce an advanced qPCR with primer-incorporated network (PIN). One directional primers are immobilized in the porous hydrogel particle by covalent bond and the other direction of primers are temporarily immobilized at so-called 'Supplimers'. Supplimers released the primers to aqueous phase in the hydrogel at the thermal cycling of PCR. It induced the high PCR efficiency over 92% with high reliability. It reduced the formation of primer dimers and improved the selectivity of qPCR thanks to the strategy of 'right primers supplied to right place only'. By conducting a six-plex qPCR of 30 minutes, we analyzed DNA samples originated from malaria patients and successfully identified malaria species in a single reaction. PMID:29293604

A Single Molecular Beacon Probe Is Sufficient for the Analysis of Multiple Nucleic Acid Sequences

PubMed Central

Gerasimova, Yulia V.; Hayson, Aaron; Ballantyne, Jack; Kolpashchikov, Dmitry M.

2010-01-01

Molecular beacon (MB) probes are dual-labeled hairpin-shaped oligodeoxyribonucleotides that are extensively used for real-time detection of specific RNA/DNA analytes. In the MB probe, the loop fragment is complementary to the analyte: therefore, a unique probe is required for the analysis of each new analyte sequence. The conjugation of an oligonucleotide with two dyes and subsequent purification procedures add to the cost of MB probes, thus reducing their application in multiplex formats. Here we demonstrate how one MB probe can be used for the analysis of an arbitrary nucleic acid. The approach takes advantage of two oligonucleotide adaptor strands, each of which contains a fragment complementary to the analyte and a fragment complementary to an MB probe. The presence of the analyte leads to association of MB probe and the two DNA strands in quadripartite complex. The MB probe fluorescently reports the formation of this complex. In this design, the MB does not bind the analyte directly; therefore, the MB sequence is independent of the analyte. In this study one universal MB probe was used to genotype three human polymorphic sites. This approach promises to reduce the cost of multiplex real-time assays and improve the accuracy of single-nucleotide polymorphism genotyping. PMID:20665615

Designing Two-Layer Optical Networks with Statistical Multiplexing

NASA Astrophysics Data System (ADS)

Addis, B.; Capone, A.; Carello, G.; Malucelli, F.; Fumagalli, M.; Pedrin Elli, E.

The possibility of adding multi-protocol label switching (MPLS) support to transport networks is considered an important opportunity by telecom carriers that want to add packet services and applications to their networks. However, the question that arises is whether it is suitable to have MPLS nodes just at the edge of the network to collect packet traffic from users, or also to introduce MPLS facilities on a subset of the core nodes in order to exploit packet switching flexibility and multiplexing, thus providing induction of a better bandwidth allocation. In this article, we address this complex decisional problem with the support of a mathematical programming approach. We consider two-layer networks where MPLS is overlaid on top of transport networks-synchronous digital hierarchy (SDH) or wavelength division multiplexing (WDM)-depending on the required link speed. The discussions' decisions take into account the trade-off between the cost of adding MPLS support in the core nodes and the savings in the link bandwidth allocation due to the statistical multiplexing and the traffic grooming effects induced by MPLS nodes. The traffic matrix specifies for each point-to-point request a pair of values: a mean traffic value and an additional one. Using this traffic model, the effect of statistical multiplexing on a link allows the allocation of a capacity equal to the sum of all the mean values of the traffic demands routed on the link and only the highest additional one. The proposed approach is suitable to solve real instances in reasonable time.

Detection of enteroviruses and hepatitis a virus in water by consensus primer multiplex RT-PCR

PubMed Central

Li, Jun-Wen; Wang, Xin-Wei; Yuan, Chang-Qing; Zheng, Jin-Lai; Jin, Min; Song, Nong; Shi, Xiu-Quan; Chao, Fu-Huan

2002-01-01

AIM: To develop a rapid detection method of enteroviruses and Hepatitis A virus (HAV). METHODS: A one-step, single-tube consensus primers multiplex RT-PCR was developed to simultaneously detect Poliovirus, Coxsackie virus, Echovirus and HAV. A general upstream primer and a HAV primer and four different sets of primers (5 primers) specific for Poliovirus, Coxsacki evirus, Echovirus and HAV cDNA were mixed in the PCR mixture to reverse transcript and amplify the target DNA. Four distinct amplified DNA segments representing Poliovirus, Coxsackie virus, Echovirus and HAV were identified by gel electrophoresis as 589-, 671-, 1084-, and 1128 bp sequences, respectively. Semi-nested PCR was used to confirm the amplified products for each enterovirus and HAV. RESULTS: All four kinds of viral genome RNA were detected, and producing four bands which could be differentiated by the band size on the gel. To confirm the specificity of the multiplex PCR products, semi-nested PCR was performed. For all the four strains tested gave positive results. The detection sensitivity of multiplex PCR was similar to that of monoplex RT-PCR which was 24 PFU for Poliovrus, 21 PFU for Coxsackie virus, 60 PFU for Echovirus and 105 TCID50 for HAV. The minimum amount of enteric viral RNA detected by semi-nested PCR was equivalent to 2.4 PFU for Poliovrus, 2.1 PFU for Coxsackie virus, 6.0 PFU for Echovirus and 10.5 TCID50 for HAV. CONCLUSION: The consensus primers multiplex RT-PCR has more advantages over monoplex RT-PCR for enteric viruses detection, namely, the rapid turnaround time and cost effectiveness. PMID:12174381

Reconstructing the world trade multiplex: The role of intensive and extensive biases

NASA Astrophysics Data System (ADS)

Mastrandrea, Rossana; Squartini, Tiziano; Fagiolo, Giorgio; Garlaschelli, Diego

2014-12-01

In economic and financial networks, the strength of each node has always an important economic meaning, such as the size of supply and demand, import and export, or financial exposure. Constructing null models of networks matching the observed strengths of all nodes is crucial in order to either detect interesting deviations of an empirical network from economically meaningful benchmarks or reconstruct the most likely structure of an economic network when the latter is unknown. However, several studies have proved that real economic networks and multiplexes topologically differ from configurations inferred only from node strengths. Here we provide a detailed analysis of the world trade multiplex by comparing it to an enhanced null model that simultaneously reproduces the strength and the degree of each node. We study several temporal snapshots and almost 100 layers (commodity classes) of the multiplex and find that the observed properties are systematically well reproduced by our model. Our formalism allows us to introduce the (static) concept of extensive and intensive bias, defined as a measurable tendency of the network to prefer either the formation of extra links or the reinforcement of link weights, with respect to a reference case where only strengths are enforced. Our findings complement the existing economic literature on (dynamic) intensive and extensive trade margins. More generally, they show that real-world multiplexes can be strongly shaped by layer-specific local constraints.

A novel approach for copy number variation analysis by combining multiplex PCR with matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

Gao, Yonghui; Chen, Xiaoli; Wang, Jianhua; Shangguan, Shaofang; Dai, Yaohua; Zhang, Ting; Liu, Junling

2013-06-20

With the increasing interest in copy number variation as it pertains to human genomic variation, common phenotypes, and disease susceptibility, there is a pressing need for methods to accurately identify copy number. In this study, we developed a simple approach that combines multiplex PCR with matrix-assisted laser desorption ionization time-of-flight mass spectrometry for submicroscopic copy number variation detection. Two pairs of primers were used to simultaneously amplify query and endogenous control regions in the same reaction. Using a base extension reaction, the two amplicons were then distinguished and quantified in a mass spectrometry map. The peak ratio between the test region and the endogenous control region was manually calculated. The relative copy number could be determined by comparing the peak ratio between the test and control samples. This method generated a copy number measurement comparable to those produced by two other commonly used methods - multiplex ligation-dependent probe amplification and quantitative real-time PCR. Furthermore, it can discriminate a wide range of copy numbers. With a typical 384-format SpectroCHIP, at least six loci on 384 samples can be analyzed simultaneously in a hexaplex assay, making this assay adaptable for high throughput, and potentially applicable for large-scale association studies. Copyright © 2013 Elsevier B.V. All rights reserved.

GMR biosensor arrays: a system perspective.

PubMed

Hall, D A; Gaster, R S; Lin, T; Osterfeld, S J; Han, S; Murmann, B; Wang, S X

2010-05-15

Giant magnetoresistive biosensors are becoming more prevalent for sensitive, quantifiable biomolecular detection. However, in order for magnetic biosensing to become competitive with current optical protein microarray technology, there is a need to increase the number of sensors while maintaining the high sensitivity and fast readout time characteristic of smaller arrays (1-8 sensors). In this paper, we present a circuit architecture scalable for larger sensor arrays (64 individually addressable sensors) while maintaining a high readout rate (scanning the entire array in less than 4s). The system utilizes both time domain multiplexing and frequency domain multiplexing in order to achieve this scan rate. For the implementation, we propose a new circuit architecture that does not use a classical Wheatstone bridge to measure the small change in resistance of the sensor. Instead, an architecture designed around a transimpedance amplifier is employed. A detailed analysis of this architecture including the noise, distortion, and potential sources of errors is presented, followed by a global optimization strategy for the entire system comprising the magnetic tags, sensors, and interface electronics. To demonstrate the sensitivity, quantifiable detection of two blindly spiked samples of unknown concentrations has been performed at concentrations below the limit of detection for the enzyme-linked immunosorbent assay. Lastly, the multiplexing capability and reproducibility of the system was demonstrated by simultaneously monitoring sensors functionalized with three unique proteins at different concentrations in real-time. 2010 Elsevier B.V. All rights reserved.

Health monitoring of unmanned aerial vehicle based on optical fiber sensor array

NASA Astrophysics Data System (ADS)

Luo, Yuxiang; Shen, Jingshi; Shao, Fei; Guo, Chunhui; Yang, Ning; Zhang, Jiande

2017-10-01

The unmanned aerial vehicle (UAV) in flight needs to face the complicated environment, especially to withstand harsh weather conditions, such as the temperature and pressure. Compared with conventional sensors, fiber Bragg grating (FBG) sensor has the advantages of small size, light weight, high reliability, high precision, anti-electromagnetic interference, long lift-span, moistureproof and good resistance to causticity. It's easy to be embedded in composite structural components of UAVs. In the paper, over 1000 FBG sensors distribute regularly on a wide range of UAVs body, combining wavelength division multiplexing (WDM), time division multiplexing (TDM) and multichannel parallel architecture. WDM has the advantage of high spatial resolution. TDM has the advantage of large capacity and wide range. It is worthful to constitute a sensor network by different technologies. For the signal demodulation of FBG sensor array, WDM works by means of wavelength scanning light sources and F-P etalon. TDM adopts the technology of optical time-domain reflectometry. In order to demodulate efficiently, the most proper sensor multiplex number with some reflectivity is given by the curves fitting. Due to the regular array arrangement of FBG sensors on the UAVs, we can acquire the health state of UAVs in the form of 3D visualization. It is helpful to master the information of health status rapidly and give a real-time health evaluation.

Multiplex reverse transcription polymerase chain reaction to study the expression of virulence and stress response genes in Staphylococcus aureus.

PubMed

Shrihari, Rohinishree Yadahalli; Singh, Negi Pradeep

2012-02-01

Staphylococcus aureus survives well in different stress conditions. The ability of this organism to adapt to various stresses is the result of a complex regulatory response, which is attributed to regulation of multiple genes. The aims of the present study were (1) to develop a multiplex PCR for the detection of genes which are involved in stress adaptation (asp23, dnaK, and groEL); alternative sigma factor (sigB) and virulence determination (entB and spa) and (2) to study the expression of these genes during stress conditions for S. aureus culture collection strains (FRI 722 and ATCC 6538) and S. aureus food isolates at mRNA level using multiplex reverse transcription polymerase chain reaction (RT-PCR). During heat shock treatment groEL, dnaK, asp23, sodA, entB, spa, and sigB genes were up regulated up to 2.58, 2.07, 2.76, 2.55, 3.55, 2.71, and 2.62- folds, respectively, whereas in acid shock treatment, sodA and groEL were up regulated; dnaK was downregulated; and entB and sigB genes were not expressed in food isolates. Multiplex PCR assay standardized in this study offers an inexpensive alternative to uniplex PCR for detection of various virulence and stress response genes. This study is relevant to rapid and accurate detection of potential pathogenic S. aureus in foods. © 2012 Institute of Food Technologists®

Multiplex visibility graphs to investigate recurrent neural network dynamics

NASA Astrophysics Data System (ADS)

Bianchi, Filippo Maria; Livi, Lorenzo; Alippi, Cesare; Jenssen, Robert

2017-03-01

A recurrent neural network (RNN) is a universal approximator of dynamical systems, whose performance often depends on sensitive hyperparameters. Tuning them properly may be difficult and, typically, based on a trial-and-error approach. In this work, we adopt a graph-based framework to interpret and characterize internal dynamics of a class of RNNs called echo state networks (ESNs). We design principled unsupervised methods to derive hyperparameters configurations yielding maximal ESN performance, expressed in terms of prediction error and memory capacity. In particular, we propose to model time series generated by each neuron activations with a horizontal visibility graph, whose topological properties have been shown to be related to the underlying system dynamics. Successively, horizontal visibility graphs associated with all neurons become layers of a larger structure called a multiplex. We show that topological properties of such a multiplex reflect important features of ESN dynamics that can be used to guide the tuning of its hyperparamers. Results obtained on several benchmarks and a real-world dataset of telephone call data records show the effectiveness of the proposed methods.

Multiplexed phase-space imaging for 3D fluorescence microscopy.

PubMed

Liu, Hsiou-Yuan; Zhong, Jingshan; Waller, Laura

2017-06-26

Optical phase-space functions describe spatial and angular information simultaneously; examples of optical phase-space functions include light fields in ray optics and Wigner functions in wave optics. Measurement of phase-space enables digital refocusing, aberration removal and 3D reconstruction. High-resolution capture of 4D phase-space datasets is, however, challenging. Previous scanning approaches are slow, light inefficient and do not achieve diffraction-limited resolution. Here, we propose a multiplexed method that solves these problems. We use a spatial light modulator (SLM) in the pupil plane of a microscope in order to sequentially pattern multiplexed coded apertures while capturing images in real space. Then, we reconstruct the 3D fluorescence distribution of our sample by solving an inverse problem via regularized least squares with a proximal accelerated gradient descent solver. We experimentally reconstruct a 101 Megavoxel 3D volume (1010×510×500µm with NA 0.4), demonstrating improved acquisition time, light throughput and resolution compared to scanning aperture methods. Our flexible patterning scheme further allows sparsity in the sample to be exploited for reduced data capture.

A Real-Time Clinical Endoscopic System for Intraluminal, Multiplexed Imaging of Surface-Enhanced Raman Scattering Nanoparticles

PubMed Central

Garai, Ellis; Loewke, Nathan O.; Rogalla, Stephan; Mandella, Michael J.; Felt, Stephen A.; Friedland, Shai; Liu, Jonathan T. C.; Gambhir, Sanjiv S.; Contag, Christopher H.

2015-01-01

The detection of biomarker-targeting surface-enhanced Raman scattering (SERS) nanoparticles (NPs) in the human gastrointestinal tract has the potential to improve early cancer detection; however, a clinically relevant device with rapid Raman-imaging capability has not been described. Here we report the design and in vivo demonstration of a miniature, non-contact, opto-electro-mechanical Raman device as an accessory to clinical endoscopes that can provide multiplexed molecular data via a panel of SERS NPs. This device enables rapid circumferential scanning of topologically complex luminal surfaces of hollow organs (e.g., colon and esophagus) and produces quantitative images of the relative concentrations of SERS NPs that are present. Human and swine studies have demonstrated the speed and simplicity of this technique. This approach also offers unparalleled multiplexing capabilities by simultaneously detecting the unique spectral fingerprints of multiple SERS NPs. Therefore, this new screening strategy has the potential to improve diagnosis and to guide therapy by enabling sensitive quantitative molecular detection of small and otherwise hard-to-detect lesions in the context of white-light endoscopy. PMID:25923788

Multiplex visibility graphs to investigate recurrent neural network dynamics

PubMed Central

Bianchi, Filippo Maria; Livi, Lorenzo; Alippi, Cesare; Jenssen, Robert

2017-01-01

A recurrent neural network (RNN) is a universal approximator of dynamical systems, whose performance often depends on sensitive hyperparameters. Tuning them properly may be difficult and, typically, based on a trial-and-error approach. In this work, we adopt a graph-based framework to interpret and characterize internal dynamics of a class of RNNs called echo state networks (ESNs). We design principled unsupervised methods to derive hyperparameters configurations yielding maximal ESN performance, expressed in terms of prediction error and memory capacity. In particular, we propose to model time series generated by each neuron activations with a horizontal visibility graph, whose topological properties have been shown to be related to the underlying system dynamics. Successively, horizontal visibility graphs associated with all neurons become layers of a larger structure called a multiplex. We show that topological properties of such a multiplex reflect important features of ESN dynamics that can be used to guide the tuning of its hyperparamers. Results obtained on several benchmarks and a real-world dataset of telephone call data records show the effectiveness of the proposed methods. PMID:28281563

Crosstalk analyse of DFB fiber laser hydrophone array based on time division multiplexing

NASA Astrophysics Data System (ADS)

Li, Yu; Huang, Junbin; Gu, Hongcan; Tang, Bo; Wu, Jing

2014-12-01

In this paper, the crosstalk of a time division multiplexed (TDM) system of distributed feedback (DFB) fiber laser (FL)hydrophones based on optical switch using Phase Generated Carrier (PGC) method was quantitatively analyzed. After mathematical deduction, the relationship among crosstalk, multiplexing scale and extinction ratio of optical switch was given. The simulation results show that to realize a TDM system of DFB fiber laser hydrophones with crosstalk lower than -40dB, the average extinction ratio should be higher than 24.78dB for a 4- channel system, while higher than 28.45dB for an 8- channel system. Two experiments to analyze the array crosstalk to a certain channel in an 8- channel array were conducted in this paper. Firstly, by testing the powers of leak laser to a certain channel from others, the array crosstalk to this channel was obtained according to the equation mathematically deduced in this paper. The result shows the array crosstalk to a certain channel of the 8-channel array was -7.6dB. An experiment of underwater acoustic detection was carried out finally to get the real array crosstalk to this certain channel, and the experimental result shows that the array crosstalk to this channel is -8.8dB, which is close to the calculated result.

Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species

PubMed Central

Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun

2017-01-01

ABSTRACT The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis (M. tuberculosis) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680, and (iv) simultaneously detect five clinically important NTM (M. avium, M. intracellulare, M. abscessus, M. massiliense, and M. kansasii) by targeting IS1311, DT1, mass_3210, and mkan_rs12360. An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 103 and 104 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis, M. tuberculosis Beijing genotype, and major NTM species. PMID:28659320

Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species.

PubMed

Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun; Koh, Won-Jung; Shin, Sung Jae

2017-09-01

The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis ( M. tuberculosis ) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680 , and (iv) simultaneously detect five clinically important NTM ( M. avium , M. intracellulare , M. abscessus , M. massiliense , and M. kansasii ) by targeting IS 1311 , DT1, mass_3210 , and mkan_rs12360 An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 10 3 and 10 4 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis , M. tuberculosis Beijing genotype, and major NTM species. Copyright © 2017 American Society for Microbiology.

Sensitive multiplex PCR assay to differentiate Lyme spirochetes and emerging pathogens Anaplasma phagocytophilum and Babesia microti.

PubMed

Chan, Kamfai; Marras, Salvatore A E; Parveen, Nikhat

2013-12-20

The infection with Borrelia burgdorferi can result in acute to chronic Lyme disease. In addition, coinfection with tick-borne pathogens, Babesia species and Anaplasma phagocytophilum has been increasing in endemic regions of the USA and Europe. The currently used serological diagnostic tests are often difficult to interpret and, moreover, antibodies against the pathogens persist for a long time making it difficult to confirm the cure of the disease. In addition, these tests cannot be used for diagnosis of early disease state before the adaptive immune response is established. Since nucleic acids of the pathogens do not persist after the cure, DNA-based diagnostic tests are becoming highly useful for detecting infectious diseases. In this study, we describe a real-time multiplex PCR assay to detect the presence of B. burgdorferi, B. microti and A. phagocytophilum simultaneously even when they are present in very low copy numbers. Interestingly, this quantitative PCR technique is also able to differentiate all three major Lyme spirochete species, B. burgdorferi, B. afzelii, and B. garinii by utilizing a post-PCR denaturation profile analysis and a single molecular beacon probe. This could be very useful for diagnosis and discrimination of various Lyme spirochetes in European countries where all three Lyme spirochete species are prevalent. As proof of the principle for patient samples, we detected the presence of low number of Lyme spirochetes spiked in the human blood using our assay. Finally, our multiplex assay can detect all three tick-borne pathogens in a sensitive and specific manner irrespective of the level of each pathogen present in the sample. We anticipate that this novel diagnostic method will be able to simultaneously diagnose early to chronic stages of Lyme disease, babesiosis and anaplasmosis using the patients' blood samples. Real-time quantitative PCR using specific primers and molecular beacon probes for the selected amplicon described in this study can detect three tick-borne pathogens simultaneously in an accurate manner.

Sensitive multiplex PCR assay to differentiate Lyme spirochetes and emerging pathogens Anaplasma phagocytophilum and Babesia microti

PubMed Central

2013-01-01

Background The infection with Borrelia burgdorferi can result in acute to chronic Lyme disease. In addition, coinfection with tick-borne pathogens, Babesia species and Anaplasma phagocytophilum has been increasing in endemic regions of the USA and Europe. The currently used serological diagnostic tests are often difficult to interpret and, moreover, antibodies against the pathogens persist for a long time making it difficult to confirm the cure of the disease. In addition, these tests cannot be used for diagnosis of early disease state before the adaptive immune response is established. Since nucleic acids of the pathogens do not persist after the cure, DNA-based diagnostic tests are becoming highly useful for detecting infectious diseases. Results In this study, we describe a real-time multiplex PCR assay to detect the presence of B. burgdorferi, B. microti and A. phagocytophilum simultaneously even when they are present in very low copy numbers. Interestingly, this quantitative PCR technique is also able to differentiate all three major Lyme spirochete species, B. burgdorferi, B. afzelii, and B. garinii by utilizing a post-PCR denaturation profile analysis and a single molecular beacon probe. This could be very useful for diagnosis and discrimination of various Lyme spirochetes in European countries where all three Lyme spirochete species are prevalent. As proof of the principle for patient samples, we detected the presence of low number of Lyme spirochetes spiked in the human blood using our assay. Finally, our multiplex assay can detect all three tick-borne pathogens in a sensitive and specific manner irrespective of the level of each pathogen present in the sample. We anticipate that this novel diagnostic method will be able to simultaneously diagnose early to chronic stages of Lyme disease, babesiosis and anaplasmosis using the patients’ blood samples. Conclusion Real-time quantitative PCR using specific primers and molecular beacon probes for the selected amplicon described in this study can detect three tick-borne pathogens simultaneously in an accurate manner. PMID:24359556

Digital image compression for a 2f multiplexing optical setup

NASA Astrophysics Data System (ADS)

Vargas, J.; Amaya, D.; Rueda, E.

2016-07-01

In this work a virtual 2f multiplexing system was implemented in combination with digital image compression techniques and redundant information elimination. Depending on the image type to be multiplexed, a memory-usage saving of as much as 99% was obtained. The feasibility of the system was tested using three types of images, binary characters, QR codes, and grey level images. A multiplexing step was implemented digitally, while a demultiplexing step was implemented in a virtual 2f optical setup following real experimental parameters. To avoid cross-talk noise, each image was codified with a specially designed phase diffraction carrier that would allow the separation and relocation of the multiplexed images on the observation plane by simple light propagation. A description of the system is presented together with simulations that corroborate the method. The present work may allow future experimental implementations that will make use of all the parallel processing capabilities of optical systems.

Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

PubMed Central

Abate, Teresa; Cayo, Nelly M.; Parrado, Rudy; Bello, Zoraida Diaz; Velazquez, Elsa; Muñoz-Calderon, Arturo; Juiz, Natalia A.; Basile, Joaquín; Garcia, Lineth; Riarte, Adelina; Nasser, Julio R.; Ocampo, Susana B.; Yadon, Zaida E.; Torrico, Faustino; de Noya, Belkisyole Alarcón; Ribeiro, Isabela; Schijman, Alejandro G.

2013-01-01

Background The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. Methods/Principal Findings We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. Conclusions/Significance The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment. PMID:23350002

One-step cross-genogroup multiplex RT-qPCR with an internal control system for the detection of infectious pancreatic necrosis virus (IPNV).

PubMed

Hoferer, Marc; Braun, Anne; Skrypski, Julia; Bock, Sabine; Thalheim, Sabine; Sting, Reinhard

2017-09-01

Infectious pancreatic necrosis virus (IPNV) causes great losses in fish hatcheries world-wide. The detection of IPNV can be challenging in certain circumstances, particularly due to low viral load and the genetic variability of this RNA virus. For the first time, this project created a quantitative triplex real-time reverse transcription PCR (RT-qPCR), including an endogenous control system, for specific, sensitive and rapid detection of IPNV in routine diagnostics. Multiple sequence alignment of 46 nucleotide sequences of the segment A genome obtained from the NCBI database allowed the design of two RT-qPCR systems covering the IPNV genogroup 1 and genogroups 2-5, respectively. The completed triplex RT-qPCR including a salmonid-specific endogenous control showed high specificity and an analytical sensitivity of 20-40 oligonucleotide copies. Testing of dilution series of virus-loaded cell culture suspensions proved equality of the triplex RT-qPCR with virus detection in cell culture and a higher sensitivity than conventional RT-PCR in field samples. In comparative studies of a total of 77 field samples tested, 51 showed identical positive and 19 identical negative results in cell culture and the triplex RT-qPCR. However, seven other samples yielded positive results in the triplex RT-qPCR, but negative results in cell culture. Copyright © 2017 Elsevier B.V. All rights reserved.

Fiber optic oxygen sensor leak detection system for space applications

NASA Astrophysics Data System (ADS)

Kazemi, Alex A.; Goswami, Kish; Mendoza, Edgar A.; Kempen, Lothar U.

2007-09-01

This paper describes the successful test of a multi-point fiber optic oxygen sensor system during the static firing of an Evolved Expandable Launch Vehicle (EELV)/Delta IV common booster core (CBC) rocket engine at NASA's Stennis Flight Center. The system consisted of microsensors (optrodes) using an oxygen gas sensitive indicator incorporated onto an optically transparent porous substrate. The modular optoelectronics and multiplexing network system was designed and assembled utilizing a multi-channel opto-electronic sensor readout unit that monitored the oxygen and temperature response of the individual optrodes in real-time and communicated this information via a serial communication port to a remote laptop computer. The sensor packaging for oxygen consisted of two optrodes - one doped with an indicator sensitive to oxygen, and the other doped with an indicator sensitive to temperature. The multichannel oxygen sensor system is fully reversible. It has demonstrated a dynamic response to oxygen gas in the range of 0% to 100% with 0.1% resolution and a response time of <=10 seconds. The sensor package was attached to a custom fiber optic ribbon cable, which was then connected to a fiber optic trunk communications cable (standard telecommunications-grade fiber) that connected to the optoelectronics module. Each board in the expandable module included light sources, photo-detectors, and associated electronics required for detecting oxygen and temperature. The paper illustrates the sensor design and performance data under field deployment conditions.

Dynamic, in vivo, real-time detection of retinal oxidative status in a model of elevated intraocular pressure using a novel, reversibly responsive, profluorescent nitroxide probe.

PubMed

Rayner, Cassie L; Gole, Glen A; Bottle, Steven E; Barnett, Nigel L

2014-12-01

Changes to the redox status of biological systems have been implicated in the pathogenesis of a wide variety of disorders including cancer, Ischemia-reperfusion (I/R) injury and neurodegeneration. In times of metabolic stress e.g. ischaemia/reperfusion, reactive oxygen species (ROS) production overwhelms the intrinsic antioxidant capacity of the cell, damaging vital cellular components. The ability to quantify ROS changes in vivo, is therefore essential to understanding their biological role. Here we evaluate the suitability of a novel reversible profluorescent probe containing a redox-sensitive nitroxide moiety (methyl ester tetraethylrhodamine nitroxide, ME-TRN), as an in vivo, real-time reporter of retinal oxidative status. The reversible nature of the probe's response offers the unique advantage of being able to monitor redox changes in both oxidizing and reducing directions in real time. After intravitreal administration of the ME-TRN probe, we induced ROS production in rat retina using an established model of complete, acute retinal ischaemia followed by reperfusion. After restoration of blood flow, retinas were imaged using a Micron III rodent fundus fluorescence imaging system, to quantify the redox-response of the probe. Fluorescent intensity declined during the first 60 min of reperfusion. The ROS-induced change in probe fluorescence was ameliorated with the retinal antioxidant, lutein. Fluorescence intensity in non-Ischemia eyes did not change significantly. This new probe and imaging technology provide a reversible and real-time response to oxidative changes and may allow the in vivo testing of antioxidant therapies of potential benefit to a range of diseases linked to oxidative stress. Copyright © 2014 Elsevier Ltd. All rights reserved.

Development of a multiplex real time PCR to detect thermophilic lactic acid bacteria in natural whey starters.

PubMed

Bottari, Benedetta; Agrimonti, Caterina; Gatti, Monica; Neviani, Erasmo; Marmiroli, Nelson

2013-01-01

A multiplex real time PCR (mRealT-PCR) useful to rapidly screen microbial composition of thermophilic starter cultures for hard cooked cheeses and to compare samples with potentially different technological properties was developed. Novel primers directed toward pheS gene were designed and optimized for multiple detection of Lactobacillus helveticus, Lactobacillus delbrueckii, Streptococcus thermophilus and Lactobacillus fermentum. The assay was based on SYBR Green chemistry followed by melting curves analysis. The method was then evaluated for applications in the specific detection of the 4 lactic acid bacteria (LAB) in 29 different natural whey starters for Parmigiano Reggiano cheese production. The results obtained by mRealT-PCR were also compared with those obtained on the same samples by Fluorescence in Situ Hybridization (FISH) and Length-Heterogeneity PCR (LH-PCR). The mRealT-PCR developed in this study, was found to be effective for analyzing species present in the samples with an average sensitivity down to less than 600 copies of DNA and therefore sensitive enough to detect even minor LAB community members of thermophilic starter cultures. The assay was able to describe the microbial population of all the different natural whey starter samples analyzed, despite their natural variability. A higher number of whey starter samples with S. thermophilus and L. fermentum present in their microbial community were revealed, suggesting that these species could be more frequent in Parmigiano Reggiano natural whey starter samples than previously shown. The method was more effective than LH-PCR and FISH and, considering that these two techniques have to be used in combination to detect the less abundant species, the mRealT-PCR was also faster. Providing a single step sensitive detection of L. helveticus, L. delbrueckii, S. thermophilus and L. fermentum, the developed mRealT-PCR could be used for screening thermophilic starter cultures and to follow the presence of those species during ripening of derived dairy products. A major increase in understanding the starter culture contribution to cheese ecosystem could be harnessed to control cheese ripening and flavor formation. Copyright © 2012 Elsevier B.V. All rights reserved.

Multiplex Reverse Transcription-PCR for Simultaneous Surveillance of Influenza A and B Viruses

PubMed Central

Zhou, Bin; Barnes, John R.; Sessions, October M.; Chou, Tsui-Wen; Wilson, Malania; Stark, Thomas J.; Volk, Michelle; Spirason, Natalie; Halpin, Rebecca A.; Kamaraj, Uma Sangumathi; Ding, Tao; Stockwell, Timothy B.; Ghedin, Elodie; Barr, Ian G.

2017-01-01

ABSTRACT Influenza A and B viruses are the causative agents of annual influenza epidemics that can be severe, and influenza A viruses intermittently cause pandemics. Sequence information from influenza virus genomes is instrumental in determining mechanisms underpinning antigenic evolution and antiviral resistance. However, due to sequence diversity and the dynamics of influenza virus evolution, rapid and high-throughput sequencing of influenza viruses remains a challenge. We developed a single-reaction influenza A/B virus (FluA/B) multiplex reverse transcription-PCR (RT-PCR) method that amplifies the most critical genomic segments (hemagglutinin [HA], neuraminidase [NA], and matrix [M]) of seasonal influenza A and B viruses for next-generation sequencing, regardless of viral type, subtype, or lineage. Herein, we demonstrate that the strategy is highly sensitive and robust. The strategy was validated on thousands of seasonal influenza A and B virus-positive specimens using multiple next-generation sequencing platforms. PMID:28978683

Application of anti-listerial bacteriocins: monitoring enterocin expression by multiplex relative reverse transcription-PCR.

PubMed

Williams, D Ross; Chanos, Panagiotis

2012-12-01

Listeriosis is a deadly food-borne disease, and its incidence may be limited through the biotechnological exploitation of a number of anti-listerial biocontrol agents. The most widely used of these agents are bacteriocins and the Class II enterocins are characterized by their activity against Listeria. Enterocins are primarily produced by enterococci, particularly Enterococcus faecium and many strains have been described, often encoding multiple bacteriocins. The use of these strains in food will require that they are free of virulence functions and that they exhibit a high level expression of anti-listerial enterocins in fermentation conditions. Multiplex relative RT (reverse transcription)-PCR is a technique that is useful in the discovery of advantageous expression characteristics among enterocin-producing strains. It allows the levels of individual enterocin gene expression to be monitored and determination of how expression is altered under different growth conditions.

Digitally synthesized beat frequency-multiplexed fluorescence lifetime spectroscopy

PubMed Central

Chan, Jacky C. K.; Diebold, Eric D.; Buckley, Brandon W.; Mao, Sien; Akbari, Najva; Jalali, Bahram

2014-01-01

Frequency domain fluorescence lifetime imaging is a powerful technique that enables the observation of subtle changes in the molecular environment of a fluorescent probe. This technique works by measuring the phase delay between the optical emission and excitation of fluorophores as a function of modulation frequency. However, high-resolution measurements are time consuming, as the excitation modulation frequency must be swept, and faster low-resolution measurements at a single frequency are prone to large errors. Here, we present a low cost optical system for applications in real-time confocal lifetime imaging, which measures the phase vs. frequency spectrum without sweeping. Deemed Lifetime Imaging using Frequency-multiplexed Excitation (LIFE), this technique uses a digitally-synthesized radio frequency comb to drive an acousto-optic deflector, operated in a cat’s-eye configuration, to produce a single laser excitation beam modulated at multiple beat frequencies. We demonstrate simultaneous fluorescence lifetime measurements at 10 frequencies over a bandwidth of 48 MHz, enabling high speed frequency domain lifetime analysis of single- and multi-component sample mixtures. PMID:25574449

Parallel implementation of all-digital timing recovery for high-speed and real-time optical coherent receivers.

PubMed

Zhou, Xian; Chen, Xue

2011-05-09

The digital coherent receivers combine coherent detection with digital signal processing (DSP) to compensate for transmission impairments, and therefore are a promising candidate for future high-speed optical transmission system. However, the maximum symbol rate supported by such real-time receivers is limited by the processing rate of hardware. In order to cope with this difficulty, the parallel processing algorithms is imperative. In this paper, we propose a novel parallel digital timing recovery loop (PDTRL) based on our previous work. Furthermore, for increasing the dynamic dispersion tolerance range of receivers, we embed a parallel adaptive equalizer in the PDTRL. This parallel joint scheme (PJS) can be used to complete synchronization, equalization and polarization de-multiplexing simultaneously. Finally, we demonstrate that PDTRL and PJS allow the hardware to process 112 Gbit/s POLMUX-DQPSK signal at the hundreds MHz range. © 2011 Optical Society of America

Reversible Oxygen Gas Sensor Based On Electrochemiluminescence

PubMed Central

Zhang, Lihua; Tsow, Francis

2013-01-01

A novel and robust oxygen gas sensor based on electrochemiluminescence of Ru(bpy)33+/+ ion annihilation in an ionic liquid is presented. Real-time detection of environmental oxygen concentration together with selective, sensitive and reversible performance is demonstrated. PMID:20386795

Multiplexer and time duration measuring circuit

DOEpatents

Gray, Jr., James

1980-01-01

A multiplexer device is provided for multiplexing data in the form of randomly developed, variable width pulses from a plurality of pulse sources to a master storage. The device includes a first multiplexer unit which includes a plurality of input circuits each coupled to one of the pulse sources, with all input circuits being disabled when one input circuit receives an input pulse so that only one input pulse is multiplexed by the multiplexer unit at any one time.

Delay grid multiplexing: simple time-based multiplexing and readout method for silicon photomultipliers

NASA Astrophysics Data System (ADS)

Won, Jun Yeon; Ko, Guen Bae; Lee, Jae Sung

2016-10-01

In this paper, we propose a fully time-based multiplexing and readout method that uses the principle of the global positioning system. Time-based multiplexing allows simplifying the multiplexing circuits where the only innate traces that connect the signal pins of the silicon photomultiplier (SiPM) channels to the readout channels are used as the multiplexing circuit. Every SiPM channel is connected to the delay grid that consists of the traces on a printed circuit board, and the inherent transit times from each SiPM channel to the readout channels encode the position information uniquely. Thus, the position of each SiPM can be identified using the time difference of arrival (TDOA) measurements. The proposed multiplexing can also allow simplification of the readout circuit using the time-to-digital converter (TDC) implemented in a field-programmable gate array (FPGA), where the time-over-threshold (ToT) is used to extract the energy information after multiplexing. In order to verify the proposed multiplexing method, we built a positron emission tomography (PET) detector that consisted of an array of 4  ×  4 LGSO crystals, each with a dimension of 3  ×  3  ×  20 mm3, and one- to-one coupled SiPM channels. We first employed the waveform sampler as an initial study, and then replaced the waveform sampler with an FPGA-TDC to further simplify the readout circuits. The 16 crystals were clearly resolved using only the time information obtained from the four readout channels. The coincidence resolving times (CRTs) were 382 and 406 ps FWHM when using the waveform sampler and the FPGA-TDC, respectively. The proposed simple multiplexing and readout methods can be useful for time-of-flight (TOF) PET scanners.

Multi-granularity Bandwidth Allocation for Large-Scale WDM/TDM PON

NASA Astrophysics Data System (ADS)

Gao, Ziyue; Gan, Chaoqin; Ni, Cuiping; Shi, Qiongling

2017-12-01

WDM (wavelength-division multiplexing)/TDM (time-division multiplexing) PON (passive optical network) is being viewed as a promising solution for delivering multiple services and applications, such as high-definition video, video conference and data traffic. Considering the real-time transmission, QoS (quality of services) requirements and differentiated services model, a multi-granularity dynamic bandwidth allocation (DBA) in both domains of wavelengths and time for large-scale hybrid WDM/TDM PON is proposed in this paper. The proposed scheme achieves load balance by using the bandwidth prediction. Based on the bandwidth prediction, the wavelength assignment can be realized fairly and effectively to satisfy the different demands of various classes. Specially, the allocation of residual bandwidth further augments the DBA and makes full use of bandwidth resources in the network. To further improve the network performance, two schemes named extending the cycle of one free wavelength (ECoFW) and large bandwidth shrinkage (LBS) are proposed, which can prevent transmission from interruption when the user employs more than one wavelength. The simulation results show the effectiveness of the proposed scheme.

Performance analysis of cooperative virtual MIMO systems for wireless sensor networks.

PubMed

Rafique, Zimran; Seet, Boon-Chong; Al-Anbuky, Adnan

2013-05-28

Multi-Input Multi-Output (MIMO) techniques can be used to increase the data rate for a given bit error rate (BER) and transmission power. Due to the small form factor, energy and processing constraints of wireless sensor nodes, a cooperative Virtual MIMO as opposed to True MIMO system architecture is considered more feasible for wireless sensor network (WSN) applications. Virtual MIMO with Vertical-Bell Labs Layered Space-Time (V-BLAST) multiplexing architecture has been recently established to enhance WSN performance. In this paper, we further investigate the impact of different modulation techniques, and analyze for the first time, the performance of a cooperative Virtual MIMO system based on V-BLAST architecture with multi-carrier modulation techniques. Through analytical models and simulations using real hardware and environment settings, both communication and processing energy consumptions, BER, spectral efficiency, and total time delay of multiple cooperative nodes each with single antenna are evaluated. The results show that cooperative Virtual-MIMO with Binary Phase Shift Keying-Wavelet based Orthogonal Frequency Division Multiplexing (BPSK-WOFDM) modulation is a promising solution for future high data-rate and energy-efficient WSNs.

Performance Analysis of Cooperative Virtual MIMO Systems for Wireless Sensor Networks

PubMed Central

Rafique, Zimran; Seet, Boon-Chong; Al-Anbuky, Adnan

2013-01-01

Multi-Input Multi-Output (MIMO) techniques can be used to increase the data rate for a given bit error rate (BER) and transmission power. Due to the small form factor, energy and processing constraints of wireless sensor nodes, a cooperative Virtual MIMO as opposed to True MIMO system architecture is considered more feasible for wireless sensor network (WSN) applications. Virtual MIMO with Vertical-Bell Labs Layered Space-Time (V-BLAST) multiplexing architecture has been recently established to enhance WSN performance. In this paper, we further investigate the impact of different modulation techniques, and analyze for the first time, the performance of a cooperative Virtual MIMO system based on V-BLAST architecture with multi-carrier modulation techniques. Through analytical models and simulations using real hardware and environment settings, both communication and processing energy consumptions, BER, spectral efficiency, and total time delay of multiple cooperative nodes each with single antenna are evaluated. The results show that cooperative Virtual-MIMO with Binary Phase Shift Keying-Wavelet based Orthogonal Frequency Division Multiplexing (BPSK-WOFDM) modulation is a promising solution for future high data-rate and energy-efficient WSNs. PMID:23760087

Time-programmable drug dosing allows the manipulation, suppression and reversal of antibiotic drug resistance in vitro

NASA Astrophysics Data System (ADS)

Yoshida, Mari; Reyes, Sabrina Galiñanes; Tsuda, Soichiro; Horinouchi, Takaaki; Furusawa, Chikara; Cronin, Leroy

2017-06-01

Multi-drug strategies have been attempted to prolong the efficacy of existing antibiotics, but with limited success. Here we show that the evolution of multi-drug-resistant Escherichia coli can be manipulated in vitro by administering pairs of antibiotics and switching between them in ON/OFF manner. Using a multiplexed cell culture system, we find that switching between certain combinations of antibiotics completely suppresses the development of resistance to one of the antibiotics. Using this data, we develop a simple deterministic model, which allows us to predict the fate of multi-drug evolution in this system. Furthermore, we are able to reverse established drug resistance based on the model prediction by modulating antibiotic selection stresses. Our results support the idea that the development of antibiotic resistance may be potentially controlled via continuous switching of drugs.

Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas▿ †

PubMed Central

Hodgetts, Jennifer; Boonham, Neil; Mumford, Rick; Dickinson, Matthew

2009-01-01

Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays. PMID:19270148

MULTIPLEX SYBR® GREEN-REAL TIME PCR (qPCR) ASSAY FOR THE DETECTION AND DIFFERENTIATION OF Bartonella henselae AND Bartonella clarridgeiae IN CATS

PubMed Central

Staggemeier, Rodrigo; Pilger, Diogo André; Spilki, Fernando Rosado; Cantarelli, Vlademir Vicente

2014-01-01

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonella species, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species. PMID:24626408

Multiplex SYBR® green-real time PCR (qPCR) assay for the detection and differentiation of Bartonella henselae and Bartonella clarridgeiae in cats.

PubMed

Staggemeier, Rodrigo; Pilger, Diogo André; Spilki, Fernando Rosado; Cantarelli, Vlademir Vicente

2014-01-01

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonella species, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

41 CFR 102-74.520 - How much time does the Regional Officer have to affirm or reverse the Federal agency buildings...

Code of Federal Regulations, 2011 CFR

2011-01-01

... 41 Public Contracts and Property Management 3 2011-01-01 2011-01-01 false How much time does the...) FEDERAL MANAGEMENT REGULATION REAL PROPERTY 74-FACILITY MANAGEMENT Occasional Use of Public Buildings Appeals § 102-74.520 How much time does the Regional Officer have to affirm or reverse the Federal agency...

Variation in Bluetongue virus real-time reverse transcription polymerase chain reaction assay results in blood samples of sheep, cattle, and alpaca.

PubMed

Brito, Barbara P; Gardner, Ian A; Hietala, Sharon K; Crossley, Beate M

2011-07-01

Bluetongue is a vector-borne viral disease that affects domestic and wild ruminants. The epidemiology of this disease has recently changed, with occurrence in new geographic areas. Various real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) assays are used to detect Bluetongue virus (BTV); however, the impact of biologic differences between New World camelids and domestic ruminant samples on PCR efficiency, for which the BTV real-time qRT-PCR was initially validated are unknown. New world camelids are known to have important biologic differences in whole blood composition, including hemoglobin concentration, which can alter PCR performance. In the present study, sheep, cattle, and alpaca blood were spiked with BTV serotypes 10, 11, 13, and 17 and analyzed in 10-fold dilutions by real-time qRT-PCR to determine if species affected nucleic acid recovery and assay performance. A separate experiment was performed using spiked alpaca blood subsequently diluted in 10-fold series in sheep blood to assess the influence of alpaca blood on performance efficiency of the BTV real-time qRT-PCR assay. Results showed that BTV-specific nucleic acid detection from alpaca blood was consistently 1-2 logs lower than from sheep and cattle blood, and results were similar for each of the 4 BTV serotypes analyzed.

MACRO: a combined microchip-PCR and microarray system for high-throughput monitoring of genetically modified organisms.

PubMed

Shao, Ning; Jiang, Shi-Meng; Zhang, Miao; Wang, Jing; Guo, Shu-Juan; Li, Yang; Jiang, He-Wei; Liu, Cheng-Xi; Zhang, Da-Bing; Yang, Li-Tao; Tao, Sheng-Ce

2014-01-21

The monitoring of genetically modified organisms (GMOs) is a primary step of GMO regulation. However, there is presently a lack of effective and high-throughput methodologies for specifically and sensitively monitoring most of the commercialized GMOs. Herein, we developed a multiplex amplification on a chip with readout on an oligo microarray (MACRO) system specifically for convenient GMO monitoring. This system is composed of a microchip for multiplex amplification and an oligo microarray for the readout of multiple amplicons, containing a total of 91 targets (18 universal elements, 20 exogenous genes, 45 events, and 8 endogenous reference genes) that covers 97.1% of all GM events that have been commercialized up to 2012. We demonstrate that the specificity of MACRO is ~100%, with a limit of detection (LOD) that is suitable for real-world applications. Moreover, the results obtained of simulated complex samples and blind samples with MACRO were 100% consistent with expectations and the results of independently performed real-time PCRs, respectively. Thus, we believe MACRO is the first system that can be applied for effectively monitoring the majority of the commercialized GMOs in a single test.

Development and validation of duplex, triplex, and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed.

PubMed

Huber, Ingrid; Block, Annette; Sebah, Daniela; Debode, Frédéric; Morisset, Dany; Grohmann, Lutz; Berben, Gilbert; Stebih, Dejan; Milavec, Mojca; Zel, Jana; Busch, Ulrich

2013-10-30

Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis.

Pair-barcode high-throughput sequencing for large-scale multiplexed sample analysis

PubMed Central

2012-01-01

Background The multiplexing becomes the major limitation of the next-generation sequencing (NGS) in application to low complexity samples. Physical space segregation allows limited multiplexing, while the existing barcode approach only permits simultaneously analysis of up to several dozen samples. Results Here we introduce pair-barcode sequencing (PBS), an economic and flexible barcoding technique that permits parallel analysis of large-scale multiplexed samples. In two pilot runs using SOLiD sequencer (Applied Biosystems Inc.), 32 independent pair-barcoded miRNA libraries were simultaneously discovered by the combination of 4 unique forward barcodes and 8 unique reverse barcodes. Over 174,000,000 reads were generated and about 64% of them are assigned to both of the barcodes. After mapping all reads to pre-miRNAs in miRBase, different miRNA expression patterns are captured from the two clinical groups. The strong correlation using different barcode pairs and the high consistency of miRNA expression in two independent runs demonstrates that PBS approach is valid. Conclusions By employing PBS approach in NGS, large-scale multiplexed pooled samples could be practically analyzed in parallel so that high-throughput sequencing economically meets the requirements of samples which are low sequencing throughput demand. PMID:22276739

Pair-barcode high-throughput sequencing for large-scale multiplexed sample analysis.

PubMed

Tu, Jing; Ge, Qinyu; Wang, Shengqin; Wang, Lei; Sun, Beili; Yang, Qi; Bai, Yunfei; Lu, Zuhong

2012-01-25

The multiplexing becomes the major limitation of the next-generation sequencing (NGS) in application to low complexity samples. Physical space segregation allows limited multiplexing, while the existing barcode approach only permits simultaneously analysis of up to several dozen samples. Here we introduce pair-barcode sequencing (PBS), an economic and flexible barcoding technique that permits parallel analysis of large-scale multiplexed samples. In two pilot runs using SOLiD sequencer (Applied Biosystems Inc.), 32 independent pair-barcoded miRNA libraries were simultaneously discovered by the combination of 4 unique forward barcodes and 8 unique reverse barcodes. Over 174,000,000 reads were generated and about 64% of them are assigned to both of the barcodes. After mapping all reads to pre-miRNAs in miRBase, different miRNA expression patterns are captured from the two clinical groups. The strong correlation using different barcode pairs and the high consistency of miRNA expression in two independent runs demonstrates that PBS approach is valid. By employing PBS approach in NGS, large-scale multiplexed pooled samples could be practically analyzed in parallel so that high-throughput sequencing economically meets the requirements of samples which are low sequencing throughput demand.

Evaluation of a multiplex real-time PCR method for detecting Shiga toxin-producing Escherichia coli in beef and comparison to the FSIS microbiology laboratory guidebook method

USDA-ARS?s Scientific Manuscript database

The “top-six” non-O157 STEC (O26, O45, O103, O111, O121, and O145) most frequently associated with outbreaks and cases of food-borne illnesses have been declared as adulterants in beef by the USDA Food Safety and Inspection Service (FSIS), and regulatory testing for these serogroups in beef began in...

Species-specific diagnostic assays for Bonamia ostreae and B. exitiosa in European flat oyster Ostrea edulis: conventional, real-time and multiplex PCR.

PubMed

Ramilo, Andrea; Navas, J Ignacio; Villalba, Antonio; Abollo, Elvira

2013-05-27

Bonamia ostreae and B. exitiosa have caused mass mortalities of various oyster species around the world and co-occur in some European areas. The World Organisation for Animal Health (OIE) has included infections with both species in the list of notifiable diseases. However, official methods for species-specific diagnosis of either parasite have certain limitations. In this study, new species-specific conventional PCR (cPCR) and real-time PCR techniques were developed to diagnose each parasite species. Moreover, a multiplex PCR method was designed to detect both parasites in a single assay. The analytical sensitivity and specificity of each new method were evaluated. These new procedures were compared with 2 OIE-recommended methods, viz. standard histology and PCR-RFLP. The new procedures showed higher sensitivity than the OIE recommended ones for the diagnosis of both species. The sensitivity of tests with the new primers was higher using oyster gills and gonad tissue, rather than gills alone. The lack of a 'gold standard' prevented accurate estimation of sensitivity and specificity of the new methods. The implementation of statistical tools (maximum likelihood method) for the comparison of the diagnostic tests showed the possibility of false positives with the new procedures, although the absence of a gold standard precluded certainty. Nevertheless, all procedures showed negative results when used for the analysis of oysters from a Bonamia-free area.

Molecular diagnostic of cytomegalovirus, Epstein Barr virus and Herpes virus 6 infections among blood donors by multiplex real-time PCR in Ouagadougou, Burkina Faso.

PubMed

Traore, Lassina; Tao, Issoufou; Bisseye, Cyrille; Diarra, Birama; Compaore, Tegwindé Rebeca; Nebie, Yacouba; Assih, Maleki; Ouedraogo, Alice; Zohoncon, Theodora; Djigma, Florencia; Ouermi, Djénéba; Barro, Nicolas; Sanou, Mahamoudou; Ouedraogo, Rasmata Traore; Simpore, Jacques

2016-01-01

In most developing countries, Cytomegalovirus (CMV), Epstein Barr virus (EBV) and Herpes virus 6 (HHV-6) are not diagnosed in blood donors. The aim of this study is to determine the prevalence of these viruses in blood donors from the city of Ouagadougou, Burkina Faso. The study included 198 blood donors of the Regional Blood Transfusion Centre of Ouagadougou. Multiplex real time PCR was used to diagnose the three viruses. Statistical analysis was performed with the software EpiInfo version 6 and SPSS version 17. P values ≤ 0.05 were considered significant. Of 198 samples tested, 18 (9.1%) were positive to at least one of the three viruses. In fact, 10 (5.1%) were positive for EBV, 10 (5.1%) positive for CMV and 12 (6.1%) positive for HHV-6. Viral infections were higher in women than in men, EBV (8,6% versus 4.3%), CMV (8.6% versus 3.7%) and HHV-6 (11.4% versus 4.9%). EBV / CMV / HHV-6 co-infection was found in 3.5% (7/198) of blood donors. The prevalence recorded in this study is low compared to those found in previous studies from the sub-region among blood donors. The molecular diagnostic test used in our study could explain the differences with previous studies.

A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2.

PubMed

Gatto, Francesca; Cassina, Giulia; Broccolo, Francesco; Morreale, Giuseppe; Lanino, Edoardo; Di Marco, Eddi; Vardas, Efthiya; Bernasconi, Daniela; Buttò, Stefano; Principi, Nicola; Esposito, Susanna; Scarlatti, Gabriella; Lusso, Paolo; Malnati, Mauro S

2011-12-01

Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples. Copyright © 2011 Elsevier B.V. All rights reserved.

Allele-specific methylated multiplex real-time quantitative PCR (ASMM RTQ-PCR), a powerful method for diagnosing loss of imprinting of the 11p15 region in Russell Silver and Beckwith Wiedemann syndromes.

PubMed

Azzi, Salah; Steunou, Virginie; Rousseau, Alexandra; Rossignol, Sylvie; Thibaud, Nathalie; Danton, Fabienne; Le Jule, Marilyne; Gicquel, Christine; Le Bouc, Yves; Netchine, Irène

2011-02-01

Many human syndromes involve a loss of imprinting (LOI) due to a loss (LOM) or a gain of DNA methylation (GOM). Most LOI occur as mosaics and can therefore be difficult to detect with conventional methods. The human imprinted 11p15 region is crucial for the control of fetal growth, and LOI at this locus is associated with two clinical disorders with opposite phenotypes: Beckwith-Wiedemann syndrome (BWS), characterized by fetal overgrowth and a high risk of tumors, and Russell-Silver syndrome (RSS), characterized by intrauterine and postnatal growth restriction. Until recently, we have been using Southern blotting for the diagnosis of RSS and BWS. We describe here a powerful quantitative technique, allele-specific methylated multiplex real-time quantitative PCR (ASMM RTQ-PCR), for the diagnosis of these two complex disorders. We first checked the specificity of the probes and primers used for ASMM RTQ-PCR. We then carried out statistical validation for this method, on both retrospective and prospective populations of patients. This analysis demonstrated that ASMM RTQ-PCR is more sensitive than Southern blotting for detecting low degree of LOI. Moreover, ASMM RTQ-PCR is a very rapid, reliable, simple, safe, and cost effective method. © 2011 Wiley-Liss, Inc.

High-risk human papillomavirus infection involving multiple anatomic sites of the female lower genital tract: a multiplex real-time polymerase chain reaction-based study.

PubMed

Hui, Yiang; Manna, Pradip; Ou, Joyce J; Kerley, Spencer; Zhang, Cunxian; Sung, C James; Lawrence, W Dwayne; Quddus, M Ruhul

2015-09-01

High-risk human papillomavirus infection usually is seen at one anatomic site in an individual. Rarely, infection at multiple anatomic sites of the female lower genital tract in the same individual is encountered either simultaneously and/or at a later date. The current study identifies the various subtypes of high-risk human papillomavirus infection in these scenarios and analyzes the potential significance of these findings. High-risk human papillomavirus infection involving 22 anatomic sites from 7 individuals was identified after institutional review board approval. Residual paraffin-embedded tissue samples were retrieved, and all 15 high-risk human papillomavirus were identified and viral load quantified using multiplex real-time polymerase chain reaction-based method. Multiple high-risk human papillomavirus subtypes were identified in 32% of the samples and as many as 5 different subtypes of high-risk human papillomavirus infection in a single anatomic site. In general, each anatomic site has unique combination of viral subtypes, although one individual showed overlapping subtypes in the vagina, cervix, and vulvar samples. Higher viral load and rare subtypes are more frequent in younger patients and in dysplasia compared with carcinoma. Follow-up ranging from 3 to 84 months revealed persistent high-risk human papillomavirus infection in 60% of cases. Copyright © 2015 Elsevier Inc. All rights reserved.

Eyeglasses based wireless electrolyte and metabolite sensor platform.

PubMed

Sempionatto, Juliane R; Nakagawa, Tatsuo; Pavinatto, Adriana; Mensah, Samantha T; Imani, Somayeh; Mercier, Patrick; Wang, Joseph

2017-05-16

The demand for wearable sensors has grown rapidly in recent years, with increasing attention being given to epidermal chemical sensing. Here, we present the first example of a fully integrated eyeglasses wireless multiplexed chemical sensing platform capable of real-time monitoring of sweat electrolytes and metabolites. The new concept has been realized by integrating an amperometric lactate biosensor and a potentiometric potassium ion-selective electrode into the two nose-bridge pads of the glasses and interfacing them with a wireless electronic backbone placed on the glasses' arms. Simultaneous real-time monitoring of sweat lactate and potassium levels with no apparent cross-talk is demonstrated along with wireless signal transduction. The electrochemical sensors were screen-printed on polyethylene terephthalate (PET) stickers and placed on each side of the glasses' nose pads in order to monitor sweat metabolites and electrolytes. The electronic backbone on the arms of the glasses' frame offers control of the amperometric and potentiometric transducers and enables Bluetooth wireless data transmission to the host device. The new eyeglasses system offers an interchangeable-sensor feature in connection with a variety of different nose-bridge amperometric and potentiometric sensor stickers. For example, the lactate bridge-pad sensor was replaced with a glucose one to offer convenient monitoring of sweat glucose. Such a fully integrated wireless "Lab-on-a-Glass" multiplexed biosensor platform can be readily expanded for the simultaneous monitoring of additional sweat electrolytes and metabolites.

Detection of SYT-SSX mutant transcripts in formalin-fixed paraffin-embedded sarcoma tissues using one-step reverse transcriptase real-time PCR.

PubMed

Norlelawati, A T; Mohd Danial, G; Nora, H; Nadia, O; Zatur Rawihah, K; Nor Zamzila, A; Naznin, M

2016-04-01

Synovial sarcoma (SS) is a rare cancer and accounts for 5-10% of adult soft tissue sarcomas. Making an accurate diagnosis is difficult due to the overlapping histological features of SS with other types of sarcomas and the non-specific immunohistochemistry profile findings. Molecular testing is thus considered necessary to confirm the diagnosis since more than 90% of SS cases carry the transcript of t(X;18)(p11.2;q11.2). The purpose of this study is to diagnose SS at molecular level by testing for t(X;18) fusion-transcript expression through One-step reverse transcriptase real-time Polymerase Chain Reaction (PCR). Formalin-fixed paraffin-embedded tissue blocks of 23 cases of soft tissue sarcomas, which included 5 and 8 cases reported as SS as the primary diagnosis and differential diagnosis respectively, were retrieved from the Department of Pathology, Tengku Ampuan Afzan Hospital, Kuantan, Pahang. RNA was purified from the tissue block sections and then subjected to One-step reverse transcriptase real-time PCR using sequence specific hydrolysis probes for simultaneous detection of either SYT-SSX1 or SYT-SSX2 fusion transcript. Of the 23 cases, 4 cases were found to be positive for SYT-SSX fusion transcript in which 2 were diagnosed as SS whereas in the 2 other cases, SS was the differential diagnosis. Three cases were excluded due to failure of both amplification assays SYT-SSX and control β-2-microglobulin. The remaining 16 cases were negative for the fusion transcript. This study has shown that the application of One-Step reverse transcriptase real time PCR for the detection SYT-SSX transcript is feasible as an aid in confirming the diagnosis of synovial sarcoma.

Real-Time PCR (qPCR) Primer Design Using Free Online Software

ERIC Educational Resources Information Center

Thornton, Brenda; Basu, Chhandak

2011-01-01

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms.

PubMed

Wong, Y-P; Othman, S; Lau, Y-L; Radu, S; Chee, H-Y

2018-03-01

Loop-mediated isothermal amplification (LAMP) amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions by using a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA strands. Following its first discovery by Notomi et al. ( Nucleic Acids Res 28: E63), LAMP was further developed over the years which involved the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification for the detection of infectious diseases caused by micro-organisms in humans, livestock and plants. In this review, available types of LAMP techniques will be discussed together with their applications in detection of various micro-organisms. Up to date, there are varieties of LAMP detection methods available including colorimetric and fluorescent detection, real-time monitoring using turbidity metre and detection using lateral flow device which will also be highlighted in this review. Apart from that, commercialization of LAMP technique had also been reported such as lyophilized form of LAMP reagents kit and LAMP primer sets for detection of pathogenic micro-organisms. On top of that, advantages and limitations of this molecular detection method are also described together with its future potential as a diagnostic method for infectious disease. © 2017 The Society for Applied Microbiology.

Multiplex Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Therapeutic Drug Monitoring of Ribavirin, Boceprevir, and Telaprevir

PubMed Central

Aouri, Manel; Moradpour, Darius; Cavassini, Matthias; Mercier, Thomas; Buclin, Thierry; Csajka, Chantal; Telenti, Amalio; Rauch, Andri

2013-01-01

New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy. PMID:23629707

Equivalence of time-multiplexed and frequency-multiplexed signals in digital communications.

NASA Technical Reports Server (NTRS)

Timor, U.

1972-01-01

In comparing different techniques for multiplexing N binary data signals into a single channel, time-division multiplexing (TDM) is known to have a theoretic efficiency of 100 percent (neglecting sync power) and thus seems to outperform frequency-division multiplexing systems (FDM). By considering more general FDM systems, we will show that both TDM and FDM are equivalent and have an efficiency of 100 percent. The difference between the systems is in the multiplexing and demultiplexing subsystems, but not in the performance or in the generated waveforms.

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.

PubMed

Lass, Anna; Szostakowska, Beata; Korzeniewski, Krzysztof; Karanis, Panagiotis

2017-10-01

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.

The SDF1-CXCR4 Axis Functions through p38-MAPK Signaling to Drive Breast Cancer Progression and Metastasis

DTIC Science & Technology

2008-09-01

with breast cancer cells (MCF7 cell line) could induce proliferation and lead to hormone independent tumors in vivo. Upon analysis of these tumors by...1-0694 4.B MCS induce gene expression of ER mediated genes. Endpoint tumors from above studies were harvested for use in Real-time PCR analysis ...Total RNA was isolated from tumors, reverse transcribed into cDNA and subjected to real-time PCR analysis for quantification. A. Real time PCR results

Multiplexed detection of serological cancer markers with plasmon-enhanced Raman spectro-immunoassay.

PubMed

Li, Ming; Kang, Jeon Woong; Sukumar, Saraswati; Dasari, Ramachandra Rao; Barman, Ishan

2015-07-01

Circulating biomarkers have emerged as promising non-invasive, real-time surrogates for cancer diagnosis, prognostication and monitoring of therapeutic response. Emerging data, however, suggest that single markers are inadequate in describing complex pathologic transformations. Architecting assays capable of parallel measurements of multiple biomarkers can help achieve the desired clinical sensitivity and specificity while conserving patient specimen and reducing turn-around time. Here we describe a plasmon-enhanced Raman spectroscopic assay featuring nanostructured biomolecular probes and spectroscopic imaging for multiplexed detection of disseminated breast cancer markers cancer antigen (CA) 15-3, CA 27-29 and cancer embryonic antigen (CEA). In the developed SERS assay, both the assay chip and surface-enhanced Raman spectroscopy (SERS) tags are functionalized with monoclonal antibodies against CA15-3, CA27-29 and CEA, respectively. Sequential addition of biomarkers and functionalized SERS tags onto the functionalized assay chip enable the specific recognition of these biomarkers through the antibody-antigen interactions, leading to a sandwich spectro-immunoassay. In addition to offering extensive multiplexing capability, our method provides higher sensitivity than conventional immunoassays and demonstrates exquisite specificity owing to selective formation of conjugated complexes and fingerprint spectra of the Raman reporter. We envision that clinical translation of this assay may further enable asymptomatic surveillance of cancer survivors and speedy assessment of treatment benefit through a simple blood test.

Direct Digital Demultiplexing of Analog TDM Signals for Cable Reduction in Ultrasound Imaging Catheters

PubMed Central

Carpenter, Thomas M.; Rashid, M. Wasequr; Ghovanloo, Maysam; Cowell, David M. J.; Freear, Steven; Degertekin, F. Levent

2016-01-01

In real-time catheter based 3D ultrasound imaging applications, gathering data from the transducer arrays is difficult as there is a restriction on cable count due to the diameter of the catheter. Although area and power hungry multiplexing circuits integrated at the catheter tip are used in some applications, these are unsuitable for use in small sized catheters for applications like intracardiac imaging. Furthermore, the length requirement for catheters and limited power available to on-chip cable drivers leads to limited signal strength at the receiver end. In this paper an alternative approach using Analog Time Division Multiplexing (TDM) is presented which addresses the cable restrictions of ultrasound catheters. A novel digital demultiplexing technique is also described which allows for a reduction in the number of analog signal processing stages required. The TDM and digital demultiplexing schemes are demonstrated for an intracardiac imaging system that would operate in the 4 MHz to 11 MHz range. A TDM integrated circuit (IC) with 8:1 multiplexer is interfaced with a fast ADC through a micro-coaxial catheter cable bundle, and processed with an FPGA RTL simulation. Input signals to the TDM IC are recovered with −40 dB crosstalk between channels on the same micro-coax, showing the feasibility of this system for ultrasound imaging applications. PMID:27116738

Real-time nested multiplex PCR for the detection of herpes simplex virus types 1 and 2 and varicella zoster virus.

PubMed

O'Neill, Hugh J; Wyatt, Dorothy E; Coyle, Peter V; McCaughey, Conall; Mitchell, Frederick

2003-12-01

One hundred forty-nine specimens were tested in a LightCycler nested multiplex polymerase chain reaction (LCnmPCR) for Herpes simplex virus (HSV)1, HSV2, and VZV. Eighty-one were from genitourinary medicine (GUM) patients and the other 68 specimens were from other patients with skin lesions. The results were compared to a conventional multiplex nested PCR (nmPCR) using agarose gel electrophoresis. Twenty-five specimens were positive in both assays for HSV1 and 29 were positive for VZV. For HSV2 there were 27 positive in the LCnmPCR and 26 positive in the nmPCR assay. The melting temperatures (Tms) of each target were different with a mean of 84.75 degrees C for HSV1, 88.57 degrees C for HSV2, and 83.62 degrees C for VZV. The melting curves of positive specimens directly overlaid the melting curves of the positive controls in the assay. The LCnmPCR assay is a convenient alternative to conventional PCR using agarose gel electrophoresis. It improves specimen turnaround time by eliminating the need for gel electrophoresis, transillumination, and gel photography. It also shows increased sensitivity for HSV2 over our standard assay. This LCnmPCR reduces further the possibility of amplicon contamination with nested PCR protocols. Copyright 2003 Wiley-Liss, Inc.

RaPToRS Sample Delivery System

NASA Astrophysics Data System (ADS)

Henchen, Robert; Shibata, Kye; Krieger, Michael; Pogozelski, Edward; Padalino, Stephen; Glebov, Vladimir; Sangster, Craig

2010-11-01

At various labs (NIF, LLE, NRL), activated material samples are used to measure reaction properties. The Rapid Pneumatic Transport of Radioactive Samples (RaPToRS) system quickly and safely moves these radioactive samples through a closed PVC tube via airflow. The carrier travels from the reaction chamber to the control and analysis station, pneumatically braking at the outlet. A reversible multiplexer routes samples from various locations near the shot chamber to the analysis station. Also, the multiplexer allows users to remotely load unactivated samples without manually approaching the reaction chamber. All elements of the system (pneumatic drivers, flow control valves, optical position sensors, multiplexers, Geiger counters, and release gates at the analysis station) can be controlled manually or automatically using a custom LabVIEW interface. A prototype is currently operating at NRL in Washington DC. Prospective facilities for Raptors systems include LLE and NIF.

Optical wireless communication using positive real-valued orthogonal frequency-division multiplexing and optical beamforming

NASA Astrophysics Data System (ADS)

Kim, Sung-Man; Kwon, Ki-Keun

2017-07-01

The relatively unsatisfactory performance of optical wireless communication (OWC) with respect to WiFi and millimeter-wave communications has formed a key issue preventing its commercialization. We experimentally demonstrate an OWC technology using a combination of positive real-valued orthogonal frequency-division multiplexing (OFDM) and optical beamforming (OB). Due to the intensity-modulation and direct-detection aspects of OWC systems, a positive real-valued OFDM signal can be suitably utilized to maximize the OWC data rate. Further, the OB technique, which can focus laser light on a desired target, can be utilized to increase the OWC data rate and transmission distance. Our experimental results show that the received optical signal power and electrical signal increase by up to 42 and 25 dB, respectively. Further, the data rate increases by a factor of 200 with OB over the conventional approach.

Development and Operation of Arrays of TES x-ray Microcalorimeters Suitable for Constellation-X

NASA Technical Reports Server (NTRS)

Kilbourne, C. A.; Bandler, S. R.; Brown, A. D.; Chervenak, J. A.; Eckart, M. E.; Finkbeiner, F. M.; Iyomoto, N.; Kelley, R. L.; Porter, F. S.; Smith, S. J.;

Elucidating the Burden of HIV in Tissues Using Multiplexed Immunofluorescence and In Situ Hybridization: Methods for the Single-Cell Phenotypic Characterization of Cells Harboring HIV In Situ.

PubMed

Vasquez, Joshua J; Hussien, Rajaa; Aguilar-Rodriguez, Brandon; Junger, Henrik; Dobi, Dejan; Henrich, Timothy J; Thanh, Cassandra; Gibson, Erica; Hogan, Louise E; McCune, Joseph; Hunt, Peter W; Stoddart, Cheryl A; Laszik, Zoltan G

2018-06-01

Persistent tissue reservoirs of HIV present a major barrier to cure. Defining subsets of infected cells in tissues is a major focus of HIV cure research. Herein, we describe a novel multiplexed in situ hybridization (ISH) (RNAscope) protocol to detect HIV-DNA (vDNA) and HIV-RNA (vRNA) in formalin-fixed paraffin-embedded (FFPE) human tissues in combination with immunofluorescence (IF) phenotyping of the infected cells. We show that multiplexed IF and ISH (mIFISH) is suitable for quantitative assessment of HIV vRNA and vDNA and that multiparameter IF phenotyping allows precise identification of the cellular source of the ISH signal. We also provide semi-quantitative data on the impact of various tissue fixatives on the detectability of vDNA and vRNA with RNAscope technology. Finally, we describe methods to quantitate the ISH signal on whole-slide digital images and validation of the quantitative ISH data with quantitative real-time PCR for vRNA. It is our hope that this approach will provide insight into the biology of HIV tissue reservoirs and to inform strategies aimed at curing HIV.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs.

PubMed

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Zel, Jana; Gruden, Kristina

2008-10-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1-25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

PubMed Central

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Žel, Jana; Gruden, Kristina

2008-01-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification. PMID:18710880

Highly Multiplexed, Single Cell Transcriptomic Analysis of T-Cells by Microfluidic PCR.

PubMed

Dominguez, Maria; Roederer, Mario; Chattopadhyay, Pratip K

2017-01-01

Recently, technologies have been developed to measure expression of 96 (or more) mRNA transcripts at once from a single cell. Here we describe methods and important considerations for use of Fluidigm's BioMark platform for multiplexed single cell gene expression. We describe how to qualify primer/probes, select genes to examine in 96-parameter panels, perform the reverse transcription/cDNA synthesis step, and operate the instrument. In addition, we describe data analysis considerations. This technology has enormous value for characterizing the heterogeneity of T-cells, thereby providing a useful tool for immune monitoring.

Detection of Multiple Pathogens in Serum Using Silica-Encapsulated Nanotags in a Surface-Enhanced Raman Scattering-Based Immunoassay.

PubMed

Neng, Jing; Li, Yina; Driscoll, Ashley J; Wilson, William C; Johnson, Patrick A

2018-06-06

A robust immunoassay based on surface-enhanced Raman scattering (SERS) has been developed to simultaneously detect trace quantities of multiple pathogenic antigens from West Nile virus, Rift Valley fever virus, and Yersinia pestis in fetal bovine serum. Antigens were detected by capture with silica-encapsulated nanotags and magnetic nanoparticles conjugated with polyclonal antibodies. The magnetic pull-down resulted in aggregation of the immune complexes, and the silica-encapsulated nanotags provided distinct spectra corresponding to each antigen captured. The limit of detection was ∼10 pg/mL in 20% fetal bovine serum, a significant improvement over previous studies in terms of sensitivity, level of multiplexing, and medium complexity. This highly sensitive multiplex immunoassay platform provides a promising method to detect various antigens directly in crude serum samples without the tedious process of sample preparation, which is desirable for on-site diagnostic testing and real-time disease monitoring.

Memory-based frame synchronizer. [for digital communication systems

NASA Technical Reports Server (NTRS)

Stattel, R. J.; Niswander, J. K. (Inventor)

1981-01-01

A frame synchronizer for use in digital communications systems wherein data formats can be easily and dynamically changed is described. The use of memory array elements provide increased flexibility in format selection and sync word selection in addition to real time reconfiguration ability. The frame synchronizer comprises a serial-to-parallel converter which converts a serial input data stream to a constantly changing parallel data output. This parallel data output is supplied to programmable sync word recognizers each consisting of a multiplexer and a random access memory (RAM). The multiplexer is connected to both the parallel data output and an address bus which may be connected to a microprocessor or computer for purposes of programming the sync word recognizer. The RAM is used as an associative memory or decorder and is programmed to identify a specific sync word. Additional programmable RAMs are used as counter decoders to define word bit length, frame word length, and paragraph frame length.

Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction.

PubMed

Corman, V M; Eckerle, I; Bleicker, T; Zaki, A; Landt, O; Eschbach-Bludau, M; van Boheemen, S; Gopal, R; Ballhause, M; Bestebroer, T M; Muth, D; Müller, M A; Drexler, J F; Zambon, M; Osterhaus, A D; Fouchier, R M; Drosten, C

2012-09-27

We present two real-time reverse-transcription polymerase chain reaction assays for a novel human coronavirus (CoV), targeting regions upstream of the E gene (upE) or within open reading frame (ORF)1b, respectively. Sensitivity for upE is 3.4 copies per reaction (95% confidence interval (CI): 2.5–6.9 copies) or 291 copies/mL of sample. No cross-reactivity was observed with coronaviruses OC43, NL63, 229E, SARS-CoV, nor with 92 clinical specimens containing common human respiratory viruses. We recommend using upE for screening and ORF1b for confirmation.

Testing the causality of Hawkes processes with time reversal

NASA Astrophysics Data System (ADS)

Cordi, Marcus; Challet, Damien; Muni Toke, Ioane

2018-03-01

We show that univariate and symmetric multivariate Hawkes processes are only weakly causal: the true log-likelihoods of real and reversed event time vectors are almost equal, thus parameter estimation via maximum likelihood only weakly depends on the direction of the arrow of time. In ideal (synthetic) conditions, tests of goodness of parametric fit unambiguously reject backward event times, which implies that inferring kernels from time-symmetric quantities, such as the autocovariance of the event rate, only rarely produce statistically significant fits. Finally, we find that fitting financial data with many-parameter kernels may yield significant fits for both arrows of time for the same event time vector, sometimes favouring the backward time direction. This goes to show that a significant fit of Hawkes processes to real data with flexible kernels does not imply a definite arrow of time unless one tests it.

Nanoparticle Enhancement Cascade for Sensitive Multiplex Measurements of Biomarkers in Complex Fluids with Surface Plasmon Resonance Imaging.

PubMed

Hendriks, Jan; Stojanovic, Ivan; Schasfoort, Richard B M; Saris, Daniël B F; Karperien, Marcel

2018-06-05

There is a large unmet need for reliable biomarker measurement systems for clinical application. Such systems should meet challenging requirements for large scale use, including a large dynamic detection range, multiplexing capacity, and both high specificity and sensitivity. More importantly, these requirements need to apply to complex biological samples, which require extensive quality control. In this paper, we present the development of an enhancement detection cascade for surface plasmon resonance imaging (SPRi). The cascade applies an antibody sandwich assay, followed by neutravidin and a gold nanoparticle enhancement for quantitative biomarker measurements in small volumes of complex fluids. We present a feasibility study both in simple buffers and in spiked equine synovial fluid with four cytokines, IL-1β, IL-6, IFN-γ, and TNF-α. Our enhancement cascade leads to an antibody dependent improvement in sensitivity up to 40 000 times, resulting in a limit of detection as low as 50 fg/mL and a dynamic detection range of more than 7 logs. Additionally, measurements at these low concentrations are highly reliable with intra- and interassay CVs between 2% and 20%. We subsequently showed this assay is suitable for multiplex measurements with good specificity and limited cross-reactivity. Moreover, we demonstrated robust detection of IL-6 and IL-1β in spiked undiluted equine synovial fluid with small variation compared to buffer controls. In addition, the availability of real time measurements provides extensive quality control opportunities, essential for clinical applications. Therefore, we consider this method is suitable for broad application in SPRi for multiplex biomarker detection in both research and clinical settings.

Comparison between Frame-Constrained Fix-Pixel-Value and Frame-Free Spiking-Dynamic-Pixel ConvNets for Visual Processing

PubMed Central

Farabet, Clément; Paz, Rafael; Pérez-Carrasco, Jose; Zamarreño-Ramos, Carlos; Linares-Barranco, Alejandro; LeCun, Yann; Culurciello, Eugenio; Serrano-Gotarredona, Teresa; Linares-Barranco, Bernabe

2012-01-01

Most scene segmentation and categorization architectures for the extraction of features in images and patches make exhaustive use of 2D convolution operations for template matching, template search, and denoising. Convolutional Neural Networks (ConvNets) are one example of such architectures that can implement general-purpose bio-inspired vision systems. In standard digital computers 2D convolutions are usually expensive in terms of resource consumption and impose severe limitations for efficient real-time applications. Nevertheless, neuro-cortex inspired solutions, like dedicated Frame-Based or Frame-Free Spiking ConvNet Convolution Processors, are advancing real-time visual processing. These two approaches share the neural inspiration, but each of them solves the problem in different ways. Frame-Based ConvNets process frame by frame video information in a very robust and fast way that requires to use and share the available hardware resources (such as: multipliers, adders). Hardware resources are fixed- and time-multiplexed by fetching data in and out. Thus memory bandwidth and size is important for good performance. On the other hand, spike-based convolution processors are a frame-free alternative that is able to perform convolution of a spike-based source of visual information with very low latency, which makes ideal for very high-speed applications. However, hardware resources need to be available all the time and cannot be time-multiplexed. Thus, hardware should be modular, reconfigurable, and expansible. Hardware implementations in both VLSI custom integrated circuits (digital and analog) and FPGA have been already used to demonstrate the performance of these systems. In this paper we present a comparison study of these two neuro-inspired solutions. A brief description of both systems is presented and also discussions about their differences, pros and cons. PMID:22518097

Comparison between Frame-Constrained Fix-Pixel-Value and Frame-Free Spiking-Dynamic-Pixel ConvNets for Visual Processing.

PubMed

Farabet, Clément; Paz, Rafael; Pérez-Carrasco, Jose; Zamarreño-Ramos, Carlos; Linares-Barranco, Alejandro; Lecun, Yann; Culurciello, Eugenio; Serrano-Gotarredona, Teresa; Linares-Barranco, Bernabe

2012-01-01

Most scene segmentation and categorization architectures for the extraction of features in images and patches make exhaustive use of 2D convolution operations for template matching, template search, and denoising. Convolutional Neural Networks (ConvNets) are one example of such architectures that can implement general-purpose bio-inspired vision systems. In standard digital computers 2D convolutions are usually expensive in terms of resource consumption and impose severe limitations for efficient real-time applications. Nevertheless, neuro-cortex inspired solutions, like dedicated Frame-Based or Frame-Free Spiking ConvNet Convolution Processors, are advancing real-time visual processing. These two approaches share the neural inspiration, but each of them solves the problem in different ways. Frame-Based ConvNets process frame by frame video information in a very robust and fast way that requires to use and share the available hardware resources (such as: multipliers, adders). Hardware resources are fixed- and time-multiplexed by fetching data in and out. Thus memory bandwidth and size is important for good performance. On the other hand, spike-based convolution processors are a frame-free alternative that is able to perform convolution of a spike-based source of visual information with very low latency, which makes ideal for very high-speed applications. However, hardware resources need to be available all the time and cannot be time-multiplexed. Thus, hardware should be modular, reconfigurable, and expansible. Hardware implementations in both VLSI custom integrated circuits (digital and analog) and FPGA have been already used to demonstrate the performance of these systems. In this paper we present a comparison study of these two neuro-inspired solutions. A brief description of both systems is presented and also discussions about their differences, pros and cons.

Self-calibrating multiplexer circuit

DOEpatents

Wahl, Chris P.

1997-01-01

A time domain multiplexer system with automatic determination of acceptable multiplexer output limits, error determination, or correction is comprised of a time domain multiplexer, a computer, a constant current source capable of at least three distinct current levels, and two series resistances employed for calibration and testing. A two point linear calibration curve defining acceptable multiplexer voltage limits may be defined by the computer by determining the voltage output of the multiplexer to very accurately known input signals developed from predetermined current levels across the series resistances. Drift in the multiplexer may be detected by the computer when the output voltage limits, expected during normal operation, are exceeded, or the relationship defined by the calibration curve is invalidated.

Rapid detection and subtyping of European swine influenza viruses in porcine clinical samples by haemagglutinin- and neuraminidase-specific tetra- and triplex real-time RT-PCRs.

PubMed

Henritzi, Dinah; Zhao, Na; Starick, Elke; Simon, Gaelle; Krog, Jesper S; Larsen, Lars Erik; Reid, Scott M; Brown, Ian H; Chiapponi, Chiara; Foni, Emanuela; Wacheck, Silke; Schmid, Peter; Beer, Martin; Hoffmann, Bernd; Harder, Timm C

2016-11-01

A diversifying pool of mammalian-adapted influenza A viruses (IAV) with largely unknown zoonotic potential is maintained in domestic swine populations worldwide. The most recent human influenza pandemic in 2009 was caused by a virus with genes originating from IAV isolated from swine. Swine influenza viruses (SIV) are widespread in European domestic pig populations and evolve dynamically. Knowledge regarding occurrence, spread and evolution of potentially zoonotic SIV in Europe is poorly understood. Efficient SIV surveillance programmes depend on sensitive and specific diagnostic methods which allow for cost-effective large-scale analysis. New SIV haemagglutinin (HA) and neuraminidase (NA) subtype- and lineage-specific multiplex real-time RT-PCRs (RT-qPCR) have been developed and validated with reference virus isolates and clinical samples. A diagnostic algorithm is proposed for the combined detection in clinical samples and subtyping of SIV strains currently circulating in Europe that is based on a generic, M-gene-specific influenza A virus RT-qPCR. In a second step, positive samples are examined by tetraplex HA- and triplex NA-specific RT-qPCRs to differentiate the porcine subtypes H1, H3, N1 and N2. Within the HA subtype H1, lineages "av" (European avian-derived), "hu" (European human-derived) and "pdm" (human pandemic A/H1N1, 2009) are distinguished by RT-qPCRs, and within the NA subtype N1, lineage "pdm" is differentiated. An RT-PCR amplicon Sanger sequencing method of small fragments of the HA and NA genes is also proposed to safeguard against failure of multiplex RT-qPCR subtyping. These new multiplex RT-qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe. © 2016 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

Is PCR the Next Reference Standard for the Diagnosis of Schistosoma in Stool? A Comparison with Microscopy in Senegal and Kenya.

PubMed

Meurs, Lynn; Brienen, Eric; Mbow, Moustapha; Ochola, Elizabeth A; Mboup, Souleymane; Karanja, Diana M S; Secor, W Evan; Polman, Katja; van Lieshout, Lisette

2015-01-01

The current reference test for the detection of S. mansoni in endemic areas is stool microscopy based on one or more Kato-Katz stool smears. However, stool microscopy has several shortcomings that greatly affect the efficacy of current schistosomiasis control programs. A highly specific multiplex real-time polymerase chain reaction (PCR) targeting the Schistosoma internal transcriber-spacer-2 sequence (ITS2) was developed by our group a few years ago, but so far this PCR has been applied mostly on urine samples. Here, we performed more in-depth evaluation of the ITS2 PCR as an alternative method to standard microscopy for the detection and quantification of Schistosoma spp. in stool samples. Microscopy and PCR were performed in a Senegalese community (n = 197) in an area with high S. mansoni transmission and co-occurrence of S. haematobium, and in Kenyan schoolchildren (n = 760) from an area with comparatively low S. mansoni transmission. Despite the differences in Schistosoma endemicity the PCR performed very similarly in both areas; 13-15% more infections were detected by PCR when comparing to microscopy of a single stool sample. Even when 2-3 stool samples were used for microscopy, PCR on one stool sample detected more infections, especially in people with light-intensity infections and in children from low-risk schools. The low prevalence of soil-transmitted helminthiasis in both populations was confirmed by an additional multiplex PCR. The ITS2-based PCR was more sensitive than standard microscopy in detecting Schistosoma spp. This would be particularly useful for S. mansoni detection in low transmission areas, and post-control settings, and as such improve schistosomiasis control programs, epidemiological research, and quality control of microscopy. Moreover, it can be complemented with other (multiplex real-time) PCRs to detect a wider range of helminths and thus enhance effectiveness of current integrated control and elimination strategies for neglected tropical diseases.

Acousto-ultrasonic system for the inspection of composite armored vehicles

NASA Astrophysics Data System (ADS)

Godinez, Valery F.; Carlos, Mark F.; Delamere, Michael; Hoch, William; Fotopoulos, Christos; Dai, Weiming; Raju, Basavaraju B.

2001-04-01

In this paper the design and implementation of a unique acousto-ultrasonics system for the inspection of composite armored vehicles is discussed. The system includes a multi-sensor probe with a position-tracking device mounted on a computer controlled scanning bridge. The system also includes an arbitrary waveform generator with a multiplexer and a multi-channel acoustic emission board capable of simultaneously collecting and processing up to four acoustic signals in real time. C-scans of an armored vehicle panel with defects are presented.

Rapid detection of highly pathogenic porcine reproductive and respiratory syndrome virus by a fluorescent probe-based isothermal recombinase polymerase amplification assay.

PubMed

Yang, Yang; Qin, Xiaodong; Sun, Yingjun; Chen, Ting; Zhang, Zhidong

2016-12-01

A novel fluorescent probe-based real-time reverse transcription recombinase polymerase amplification (real-time RT-RPA) assay was developed for rapid detection of highly pathogenic type 2 porcine reproductive and respiratory syndrome virus (HP-PRRSV). The sensitivity analysis showed that the detection limit of RPA was 70 copies of HP-PRRSV RNA/reaction. The real-time RT-RPA highly specific amplified HP-PRRSV with no cross-reaction with classic PRRSV, classic swine fever virus, pseudorabies virus, and foot-and-mouth disease virus. Assessment with 125 clinical samples showed that the developed real-time RT-RPA assay was well correlated with real-time RT-qPCR assays for detection of HP-PRRSV. These results suggest that the developed real-time RT-RPA assay is suitable for rapid detection of HP-PRRSV.

Dichromated polyvinyl alcohol (DC-PVA) wet processed for high index modulation

NASA Astrophysics Data System (ADS)

Rallison, Richard D.

1997-04-01

PVA films have been used as mold releases, strippable coatings, binders for photopolymers and when sensitized with metals and/or dyes they have been used as photoresists, volume HOEs, multiplexed holographic optical memory and real time non destructive holographic testing. The list goes on and includes Slime and birth control. In holography, DC-PVA is a real time photoanisotropic recording material useful for phase conjugation experiments and also a stable long term storage medium needing no processing other than heat. Now we add the capability of greatly increasing the versatility of PVA by boosting the index modulation by almost two orders of magnitude. We can add broadband display and HOE applications that were not possible before. Simple two or three step liquid processing is all that is required to make the index modulation grow.

Molecular Differentiation of Treponema pallidum Subspecies in Skin Ulceration Clinically Suspected as Yaws in Vanuatu Using Real-Time Multiplex PCR and Serological Methods

PubMed Central

Chi, Kai-Hua; Danavall, Damien; Taleo, Fasihah; Pillay, Allan; Ye, Tun; Nachamkin, Eli; Kool, Jacob L.; Fegan, David; Asiedu, Kingsley; Vestergaard, Lasse S.; Ballard, Ronald C.; Chen, Cheng-Yen

2015-01-01

We developed a TaqMan-based real-time quadriplex polymerase chain reaction (PCR) to simultaneously detect Treponema pallidum subspecies pallidum, T. pallidum subsp. pertenue, and T. pallidum subsp. endemicum, the causative agents of venereal syphilis, yaws, and bejel, respectively. The PCR assay was applied to samples from skin ulcerations of clinically presumptive yaws cases among children on Tanna Island, Vanuatu. Another real-time triplex PCR was used to screen for the point mutations in the 23S rRNA genes that have previously been associated with azithromycin resistance in T. pallidum subsp. pallidum strains. Seropositivity by the classical syphilis serological tests was 35.5% among children with skin ulcerations clinically suspected with yaws, whereas the presence of T. pallidum subsp. pertenue DNA was only found in lesions from 15.5% of children. No evidence of T. pallidum subsp. pertenue infection, by either PCR or serology was found in ∼59% of cases indicating alternative causes of yaws-like lesions in this endemic area. PMID:25404075

Implementation of a Multiplex and Quantitative Proteomics Platform for Assessing Protein Lysates Using DNA-Barcoded Antibodies.

PubMed

Lee, Jinho; Geiss, Gary K; Demirkan, Gokhan; Vellano, Christopher P; Filanoski, Brian; Lu, Yiling; Ju, Zhenlin; Yu, Shuangxing; Guo, Huifang; Bogatzki, Lisa Y; Carter, Warren; Meredith, Rhonda K; Krishnamurthy, Savitri; Ding, Zhiyong; Beechem, Joseph M; Mills, Gordon B

2018-06-01

Molecular analysis of tumors forms the basis for personalized cancer medicine and increasingly guides patient selection for targeted therapy. Future opportunities for personalized medicine are highlighted by the measurement of protein expression levels via immunohistochemistry, protein arrays, and other approaches; however, sample type, sample quantity, batch effects, and "time to result" are limiting factors for clinical application. Here, we present a development pipeline for a novel multiplexed DNA-labeled antibody platform which digitally quantifies protein expression from lysate samples. We implemented a rigorous validation process for each antibody and show that the platform is amenable to multiple protocols covering nitrocellulose and plate-based methods. Results are highly reproducible across technical and biological replicates, and there are no observed "batch effects" which are common for most multiplex molecular assays. Tests from basal and perturbed cancer cell lines indicate that this platform is comparable to orthogonal proteomic assays such as Reverse-Phase Protein Array, and applicable to measuring the pharmacodynamic effects of clinically-relevant cancer therapeutics. Furthermore, we demonstrate the potential clinical utility of the platform with protein profiling from breast cancer patient samples to identify molecular subtypes. Together, these findings highlight the potential of this platform for enhancing our understanding of cancer biology in a clinical translation setting. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

A rapid multiplex PCR assay for presumptive species identification of rhinoceros horns and its implementation in Vietnam

PubMed Central

Frankham, Greta J.; McEwing, Ross; The, Dang Tat; Hogg, Carolyn J.; Lo, Nathan; Johnson, Rebecca N.

2018-01-01

Rhinoceros (rhinos) have suffered a dramatic increase in poaching over the past decade due to the growing demand for rhino horn products in Asia. One way to reverse this trend is to enhance enforcement and intelligence gathering tools used for species identification of horns, in particular making them fast, inexpensive and accurate. Traditionally, species identification tests are based on DNA sequence data, which, depending on laboratory resources, can be either time or cost prohibitive. This study presents a rapid rhino species identification test, utilizing species-specific primers within the cytochrome b gene multiplexed in a single reaction, with a presumptive species identification based on the length of the resultant amplicon. This multiplex PCR assay can provide a presumptive species identification result in less than 24 hours. Sequence-based definitive testing can be conducted if/when required (e.g. court purposes). This work also presents an actual casework scenario in which the presumptive test was successfully utlitised, in concert with sequence-based definitive testing. The test was carried out on seized suspected rhino horns tested at the Institute of Ecology and Biological Resources, the CITES mandated laboratory in Vietnam, a country that is known to be a major source of demand for rhino horns. This test represents the basis for which future ‘rapid species identification tests’ can be trialed. PMID:29902212

Enabling multiplexed testing of pooled donor cells through whole-genome sequencing.

PubMed

Chan, Yingleong; Chan, Ying Kai; Goodman, Daniel B; Guo, Xiaoge; Chavez, Alejandro; Lim, Elaine T; Church, George M

2018-04-19

We describe a method that enables the multiplex screening of a pool of many different donor cell lines. Our method accurately predicts each donor proportion from the pool without requiring the use of unique DNA barcodes as markers of donor identity. Instead, we take advantage of common single nucleotide polymorphisms, whole-genome sequencing, and an algorithm to calculate the proportions from the sequencing data. By testing using simulated and real data, we showed that our method robustly predicts the individual proportions from a mixed-pool of numerous donors, thus enabling the multiplexed testing of diverse donor cells en masse.More information is available at https://pgpresearch.med.harvard.edu/poolseq/.

Multiplex real-time PCR for detection, identification and quantification of 'Candidatus Liberibacter solanacearum' in potato plants with zebra chip.

PubMed

Li, Wenbin; Abad, Jorge A; French-Monar, Ronald D; Rascoe, John; Wen, Aimin; Gudmestad, Neil C; Secor, Gary A; Lee, Ing-Ming; Duan, Yongping; Levy, Laurene

2009-07-01

The new Liberibacter species, 'Candidatus Liberibacter solanacearum' (Lso) recently associated with potato/tomato psyllid-transmitted diseases in tomato and capsicum in New Zealand, was found to be consistently associated with a newly emerging potato zebra chip (ZC) disease in Texas and other southwestern states in the USA. A species-specific primer LsoF was developed for both quantitative real-time PCR (qPCR) and conventional PCR (cPCR) to detect and quantify Lso in infected samples. In multiplex qPCR, a plant cytochrome oxidase (COX)-based probe-primer set was used as a positive internal control for host plants, which could be used to reliably access the DNA extraction quality and to normalize qPCR data for accurate quantification of the bacterial populations in environment samples. Neither the qPCR nor the cPCR using the primer and/or probe sets with LsoF reacted with other Liberibacter species infecting citrus or other potato pathogens. The low detection limit of the multiplex qPCR was about 20 copies of the target 16S rDNA templates per reaction for field samples. Lso was readily detected and quantified in various tissues of ZC-affected potato plants collected from fields in Texas. A thorough but uneven colonization of Lso was revealed in various tissues of potato plants. The highest Lso populations were about 3x10(8) genomes/g tissue in the root, which were 3-order higher than those in the above-ground tissues of potato plants. The Lso bacterial populations were normally distributed across the ZC-affected potato plants collected from fields in Texas, with 60% of ZC-affected potato plants harboring an average Lso population from 10(5) to 10(6) genomes/g tissue, 4% of plants hosting above 10(7) Lso genomes/g tissue, and 8% of plants holding below 10(3) Lso genomes/g tissue. The rapid, sensitive, specific and reliable multiplex qPCR showed its potential to become a powerful tool for early detection and quantification of the new Liberibacter species associated with potato ZC, and will be very useful for the potato quarantine programs and seed potato certification programs to ensure the availability of clean seed potato stocks and also for epidemiological studies on the disease.

A Nanocoaxial-Based Electrochemical Sensor for the Detection of Cholera Toxin

NASA Astrophysics Data System (ADS)

Archibald, Michelle M.; Rizal, Binod; Connolly, Timothy; Burns, Michael J.; Naughton, Michael J.; Chiles, Thomas C.

2015-03-01

Sensitive, real-time detection of biomarkers is of critical importance for rapid and accurate diagnosis of disease for point of care (POC) technologies. Current methods do not allow for POC applications due to several limitations, including sophisticated instrumentation, high reagent consumption, limited multiplexing capability, and cost. Here, we report a nanocoaxial-based electrochemical sensor for the detection of bacterial toxins using an electrochemical enzyme-linked immunosorbent assay (ELISA) and differential pulse voltammetry (DPV). Proof-of-concept was demonstrated for the detection of cholera toxin (CT). The linear dynamic range of detection was 10 ng/ml - 1 μg/ml, and the limit of detection (LOD) was found to be 2 ng/ml. This level of sensitivity is comparable to the standard optical ELISA used widely in clinical applications. In addition to matching the detection profile of the standard ELISA, the nanocoaxial array provides a simple electrochemical readout and a miniaturized platform with multiplexing capabilities for the simultaneous detection of multiple biomarkers, giving the nanocoax a desirable advantage over the standard method towards POC applications. Sensitive, real-time detection of biomarkers is of critical importance for rapid and accurate diagnosis of disease for point of care (POC) technologies. Current methods do not allow for POC applications due to several limitations, including sophisticated instrumentation, high reagent consumption, limited multiplexing capability, and cost. Here, we report a nanocoaxial-based electrochemical sensor for the detection of bacterial toxins using an electrochemical enzyme-linked immunosorbent assay (ELISA) and differential pulse voltammetry (DPV). Proof-of-concept was demonstrated for the detection of cholera toxin (CT). The linear dynamic range of detection was 10 ng/ml - 1 μg/ml, and the limit of detection (LOD) was found to be 2 ng/ml. This level of sensitivity is comparable to the standard optical ELISA used widely in clinical applications. In addition to matching the detection profile of the standard ELISA, the nanocoaxial array provides a simple electrochemical readout and a miniaturized platform with multiplexing capabilities for the simultaneous detection of multiple biomarkers, giving the nanocoax a desirable advantage over the standard method towards POC applications. This work was supported by the National Institutes of Health (National Cancer Institute award No. CA137681 and National Institute of Allergy and Infectious Diseases Award No. AI100216).

Integration of Multiplexed Microfluidic Electrokinetic Concentrators with a Morpholino Microarray via Reversible Surface Bonding for Enhanced DNA Hybridization.

PubMed

Martins, Diogo; Wei, Xi; Levicky, Rastislav; Song, Yong-Ak

2016-04-05

We describe a microfluidic concentration device to accelerate the surface hybridization reaction between DNA and morpholinos (MOs) for enhanced detection. The microfluidic concentrator comprises a single polydimethylsiloxane (PDMS) microchannel onto which an ion-selective layer of conductive polymer poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) ( PSS) was directly printed and then reversibly surface bonded onto a morpholino microarray for hybridization. Using this electrokinetic trapping concentrator, we could achieve a maximum concentration factor of ∼800 for DNA and a limit of detection of 10 nM within 15 min. In terms of the detection speed, it enabled faster hybridization by around 10-fold when compared to conventional diffusion-based hybridization. A significant advantage of our approach is that the fabrication of the microfluidic concentrator is completely decoupled from the microarray; by eliminating the need to deposit an ion-selective layer on the microarray surface prior to device integration, interfacing between both modules, the PDMS chip for electrokinetic concentration and the substrate for DNA sensing are easier and applicable to any microarray platform. Furthermore, this fabrication strategy facilitates a multiplexing of concentrators. We have demonstrated the proof-of-concept for multiplexing by building a device with 5 parallel concentrators connected to a single inlet/outlet and applying it to parallel concentration and hybridization. Such device yielded similar concentration and hybridization efficiency compared to that of a single-channel device without adding any complexity to the fabrication and setup. These results demonstrate that our concentrator concept can be applied to the development of a highly multiplexed concentrator-enhanced microarray detection system for either genetic analysis or other diagnostic assays.

Multiplexed detection of anthrax-related toxin genes.

PubMed

Moser, Michael J; Christensen, Deanna R; Norwood, David; Prudent, James R

2006-02-01

Simultaneous analysis of three targets in three colors on any real-time polymerase chain reaction (PCR) instrument would increase the flexibility of real-time PCR. For the detection of Bacillus strains that can cause inhalation anthrax-related illness, this ability would be valuable because two plasmids confer virulence, and internal positive controls are needed to monitor the testing in cases lacking target-specific signals. Using a real-time PCR platform called MultiCode-RTx, multiple assays were developed that specifically monitor the presence of Bacillus anthracis-specific virulence plasmid-associated genes. In particular for use on LightCycler-1, two triplex RTx systems demonstrated high sensitivity with limits of detection nearing single-copy levels for both plasmids. Specificity was established using a combination of Ct values and correct amplicon melting temperatures. All reactions were further verified by detection of an internal positive control. For these two triplex RTx assays, the analytical detection limit was one to nine plasmid copy equivalents, 100% analytical specificity with a 95% confidence interval (CI) of 9%, and 100% analytical sensitivity with a CI of 2%. Although further testing using clinical or environmental samples will be required to assess diagnostic sensitivity and specificity, the RTx platform achieves similar results to those of probe-based real-time systems.

Reverse transcription multiplex PCR for differentiation between polio- and enteroviruses from clinical and environmental samples.

PubMed

Egger, D; Pasamontes, L; Ostermayer, M; Bienz, K

1995-06-01

For the rapid detection of polioviruses and their differentiation from nonpoliovirus enteroviruses, we developed a protocol in which clinical or environmental specimens are first inoculated onto cell cultures in tubes. After overnight incubation, the cultures are subjected to reverse transcription multiplex PCR with a primer pair which detects all enteroviruses (T. Hyypiä, P. Auvinen, and M. Maaronen, J. Gen. Virol. 70:3261-3268 1989) and two newly designed primer pairs specific for all 36 poliovirus strains tested. The PCR products can unequivocally be identified by their lengths in agarose gels, whereas the genetic heterogeneity of the poliovirus strains precludes identification by back-hybridization with internal probes. The proposed protocol is highly insensitive to the inhibitory effects of substances in the sample (stool, sewage). It allows for the detection of polioviruses and for polioviruses to be distinguished from nonpoliovirus enteroviruses within 24 h, and it allows for the concomitant isolation of a viable strain suitable for further typing.

Serotype 3 is a common serotype causing invasive pneumococcal disease in children less than 5 years old, as identified by real-time PCR.

PubMed

Selva, L; Ciruela, P; Esteva, C; de Sevilla, M F; Codina, G; Hernandez, S; Moraga, F; García-García, J J; Planes, A; Coll, F; Jordan, I; Cardeñosa, N; Batalla, J; Salleras, L; Dominguez, A; Muñoz-Almagro, C

2012-07-01

Serotype 3 is one of the most often detected pneumococcal serotypes in adults and it is associated with serious disease. In contrast, the isolation of serotype 3 by bacterial culture is unusual in children with invasive pneumococcal disease (IPD). The purpose of this study was to learn the serotype distribution of IPD, including culture-negative episodes, by using molecular methods in normal sterile samples. We studied all children<5 years of age with IPD admitted to two paediatric hospitals in Catalonia, Spain, from 2007 to 2009. A sequential real-time polymerase chain reaction (PCR) approach was added to routine methods for the detection and serotyping of pneumococcal infection. Among 257 episodes (219 pneumonia, 27 meningitis, six bacteraemia and five others), 33.5% were identified by culture and the rest, 66.5%, were detected exclusively by real-time PCR. The most common serotypes detected by culture were serotypes 1 (26.7%) and 19A (25.6%), and by real-time PCR, serotypes 1 (19.8%) and 3 (18.1%). Theoretical coverage rates by the PCV7, PCV10 and PCV13 vaccines were 10.5, 52.3 and 87.2%, respectively, for those episodes identified by culture, compared to 5.3, 31.6 and 60.2% for those identified only by real-time PCR. Multiplex real-time PCR has been shown to be useful for surveillance studies of IPD. Serotype 3 is underdiagnosed by culture and is important in paediatric IPD.

Evaluation of a new commercial real-time PCR assay for diagnosis of Pneumocystis jirovecii pneumonia and identification of dihydropteroate synthase (DHPS) mutations.

PubMed

Montesinos, Isabel; Delforge, Marie-Luce; Ajjaham, Farida; Brancart, Françoise; Hites, Maya; Jacobs, Frederique; Denis, Olivier

2017-01-01

The PneumoGenius® real-time PCR assay is a new commercial multiplex real-time PCR method, which detects the Pneumocystis mitochondrial ribosomal large subunit (mtLSU) and two dihydropteroate synthase (DHPS) point mutations. To evaluate the clinical performance of this new real-time PCR assay we tested 120 extracted DNA samples from bronchoalveolar lavage specimens. These set of extracted DNA samples had already tested positive for Pneumocystis and patients had been classified in probable and unlikely PCP in a previous study. To evaluate de accuracy of the DHPS mutant's identification, an "in house" PCR and sequencing was performed. The sensitivity and specificity of PneumoGenius® PCR in discriminating between probable and unlikely Pneumocystis pneumonia (PCP) were 70% and 82% respectively. PneumoGenius® PCR was able to genotype more samples than "in house" DHPS PCR and sequencing. The same DHPS mutations were observed by both methods in four patients: two patients with a single mutation in position 171 (Pro57Ser) and two patients with a double mutation in position 165 (Thr55Ala) and in position 171 (Pro57Ser). A low rate of P. jirovecii (4.5%) harboring DHPS mutations was found, comparable to rates observed in other European countries. The PneumoGenius® real-time PCR is a suitable real-time PCR for PCP diagnosis and detection of DHPS mutants. The added value of DHPS mutation identification can assist in understanding the role of these mutations in prophylaxis failure or treatment outcome. Copyright © 2016 Elsevier Inc. All rights reserved.

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1.

PubMed

Yaren, Ozlem; Alto, Barry W; Bradley, Kevin M; Moussatche, Patricia; Glushakova, Lyudmyla; Benner, Steven A

2018-03-13

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time. Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to "approved" reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel. This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

A novel enterovirus and parechovirus multiplex one-step real-time PCR-validation and clinical experience.

PubMed

Nielsen, Alex Christian Yde; Böttiger, Blenda; Midgley, Sofie Elisabeth; Nielsen, Lars Peter

2013-11-01

As the number of new enteroviruses and human parechoviruses seems ever growing, the necessity for updated diagnostics is relevant. We have updated an enterovirus assay and combined it with a previously published assay for human parechovirus resulting in a multiplex one-step RT-PCR assay. The multiplex assay was validated by analysing the sensitivity and specificity of the assay compared to the respective monoplex assays, and a good concordance was found. Furthermore, the enterovirus assay was able to detect 42 reference strains from all 4 species, and an additional 9 genotypes during panel testing and routine usage. During 15 months of routine use, from October 2008 to December 2009, we received and analysed 2187 samples (stool samples, cerebrospinal fluids, blood samples, respiratory samples and autopsy samples) were tested, from 1546 patients and detected enteroviruses and parechoviruses in 171 (8%) and 66 (3%) of the samples, respectively. 180 of the positive samples could be genotyped by PCR and sequencing and the most common genotypes found were human parechovirus type 3, echovirus 9, enterovirus 71, Coxsackievirus A16, and echovirus 25. During 2009 in Denmark, both enterovirus and human parechovirus type 3 had a similar seasonal pattern with a peak during the summer and autumn. Human parechovirus type 3 was almost invariably found in children less than 4 months of age. In conclusion, a multiplex assay was developed allowing simultaneous detection of 2 viruses, which can cause similar clinical symptoms. Copyright © 2013 Elsevier B.V. All rights reserved.

The vTAS suite: A simulator for classical and multiplexed three-axis neutron spectrometers

NASA Astrophysics Data System (ADS)

Boehm, M.; Filhol, A.; Raoul, Y.; Kulda, J.; Schmidt, W.; Schmalzl, K.; Farhi, E.

2013-01-01

The vTAS suite provides graphical assistance to prepare and perform inelastic neutron scattering experiments on a TAS instrument, including latest multiplexed instrumental configurations, such as FlatCone, IMPS and UFO. The interactive display allows for flexible translation between instrument positions in real space and neutron scattering conditions represented in reciprocal space. It is a platform independent public domain software tool, available for download from the website of the Institut Laue Langevin (ILL).

Evaluation of Several Biochemical and Molecular Techniques for Identification of Streptococcus pneumoniae and Streptococcus pseudopneumoniae and Their Detection in Respiratory Samples

PubMed Central

Schelfaut, Jacqueline J. G.; Bernards, Alexandra T.; Claas, Eric C. J.

2012-01-01

The identification and detection of mitis group streptococci, which contain Streptococcus pneumoniae, have been hampered by the lack of sensitive and specific assays. In this study, we evaluated several biochemical and molecular assays for the identification of S. pneumoniae and Streptococcus pseudopneumoniae and their distinction from other mitis group streptococci using a collection of 54 isolates obtained by the routine culturing of 53 respiratory specimens from patients with community-acquired pneumonia. The combined results of the biochemical and molecular assays indicated the presence of 23 S. pneumoniae, 2 S. pseudopneumoniae, and 29 other mitis group streptococcal isolates. The tube bile solubility test that is considered gold standard for the identification of S. pneumoniae showed concordant results with optochin susceptibility testing (CO2 atmosphere) and a real-time multiplex PCR assay targeting the Spn9802 fragment and the autolysin gene. Optochin susceptibility testing upon incubation in an O2 atmosphere, bile solubility testing by oxgall disk, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and sequence analysis of the tuf and rpoB genes resulted in several false-positive, false-negative, or inconclusive results. The S. pseudopneumoniae isolates could be identified only by molecular assays, and the multiplex real-time PCR assay was concluded to be most convenient for the identification of S. pneumoniae and S. pseudopneumoniae isolates. Using this method, S. pneumoniae and S. pseudopneumoniae DNA could be detected in the respiratory samples from which they were isolated and in an additional 11 samples from which only other streptococci were isolated. PMID:22278834

The need for non- or minimally-invasive biomonitoring strategies and the development of pharmacokinetic/pharmacodynamic models for quantification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Timchalk, Charles; Weber, Thomas J.; Smith, Jordan N.

Advancements in Exposure Science involving the development and deployment of biomarkers of exposure and biological response are anticipated to significantly (and positively) influence health outcomes associated with occupational, environmental and clinical exposure to chemicals/drugs. To achieve this vision, innovative strategies are needed to develop multiplex sensor platforms capable of quantifying individual and mixed exposures (i.e. systemic dose) by measuring biomarkers of dose and biological response in readily obtainable (non-invasive) biofluids. Secondly, the use of saliva (alternative to blood) for biomonitoring coupled with the ability to rapidly analyze multiple samples in real-time offers an innovative opportunity to revolutionize biomonitoring assessments. Inmore » this regard, the timing and number of samples taken for biomonitoring will not be limited as is currently the case. In addition, real-time analysis will facilitate identification of work practices or conditions that are contributing to increased exposures and will make possible a more rapid and successful intervention strategy. The initial development and application of computational models for evaluation of saliva/blood analyte concentration at anticipated exposure levels represents an important opportunity to establish the limits of quantification and robustness of multiplex sensor systems by exploiting a unique computational modeling framework. The use of these pharmacokinetic models will also enable prediction of an exposure dose based on the saliva/blood measurement. This novel strategy will result in a more accurate prediction of exposures and, once validated, can be employed to assess dosimetry to a broad range of chemicals in support of biomonitoring and epidemiology studies.« less

Novel Quaternary Quantum Decoder, Multiplexer and Demultiplexer Circuits

NASA Astrophysics Data System (ADS)

Haghparast, Majid; Monfared, Asma Taheri

2017-05-01

Multiple valued logic is a promising approach to reduce the width of the reversible or quantum circuits, moreover, quaternary logic is considered as being a good choice for future quantum computing technology hence it is very suitable for the encoded realization of binary logic functions through its grouping of 2-bits together into quaternary values. The Quaternary decoder, multiplexer, and demultiplexer are essential units of quaternary digital systems. In this paper, we have initially designed a quantum realization of the quaternary decoder circuit using quaternary 1-qudit gates and quaternary Muthukrishnan-Stroud gates. Then we have presented quantum realization of quaternary multiplexer and demultiplexer circuits using the constructed quaternary decoder circuit and quaternary controlled Feynman gates. The suggested circuits in this paper have a lower quantum cost and hardware complexity than the existing designs that are currently used in quaternary digital systems. All the scales applied in this paper are based on Nanometric area.

A reversible fluorescent probe for real-time live-cell imaging and quantification of endogenous hydropolysulfides.

PubMed

Umezawa, Keitaro; Kamiya, Mako; Urano, Yasuteru

2018-05-23

The chemical biology of reactive sulfur species, including hydropolysulfides, has been a subject undergoing intense study in recent years, but further understanding of their 'intact' function in living cells has been limited due to a lack of appropriate analytical tools. In order to overcome this limitation, we developed a new type of fluorescent probe which reversibly and selectively reacts to hydropolysulfides. The probe enables live-cell visualization and quantification of endogenous hydropolysulfides without interference from intrinsic thiol species such as glutathione. Additionally, real-time reversible monitoring of oxidative-stress-induced fluctuation of intrinsic hydropolysulfides has been achieved with a temporal resolution in the order of seconds, a result which has not yet been realized using conventional methods. These results reveal the probe's versatility as a new fluorescence imaging tool to understand the function of intracellular hydropolysulfides. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dynamical hologram generation for high speed optical trapping of smart droplet microtools

PubMed Central

Lanigan, P. M. P.; Munro, I.; Grace, E. J.; Casey, D. R.; Phillips, J.; Klug, D. R.; Ces, O.; Neil, M. A. A.

2012-01-01

This paper demonstrates spatially selective sampling of the plasma membrane by the implementation of time-multiplexed holographic optical tweezers for Smart Droplet Microtools (SDMs). High speed (>1000fps) dynamical hologram generation was computed on the graphics processing unit of a standard display card and controlled by a user friendly LabView interface. Time multiplexed binary holograms were displayed in real time and mirrored to a ferroelectric Spatial Light Modulator. SDMs were manufactured with both liquid cores (as previously described) and solid cores, which confer significant advantages in terms of stability, polydispersity and ease of use. These were coated with a number of detergents, the most successful based upon lipids doped with transfection reagents. In order to validate these, trapped SDMs were maneuvered up to the plasma membrane of giant vesicles containing Nile Red and human biliary epithelial (BE) colon cancer cells with green fluorescent labeled protein (GFP)-labeled CAAX (a motif belonging to the Ras protein). Bright field and fluorescence images showed that successful trapping and manipulation of multiple SDMs in x, y, z was achieved with success rates of 30-50% and that subsequent membrane-SDM interactions led to the uptake of Nile Red or GFP-CAAX into the SDM. PMID:22808432

Array-scale performance of TES X-ray Calorimeters Suitable for Constellation-X

NASA Technical Reports Server (NTRS)

Kilbourne, C. A.; Bandler, S. R.; Brown, A. D.; Chervenak, J. A.; Eckart, M. E.; Finkbeiner, F. M.; Iyomoto, N.; Kelley, R. L.; Porter, F. S.; Smith, S. J.;

Development of two real-time multiplex PCR assays for the detection and quantification of eight key bacterial pathogens in lower respiratory tract infections

PubMed Central

Gadsby, N.J.; McHugh, M.P.; Russell, C.D.; Mark, H.; Conway Morris, A.; Laurenson, I.F.; Hill, A.T.; Templeton, K.E.

2015-01-01

The frequent lack of a positive and timely microbiological diagnosis in patients with lower respiratory tract infection (LRTI) is an important obstacle to antimicrobial stewardship. Patients are typically prescribed broad-spectrum empirical antibiotics while microbiology results are awaited, but, because these are often slow, negative, or inconclusive, de-escalation to narrow-spectrum agents rarely occurs in clinical practice. The aim of this study was to develop and evaluate two multiplex real-time PCR assays for the sensitive detection and accurate quantification of Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. We found that all eight bacterial targets could be reliably quantified from sputum specimens down to a concentration of 100 CFUs/reaction (8333 CFUs/mL). Furthermore, all 249 positive control isolates were correctly detected with our assay, demonstrating effectiveness on both reference strains and local clinical isolates. The specificity was 98% on a panel of nearly 100 negative control isolates. Bacterial load was quantified accurately when three bacterial targets were present in mixtures of varying concentrations, mimicking likely clinical scenarios in LRTI. Concordance with culture was 100% for culture-positive sputum specimens, and 90% for bronchoalveolar lavage fluid specimens, and additional culture-negative bacterial infections were detected and quantified. In conclusion, a quantitative molecular test for eight key bacterial causes of LRTI has the potential to provide a more sensitive decision-making tool, closer to the time-point of patient admission than current standard methods. This should facilitate de-escalation from broad-spectrum to narrow-spectrum antibiotics, substantially improving patient management and supporting efforts to curtail inappropriate antibiotic use. PMID:25980353

Cat fleas (Ctenocephalides felis) from cats and dogs in New Zealand: Molecular characterisation, presence of Rickettsia felis and Bartonella clarridgeiae and comparison with Australia.

PubMed

Chandra, Shona; Forsyth, Maureen; Lawrence, Andrea L; Emery, David; Šlapeta, Jan

2017-01-30

The cat flea (Ctenocephalides felis) is the most common flea species parasitising both domestic cats and dogs globally. Fleas are known vectors of zoonotic pathogens such as vector borne Rickettsia and Bartonella. This study compared cat fleas from domestic cats and dogs in New Zealand's North and South Islands to Australian cat fleas, using the mitochondrial DNA (mtDNA) marker, cytochrome c oxidase subunit I and II (cox1, cox2). We assessed the prevalence of Rickettsia and Bartonella using genus specific multiplexed real-time PCR assays. Morphological identification confirmed that the cat flea (C. felis) is the most common flea in New Zealand. The examined fleas (n=43) at cox1 locus revealed six closely related C. felis haplotypes (inter-haplotype distance 1.1%) across New Zealand. The New Zealand C. felis haplotypes were identical or near identical with haplotypes from southern Australia demonstrating common dispersal of haplotype lineage across both the geographical (Tasman Sea) and climate scale. New Zealand cat fleas carried Rickettsia felis (5.3%) and Bartonella clarridgeiae (18.4%). To understand the capability of C. felis to vector zoonotic pathogens, we determined flea cox1 and cox2 haplotype diversity with the tandem multiplexed real-time PCR and sequencing for Bartonella and Rickettsia. This enabled us to demonstrate highly similar cat fleas on cat and dog populations across Australia and New Zealand. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular differentiation of Opisthorchis viverrini and Clonorchis sinensis eggs by multiplex real-time PCR with high resolution melting analysis.

PubMed

Kaewkong, Worasak; Intapan, Pewpan M; Sanpool, Oranuch; Janwan, Penchom; Thanchomnang, Tongjit; Laummaunwai, Porntip; Lulitanond, Viraphong; Doanh, Pham Ngoc; Maleewong, Wanchai

2013-12-01

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4±0.09℃ and 85.9±0.08℃ for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.

Surveillance of Food- and Smear-Transmitted Pathogens in European Soldiers with Diarrhea on Deployment in the Tropics: Experience from the European Union Training Mission (EUTM) Mali

PubMed Central

Frickmann, Hagen; Warnke, Philipp; Frey, Claudia; Schmidt, Salvatore; Janke, Christian; Erkens, Kay; Schotte, Ulrich; Köller, Thomas; Maaßen, Winfried; Podbielski, Andreas; Binder, Alfred; Hinz, Rebecca; Queyriaux, Benjamin; Wiemer, Dorothea; Schwarz, Norbert Georg; Hagen, Ralf Matthias

2015-01-01

Introduction. Since 2013, European soldiers have been deployed on the European Union Training Mission (EUTM) in Mali. From the beginning, diarrhea has been among the most “urgent” concerns. Diarrhea surveillance based on deployable real-time PCR equipment was conducted between December 2013 and August 2014. Material and Methods. In total, 53 stool samples were obtained from 51 soldiers with acute diarrhea. Multiplex PCR panels comprised enteroinvasive bacteria, diarrhea-associated Escherichia coli (EPEC, ETEC, EAEC, and EIEC), enteropathogenic viruses, and protozoa. Noroviruses were characterized by sequencing. Cultural screening for Enterobacteriaceae with extended-spectrum beta-lactamases (ESBL) with subsequent repetitive sequence-based PCR (rep-PCR) typing was performed. Clinical information was assessed. Results. Positive PCR results for diarrhea-associated pathogens were detected in 43/53 samples, comprising EPEC (n = 21), ETEC (n = 19), EAEC (n = 15), Norovirus (n = 10), Shigella spp./EIEC (n = 6), Cryptosporidium parvum (n = 3), Giardia duodenalis (n = 2), Salmonella spp. (n = 1), Astrovirus (n = 1), Rotavirus (n = 1), and Sapovirus (n = 1). ESBL-positive Enterobacteriaceae were grown from 13 out of 48 samples. Simultaneous infections with several enteropathogenic agents were observed in 23 instances. Symptoms were mild to moderate. There were hints of autochthonous transmission. Conclusions. Multiplex real-time PCR proved to be suitable for diarrhea surveillance on deployment. Etiological attribution is challenging in cases of detection of multiple pathogens. PMID:26525953

[Difference of three standard curves of real-time reverse-transcriptase PCR in viable Vibrio parahaemolyticus quantification].

PubMed

Jin, Mengtong; Sun, Wenshuo; Li, Qin; Sun, Xiaohong; Pan, Yingjie; Zhao, Yong

2014-04-04

We evaluated the difference of three standard curves in quantifying viable Vibrio parahaemolyticus in samples by real-time reverse-transcriptase PCR (Real-time RT-PCR). The standard curve A was established by 10-fold diluted cDNA. The cDNA was reverse transcripted after RNA synthesized in vitro. The standard curve B and C were established by 10-fold diluted cDNA. The cDNA was synthesized after RNA isolated from Vibrio parahaemolyticus in pure cultures (10(8) CFU/mL) and shrimp samples (10(6) CFU/g) (Standard curve A and C were proposed for the first time). Three standard curves were performed to quantitatively detect V. parahaemolyticus in six samples, respectively (Two pure cultured V. parahaemolyticus samples, two artificially contaminated cooked Litopenaeus vannamei samples and two artificially contaminated Litopenaeus vannamei samples). Then we evaluated the quantitative results of standard curve and the plate counting results and then analysed the differences. The three standard curves all show a strong linear relationship between the fractional cycle number and V. parahaemolyticus concentration (R2 > 0.99); The quantitative results of Real-time PCR were significantly (p < 0.05) lower than the results of plate counting. The relative errors compared with the results of plate counting ranked standard curve A (30.0%) > standard curve C (18.8%) > standard curve B (6.9%); The average differences between standard curve A and standard curve B and C were - 2.25 Lg CFU/mL and - 0.75 Lg CFU/mL, respectively, and the mean relative errors were 48.2% and 15.9%, respectively; The average difference between standard curve B and C was among (1.47 -1.53) Lg CFU/mL and the average relative errors were among 19.0% - 23.8%. Standard curve B could be applied to Real-time RT-PCR when quantify the number of viable microorganisms in samples.

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

PubMed Central

Cura, Carolina I.; Duffy, Tomas; Lucero, Raúl H.; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J.; Valencia Ayala, Edward; Kjos, Sonia A.; Santalla, José; Mahaney, Susan M.; Cayo, Nelly M.; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S.; Acosta Viana, Karla Y.; Brutus, Laurent; Ocampo, Susana B.; Aznar, Christine; Cuba Cuba, Cesar A.; Gürtler, Ricardo E.; Ramsey, Janine M.; Ribeiro, Isabela; VandeBerg, John L.; Yadon, Zaida E.; Osuna, Antonio; Schijman, Alejandro G.

2015-01-01

Background Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). Methods/Principal Findings The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Conclusions/Significance Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. PMID:25993316

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples.

PubMed

Cura, Carolina I; Duffy, Tomas; Lucero, Raúl H; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J; Valencia Ayala, Edward; Kjos, Sonia A; Santalla, José; Mahaney, Susan M; Cayo, Nelly M; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S; Acosta Viana, Karla Y; Brutus, Laurent; Ocampo, Susana B; Aznar, Christine; Cuba Cuba, Cesar A; Gürtler, Ricardo E; Ramsey, Janine M; Ribeiro, Isabela; VandeBerg, John L; Yadon, Zaida E; Osuna, Antonio; Schijman, Alejandro G

2015-05-01

Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.

Identification of forensic samples by using an infrared-based automatic DNA sequencer.

PubMed

Ricci, Ugo; Sani, Ilaria; Klintschar, Michael; Cerri, Nicoletta; De Ferrari, Francesco; Giovannucci Uzielli, Maria Luisa

2003-06-01

We have recently introduced a new protocol for analyzing all core loci of the Federal Bureau of Investigation's (FBI) Combined DNA Index System (CODIS) with an infrared (IR) automatic DNA sequencer (LI-COR 4200). The amplicons were labeled with forward oligonucleotide primers, covalently linked to a new infrared fluorescent molecule (IRDye 800). The alleles were displayed as familiar autoradiogram-like images with real-time detection. This protocol was employed for paternity testing, population studies, and identification of degraded forensic samples. We extensively analyzed some simulated forensic samples and mixed stains (blood, semen, saliva, bones, and fixed archival embedded tissues), comparing the results with donor samples. Sensitivity studies were also performed for the four multiplex systems. Our results show the efficiency, reliability, and accuracy of the IR system for the analysis of forensic samples. We also compared the efficiency of the multiplex protocol with ultraviolet (UV) technology. Paternity tests, undegraded DNA samples, and real forensic samples were analyzed with this approach based on IR technology and with UV-based automatic sequencers in combination with commercially-available kits. The comparability of the results with the widespread UV methods suggests that it is possible to exchange data between laboratories using the same core group of markers but different primer sets and detection methods.

A new precoding scheme for spectral efficient optical OFDM systems

NASA Astrophysics Data System (ADS)

Hardan, Saad Mshhain; Bayat, Oguz; Abdulkafi, Ayad Atiyah

2018-07-01

Achieving high spectral efficiency is the key requirement of 5G and optical wireless communication systems and has recently attracted much attention, aiming to satisfy the ever increasing demand for high data rates in communications systems. In this paper, we propose a new precoding/decoding algorithm for spectral efficient optical orthogonal frequency division multiplexing (OFDM) scheme based visible light communication (VLC) systems. The proposed coded modulated optical (CMO) based OFDM system can be applied for both single input single output (SISO) and multiple input multiple-output (MIMO) architectures. Firstly, the real OFDM time domain signal is obtained through invoking the precoding/decoding algorithm without the Hermitian symmetry. After that, the positive signal is achieved either by adding a DC-bias or by using the spatial multiplexing technique. The proposed CMO-OFDM scheme efficiently improves the spectral efficiency of the VLC system as it does not require the Hermitian symmetry constraint to yield real signals. A comparison of the performance improvement of the proposed scheme with other OFDM approaches is also presented in this work. Simulation results show that the proposed CMO-OFDM scheme can not only enhance the spectral efficiency of OFDM-based VLC systems but also improve bit error rate (BER) performance compared with other optical OFDM schemes.

Real-time experimental demonstrations of software reconfigurable optical OFDM transceivers utilizing DSP-based digital orthogonal filters for SDN PONs.

PubMed

Duan, X; Giddings, R P; Bolea, M; Ling, Y; Cao, B; Mansoor, S; Tang, J M

2014-08-11

Real-time optical OFDM (OOFDM) transceivers with on-line software-controllable channel reconfigurability and transmission performance adaptability are experimentally demonstrated, for the first time, utilizing Hilbert-pair-based 32-tap digital orthogonal filters implemented in FPGAs. By making use of an 8-bit DAC/ADC operating at 2GS/s, an oversampling factor of 2 and an EML intensity modulator, the demonstrated RF conversion-free transceiver supports end-to-end real-time simultaneous adaptive transmissions, within a 1GHz signal spectrum region, of a 2.03Gb/s in-phase OOFDM channel and a 1.41Gb/s quadrature-phase OOFDM channel over a 25km SSMF IMDD system. In addition, detailed experimental explorations are also undertaken of key physical mechanisms limiting the maximum achievable transmission performance, impacts of transceiver's channel multiplexing/demultiplexing operations on the system BER performance, and the feasibility of utilizing adaptive modulation to combat impairments associated with low-complexity digital filter designs. Furthermore, experimental results indicate that the transceiver incorporating a fixed digital orthogonal filter DSP architecture can be made transparent to various signal modulation formats up to 64-QAM.

Recent Progress in Biosensors for Environmental Monitoring: A Review

PubMed Central

2017-01-01

The environmental monitoring has been one of the priorities at the European and global scale due to the close relationship between the environmental pollution and the human health/socioeconomic development. In this field, the biosensors have been widely employed as cost-effective, fast, in situ, and real-time analytical techniques. The need of portable, rapid, and smart biosensing devices explains the recent development of biosensors with new transduction materials, obtained from nanotechnology, and for multiplexed pollutant detection, involving multidisciplinary experts. This review article provides an update on recent progress in biosensors for the monitoring of air, water, and soil pollutants in real conditions such as pesticides, potentially toxic elements, and small organic molecules including toxins and endocrine disrupting chemicals. PMID:29244756

Wireless OAM transmission system based on elliptical microstrip patch antenna.

PubMed

Chen, Jia Jia; Lu, Qian Nan; Dong, Fei Fei; Yang, Jing Jing; Huang, Ming

2016-05-30

The multiplexing transmission has always been a focus of attention for communication technology. In this paper, the radiation characteristics of circular microstrip patch antenna was firstly analyzed based on cavity model theory, and then spiral beams carrying orbital angular momentum (OAM) were generated, using elliptical microstrip patch antenna, with a single feed probe instead of a standard circular patch with two feedpoints. Moreover, by combining the proposed elliptic microstrip patch antenna with Universal Software Radio Peripheral (USRP), a wireless OAM transmission system was established and the real-time transmission of text, image and video in a real channel environment was realized. Since the wireless OAM transmission has the advantage of good safety and high spectrum utilization efficiency, this work has theoretical significance and potential application.

Recent Progress in Biosensors for Environmental Monitoring: A Review.

PubMed

Justino, Celine I L; Duarte, Armando C; Rocha-Santos, Teresa A P

2017-12-15

The environmental monitoring has been one of the priorities at the European and global scale due to the close relationship between the environmental pollution and the human health/socioeconomic development. In this field, the biosensors have been widely employed as cost-effective, fast, in situ, and real-time analytical techniques. The need of portable, rapid, and smart biosensing devices explains the recent development of biosensors with new transduction materials, obtained from nanotechnology, and for multiplexed pollutant detection, involving multidisciplinary experts. This review article provides an update on recent progress in biosensors for the monitoring of air, water, and soil pollutants in real conditions such as pesticides, potentially toxic elements, and small organic molecules including toxins and endocrine disrupting chemicals.

Chopped molecular beam multiplexing system

NASA Technical Reports Server (NTRS)

Adams, Billy R. (Inventor)

1986-01-01

The integration of a chopped molecular beam mass spectrometer with a time multiplexing system is described. The chopping of the molecular beam is synchronized with the time intervals by a phase detector and a synchronous motor. Arithmetic means are generated for phase shifting the chopper with respect to the multiplexer. A four channel amplifier provides the capacity to independently vary the baseline and amplitude in each channel of the multiplexing system.

Development of field-based real-time reverse transcription-polymerase chain reaction assays for detection of Chikungunya and O'nyong-nyong viruses in mosquitoes.

PubMed

Smith, Darci R; Lee, John S; Jahrling, Jordan; Kulesh, David A; Turell, Michael J; Groebner, Jennifer L; O'Guinn, Monica L

2009-10-01

Chikungunya (CHIK) and O'nyong-nyong (ONN) are important emerging arthropod-borne diseases. Molecular diagnosis of these two viruses in mosquitoes has not been evaluated, and the effects of extraneous mosquito tissue on assay performance have not been tested. Additionally, no real-time reverse transcription-polymerase chain reaction (RT-PCR) assay exists for detecting ONN virus (ONNV) RNA. We describe the development of sensitive and specific real-time RT-PCR assays for detecting CHIK and ONN viral RNA in mosquitoes, which have application for field use. In addition, we compared three methods for primer/probe design for assay development by evaluating their sensitivity and specificity. This comparison resulted in development of virus-specific assays that could detect less than one plaque-forming unit equivalent of each of the viruses in mosquitoes. The use of these assays will aid in arthropod-borne disease surveillance and in the control of the associated diseases.

Rapid detection of avian influenza virus a and subtype H5N1 by single step multiplex reverse transcription-polymerase chain reaction.

PubMed

Wei, Hui-Ling; Bai, Gui-Rong; Mweene, Aaron S; Zhou, Ying-Chun; Cong, Yan-Long; Pu, Juan; Wang, Shuai; Kida, Hiroshi; Liu, Jin-Hua

2006-06-01

Outbreaks of H5N1 highly pathogenic avian influenza (HPAI) virus caused great economic losses to the poultry industry and resulted in human deaths in Thailand and Viet Nam in 2004. Rapid typing and subtyping of H5N1 viruses, especially from clinical specimens, are desirable for taking prompt control measures to prevent the spread of the disease. Here, we developed a set of oligonucleotide primers able to detect, type and subtype H5 and N1 influenza viruses in a single step multiplex reverse transcription-polymerase chain reaction (RT-PCR). RNA was extracted from allantoic fluid or from specimens with guanidinium isothiocyanate reagent. Reverse transcription and PCR were carried out with a mixture of primers specific for influenza viruses of type A, subtype H5 and N1 in a single reaction system under identical conditions. The amplified DNA fragments were analyzed by agarose gel electrophoresis. All the H5N1 viruses tested in the study and the experimental specimens presented three specific bands by the method established here. The results presented here suggest that the method described below is rapid and specific and, therefore, could be valuable in the rapid detection of H5N1 influenza viruses in clinics.

Multiwavelength digital holography with wavelength-multiplexed holograms and arbitrary symmetric phase shifts.

PubMed

Tahara, Tatsuki; Otani, Reo; Omae, Kaito; Gotohda, Takuya; Arai, Yasuhiko; Takaki, Yasuhiro

2017-05-15

We propose multiwavelength in-line digital holography with wavelength-multiplexed phase-shifted holograms and arbitrary symmetric phase shifts. We use phase-shifting interferometry selectively extracting wavelength information to reconstruct multiwavelength object waves separately from wavelength-multiplexed monochromatic images. The proposed technique obtains systems of equations for real and imaginary parts of multiwavelength object waves from the holograms by introducing arbitrary symmetric phase shifts. Then, the technique derives each complex amplitude distribution of each object wave selectively and analytically by solving the two systems of equations. We formulate the algorithm in the case of an arbitrary number of wavelengths and confirm its validity numerically and experimentally in the cases where the number of wavelengths is two and three.

Time Division Multiplexing of Semiconductor Qubits

NASA Astrophysics Data System (ADS)

Jarratt, Marie Claire; Hornibrook, John; Croot, Xanthe; Watson, John; Gardner, Geoff; Fallahi, Saeed; Manfra, Michael; Reilly, David

Readout chains, comprising resonators, amplifiers, and demodulators, are likely to be precious resources in quantum computing architectures. The potential to share readout resources is contingent on realising efficient means of time-division multiplexing (TDM) schemes that are compatible with quantum computing. Here, we demonstrate TDM using a GaAs quantum dot device with multiple charge sensors. Our device incorporates chip-level switches that do not load the impedance matching network. When used in conjunction with frequency multiplexing, each frequency tone addresses multiple time-multiplexed qubits, vastly increasing the capacity of a single readout line.

Quantification of meat proportions by measuring DNA contents in raw and boiled sausages using matrix-adapted calibrators and multiplex real-time PCR.

PubMed

Köppel, René; Eugster, Albert; Ruf, Jürg; Rentsch, Jürg

2012-01-01

The quantification of meat proportions in raw and boiled sausage according to the recipe was evaluated using three different calibrators. To measure the DNA contents from beef, pork, sheep (mutton), and horse, a tetraplex real-time PCR method was applied. Nineteen laboratories analyzed four meat products each made of different proportions of beef, pork, sheep, and horse meat. Three kinds of calibrators were used: raw and boiled sausages of known proportions ranging from 1 to 55% of meat, and a dilution series of DNA from muscle tissue. In general, results generated using calibration sausages were more accurate than those resulting from the use of DNA from muscle tissue, and exhibited smaller measurement uncertainties. Although differences between uses of raw and boiled calibration sausages were small, the most precise and accurate results were obtained by calibration with fine-textured boiled reference sausages.

Clinical validation of a point-of-care multiplexed in vitro immunoassay using monoclonal antibodies (the MSD influenza test) in four hospitals in Vietnam.

PubMed

van Doorn, H Rogier; Kinh, Nguyen van; Tuan, Ha Manh; Tuan, Tran Anh; Minh, Ngo Ngoc Quang; Bryant, Juliet E; Hang, Vu thi Ty; Uyen, Le thi Tham; Thinh, Le Quoc; Anh, Tran thi Ngoc; Lan, Nguyen Phu Huong; Trung, Nguyen Vu; Taylor, Walter; Merson, Laura; Wertheim, Heiman F L; Farrar, Jeremy; Wolbers, Marcel; Chau, Nguyen van Vinh; de Jong, Menno D

2012-05-01

Point-of-care (POC) diagnostic tests for influenza can considerably shorten the time to clinical decision making. An investigational POC test based on a multiplexed immunoassay was developed by Meso Scale Diagnostics, LLC (MSD), with the objective to make a more sensitive rapid test that can also subtype influenza A viruses (1977 H1, H3, and H5). Between February and November 2010, we conducted a prospective multicenter study at four hospitals in Vietnam and compared the performance of this test to that of the WHO/CDC real-time reverse transcriptase PCR (RT-PCR) on nasal and throat swab specimens from patients presenting with influenza-like illness. Five hundred sixty-three adults and children with a median age of 25 months were enrolled. Sensitivity and specificity of the test with combined results from nasal and throat swab samples were 74.0% (131/177) and 99.7% (351/352), respectively, compared to RT-PCR. The POC test was as sensitive for influenza virus B as for influenza virus A (74.4% [64/86] versus 73.6% [67/91]). The positivity rate was associated with lower cycle threshold values (a marker for higher viral loads), sample type (73.6% for nasal swab versus 52.4% for throat swab), and younger age. A total of 210 (18.7%) out of 1,126 MSD tests failed, and for 34 (6%) of patients, both test samples failed (these were excluded from the performance analysis). Subtyping could be assessed only for influenza virus A/H3N2, as 1977 H1N1 was not circulating at the time and no H5N1-infected patients were enrolled, and was successful only in 9/54 patients infected with H3 influenza virus who had a positive POC test result for influenza virus A. This novel POC test provided highly sensitive detection of influenza viruses A and B compared to the reported sensitivities of other rapid tests. However, 18.7% of tests failed for technical reasons and subtyping for H3 was poor. Drawbacks to the technology include the requirement for a dedicated reader instrument and the need for continual updating of subtyping antibodies within the test array.

A MULTIPLEX REVERSE TRANSCIPTION-PCR METHOD FOR DETECTION OF HUMAN ENTERIC VIRUSES IN GROUNDWATER

EPA Science Inventory

Untreated groundwater is responsible for about half of the waterborne disease outbreaks in the United States. Human enteric viruses are thought to be leading etiological agents of many of these outbreaks, but there is relatively little information on the types and levels of viru...

Using multiplex-staining to study changes in the maize leaf phosphoproteome in response to mechanical wounding

USDA-ARS?s Scientific Manuscript database

Mechanical wounding of 2-week old maize (Zea mays L.) leaves, one of the first steps in both pathogen infection and herbivore attack, stimulates metabolism and activates signal transduction pathways dedicated to defense and recovery. The signaling pathways include reversible protein phosphorylation...

Let there be bioluminescence – Development of a biophotonic imaging platform for in situ analyses of oral biofilms in animal models

PubMed Central

Merritt, Justin; Senpuku, Hidenobu; Kreth, Jens

2016-01-01

Summary In the current study, we describe a novel biophotonic imaging-based reporter system that is particularly useful for the study of virulence in polymicrobial infections and interspecies interactions within animal models. A suite of luciferase enzymes was compared using three early colonizing species of the human oral flora (Streptococcus mutans, Streptococcus gordonii, and Streptococcus sanguinis) to determine the utility of the different reporters for multiplexed imaging studies in vivo. Using the multiplex approach, we were able to track individual species within a dual species oral infection model in mice with both temporal and spatial resolution. We also demonstrate how biophotonic imaging of multiplexed luciferase reporters could be adapted for real-time quantification of bacterial gene expression in situ. By creating an inducible dual-luciferase expressing reporter strain of S. mutans, we were able to exogenously control and measure expression of nlmAB (encoding the bacteriocin mutacin IV) within mice to assess its importance for the persistence ability of S. mutans in the oral cavity. The imaging system described in the current study circumvents many of the inherent limitations of current animal model systems, which should now make it feasible to test hypotheses that were previously impractical to model. PMID:26119252

An Integrated Quantum Dot Barcode Smartphone Optical Device for Wireless Multiplexed Diagnosis of Infected Patients

NASA Astrophysics Data System (ADS)

Ming, Kevin

Integrating mobile-cellular devices with multiplex molecular diagnostics can potentially provide the most powerful platform for tracking, managing and preventing the transmission of infectious diseases. With over 6.9 billion subscriptions globally, handheld mobile-cellular devices can be programmed to spatially map, temporally track, and transmit information on infections over wide geographical space and boundaries. Current cell phone diagnostic technologies have poor limit of detection, dynamic range, and cannot detect multiple pathogen targets simultaneously, limiting their utility to single infections with high load. Here we combined recent advances in quantum dot barcode technology for molecular detection with smartphones to engineer a simple and low-cost chip-based wireless multiplex diagnostic device. We validated our device using a variety of synthetic genomic targets for the respiratory virus and blood-borne pathogens, and demonstrated that it could detect clinical samples after simple amplification. More importantly, we confirmed that the device is capable of detecting patients infected with a single or multiple infectious pathogens (e.g., HIV and hepatitis B) in a single test. This device advances the capacity for global surveillance of infectious diseases and has the potential to accelerate knowledge exchange-transfer of emerging or exigent disease threats with healthcare and military organizations in real-time.

Development of tailored real-time RT-PCR assays for the detection and differentiation of serotype O, A and Asia-1 foot-and-mouth disease virus lineages circulating in the Middle East.

PubMed

Reid, Scott M; Mioulet, Valerie; Knowles, Nick J; Shirazi, Nazeem; Belsham, Graham J; King, Donald P

2014-10-01

Rapid and accurate diagnosis is essential for effective control of foot-and-mouth disease (FMD). In countries where FMD is endemic, identification of the serotypes of the causative virus strains is important for vaccine selection and tracing the source of outbreaks. In this study, real-time reverse transcription polymerase chain reaction (rRT-PCR) assays using primer/probe sets designed from the VP1 coding region of the virus genomes were developed for the specific detection of serotype O, A and Asia-1 FMD viruses (FMDVs) circulating in the Middle East. These assays were evaluated using representative field samples of serotype O strains belonging exclusively to the PanAsia-2 lineage, serotype A strains of the Iran-05 lineage and serotype Asia-1 viruses from three relevant sub-groups. When RNA extracted from archival and contemporary field strains was tested using one- or two-step rRT-PCR assays, all three primer/probe sets detected the RNA from homotypic viruses and no cross-reactivity was observed with heterotypic viruses. Similar results were obtained using both single- and multiplex assay formats. Using plasmid standards, the minimum detection level of these tests was found to be lower than two copies. The results illustrate the potential of tailored rRT-PCR tools for the detection and categorization of viruses circulating in the Middle East belonging to distinct subgroups of serotypes O, A and Asia-1. These assays can also overcome the problem of serotyping samples which are found positive by the generic rRT-PCR diagnostic assays but negative by virus isolation and antigen-detection ELISA which would otherwise have to be serotyped by nucleotide sequencing. A similar approach could be used to develop serotyping assays for FMDV strains circulating in other regions of the world. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Cam-driven monochromator for QEXAFS

NASA Astrophysics Data System (ADS)

Caliebe, W. A.; So, I.; Lenhard, A.; Siddons, D. P.

2006-11-01

We have developed a cam-drive for quickly tuning the energy of an X-ray monochromator through an X-ray absorption edge for quick extended X-ray absorption spectroscopy (QEXAFS). The data are collected using a 4-channel, 12-bit multiplexed VME analog to digital converter and a VME angle encoder. The VME crate controller runs a real-time operating system. This system is capable of collecting 2 EXAFS-scans in 1 s with an energy stability of better than 1 eV. Additional improvements to increase the speed and the energy stability are under way.

AWG-based WDM-PON monitoring system using an optical switch and a WDM filter

NASA Astrophysics Data System (ADS)

Liaw, S.-K.; Lai, Y.-T.; Chang, C.-L.; Shung, O.

2008-09-01

A new WDM-PON scheme with real-time monitoring based on a time-sharing method is proposed. It uses an optical time domain reflectometer (OTDR) to monitor multiple ports by integrating an optical switch (OSW) with a dense wavelength division multiplexer (DWDM) at the optical line terminal (OLT) site. Each downstream signal and its corresponding monitoring signal are separated by m times the free-space range (FSR) of an array waveguide grating (AWG). A bit error rate (BER) test in 2.5 Gb/s × 27 km is performed with and without turning on the OTDR. A small power penalty of 0.7 dB is observed compared to the back-to-back measurement.

Low-cost standalone multi-sensor thermometer for long time measurements

NASA Astrophysics Data System (ADS)

Kumchaiseemak, Nakorn; Hormwantha, Tongchai; Wungmool, Piyachat; Suwanatus, Suchat; Kanjai, Supaporn; Lertkitthaworn, Thitima; Jutamanee, Kanapol; Luengviriya, Chaiya

2017-09-01

We present a portable device for long-time recording of the temperature at multiple measuring points. Thermocouple wires are utilized as the sensors attached to the objects. To minimize the production cost, the measured voltage signals are relayed via a multiplexer to a set of amplifiers and finally to a single microcontroller. The observed temperature and the corresponding date and time, obtained from a real-time clock circuit, are recorded in a memory card for further analysis. The device is powered by a rechargeable battery and placed in a rainproof container, thus it can operate under outdoor conditions. A demonstration of the device usage in a mandarin orange cultivation field of the Royal project, located in the northern Thailand, is illustrated.

Increased robustness of single-molecule counting with microfluidics, digital isothermal amplification, and a mobile phone versus real-time kinetic measurements.

PubMed

Selck, David A; Karymov, Mikhail A; Sun, Bing; Ismagilov, Rustem F

2013-11-19

Quantitative bioanalytical measurements are commonly performed in a kinetic format and are known to not be robust to perturbation that affects the kinetics itself or the measurement of kinetics. We hypothesized that the same measurements performed in a "digital" (single-molecule) format would show increased robustness to such perturbations. Here, we investigated the robustness of an amplification reaction (reverse-transcription loop-mediated amplification, RT-LAMP) in the context of fluctuations in temperature and time when this reaction is used for quantitative measurements of HIV-1 RNA molecules under limited-resource settings (LRS). The digital format that counts molecules using dRT-LAMP chemistry detected a 2-fold change in concentration of HIV-1 RNA despite a 6 °C temperature variation (p-value = 6.7 × 10(-7)), whereas the traditional kinetic (real-time) format did not (p-value = 0.25). Digital analysis was also robust to a 20 min change in reaction time, to poor imaging conditions obtained with a consumer cell-phone camera, and to automated cloud-based processing of these images (R(2) = 0.9997 vs true counts over a 100-fold dynamic range). Fluorescent output of multiplexed PCR amplification could also be imaged with the cell phone camera using flash as the excitation source. Many nonlinear amplification schemes based on organic, inorganic, and biochemical reactions have been developed, but their robustness is not well understood. This work implies that these chemistries may be significantly more robust in the digital, rather than kinetic, format. It also calls for theoretical studies to predict robustness of these chemistries and, more generally, to design robust reaction architectures. The SlipChip that we used here and other digital microfluidic technologies already exist to enable testing of these predictions. Such work may lead to identification or creation of robust amplification chemistries that enable rapid and precise quantitative molecular measurements under LRS. Furthermore, it may provide more general principles describing robustness of chemical and biological networks in digital formats.

A real time spectrum to dose conversion system

NASA Technical Reports Server (NTRS)

Farmer, B. J.; Johnson, J. H.; Bagwell, R. G.

1972-01-01

A system has been developed which permits the determination of dose in real time or near real time directly from the pulse-height output of a radiation spectrometer. The technique involves the use of the resolution matrix of a spectrometer, the radiation energy-to-dose conversion function, and the geometrical factors, although the order of matrix operations is reversed. The new technique yields a result which is mathematically identical to the standard method while requiring no matrix manipulations or resolution matrix storage in the remote computer. It utilizes only a single function for each type dose required and each geometric factor involved.

Real-time reverse transcription polymerase chain reaction method for detection of Canine distemper virus modified live vaccine shedding for differentiation from infection with wild-type strains.

PubMed

Wilkes, Rebecca P; Sanchez, Elena; Riley, Matthew C; Kennedy, Melissa A

2014-01-01

Canine distemper virus (CDV) remains a common cause of infectious disease in dogs, particularly in high-density housing situations such as shelters. Vaccination of all dogs against CDV is recommended at the time of admission to animal shelters and many use a modified live virus (MLV) vaccine. From a diagnostic standpoint for dogs with suspected CDV infection, this is problematic because highly sensitive diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) tests are able to detect MLV virus in clinical samples. Real-time PCR can be used to quantitate amount of virus shedding and can differentiate vaccine strains from wild-type strains when shedding is high. However, differentiation by quantitation is not possible in vaccinated animals during acute infection, when shedding is low and could be mistaken for low level vaccine virus shedding. While there are gel-based RT-PCR assays for differentiation of vaccine strains from field strains based on sequence differences, the sensitivity of these assays is unable to match that of the real-time RT-PCR assay currently used in the authors' laboratory. Therefore, a real-time RT-PCR assay was developed that detects CDV MLV vaccine strains and distinguishes them from wild-type strains based on nucleotide sequence differences, rather than the amount of viral RNA in the sample. The test is highly sensitive, with detection of as few as 5 virus genomic copies (corresponding to 10(-1) TCID(50)). Sequencing of the DNA real-time products also allows phylogenetic differentiation of the wild-type strains. This test will aid diagnosis during outbreaks of CDV in recently vaccinated animals.

Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification.

PubMed

Wang, Jianchang; Wang, Jinfeng; Li, Ruiwen; Liu, Libing; Yuan, Wanzhe

2017-08-15

Canine distemper, caused by Canine distemper virus (CDV), is a highly contagious and fatal systemic disease in free-living and captive carnivores worldwide. Recombinase polymerase amplification (RPA), as an isothermal gene amplification technique, has been explored for the molecular detection of diverse pathogens. A real-time reverse transcription RPA (RT-RPA) assay for the detection of canine distemper virus (CDV) using primers and exo probe targeting the CDV nucleocapsid protein gene was developed. A series of other viruses were tested by the RT-RPA.Thirty-two field samples were further tested by RT-RPA, and the resuts were compared with those obtained by the real-time RT-PCR. The RT-RPA assay was performed successfully at 40 °C, and the results were obtained within 3 min-12 min. The assay could detect CDV, but did not show cross-detection of canine parvovirus-2 (CPV-2), canine coronavirus (CCoV), canine parainfluenza virus (CPIV), pseudorabies virus (PRV) or Newcastle disease virus (NDV), demonstrating high specificity. The analytical sensitivity of RT-RPA was 31.8 copies in vitro transcribed CDV RNA, which is 10 times lower than the real-time RT-PCR. The assay performance was validated by testing 32 field samples and compared to real-time RT-PCR. The results indicated an excellent correlation between RT-RPA and a reference real-time RT-PCR method. Both assays provided the same results, and R 2 value of the positive results was 0.947. The results demonstrated that the RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of CDV both in the laboratory and point-of-care facility, especially in the resource-limited settings.

Real time pressure signal system for a rotary engine

NASA Technical Reports Server (NTRS)

Rice, W. J. (Inventor)

1984-01-01

A real-time IMEP signal which is a composite of those produced in any one chamber of a three-lobed rotary engine is developed by processing the signals of four transducers positioned in a Wankel engine housing such that the rotor overlaps two of the transducers for a brief period during each cycle. During the overlap period of any two transducers, their output is compared and sampled for 10 microseconds per 0.18 degree of rotation by a sampling switch and capacitive circuit. When the switch is closed, the instantaneous difference between the value of the transducer signals is provided while with the switch open the average difference is produced. This combined signal, along with the original signal of the second transducer, is fed through a multiplexer to a pressure output terminal. Timing circuits, controlled by a crank angle encoder on the engine, determine which compared transducer signals are applied to the output terminal and when, as well as the open and closed periods of the switches.

Real-time reliable determination of binding kinetics of DNA hybridization using a multi-channel graphene biosensor

NASA Astrophysics Data System (ADS)

Xu, Shicai; Zhan, Jian; Man, Baoyuan; Jiang, Shouzhen; Yue, Weiwei; Gao, Shoubao; Guo, Chengang; Liu, Hanping; Li, Zhenhua; Wang, Jihua; Zhou, Yaoqi

2017-03-01

Reliable determination of binding kinetics and affinity of DNA hybridization and single-base mismatches plays an essential role in systems biology, personalized and precision medicine. The standard tools are optical-based sensors that are difficult to operate in low cost and to miniaturize for high-throughput measurement. Biosensors based on nanowire field-effect transistors have been developed, but reliable and cost-effective fabrication remains a challenge. Here, we demonstrate that a graphene single-crystal domain patterned into multiple channels can measure time- and concentration-dependent DNA hybridization kinetics and affinity reliably and sensitively, with a detection limit of 10 pM for DNA. It can distinguish single-base mutations quantitatively in real time. An analytical model is developed to estimate probe density, efficiency of hybridization and the maximum sensor response. The results suggest a promising future for cost-effective, high-throughput screening of drug candidates, genetic variations and disease biomarkers by using an integrated, miniaturized, all-electrical multiplexed, graphene-based DNA array.

Oligonucleotide microarray analysis of gene expression profiles followed by real-time reverse-transcriptase polymerase chain reaction assay in chronic active Epstein-Barr virus infection.

PubMed

Ito, Yoshinori; Shibata-Watanabe, Yukiko; Ushijima, Yoko; Kawada, Jun-Ichi; Nishiyama, Yukihiro; Kojima, Seiji; Kimura, Hiroshi

2008-03-01

Chronic active Epstein-Barr virus infection (CAEBV) is characterized by recurrent infectious mononucleosis-like symptoms and has high mortality and morbidity. To clarify the mechanisms of CAEBV, the gene-expression profiles of peripheral blood obtained from patients with CAEBV were investigated. Twenty genes were differentially expressed in 4 patients with CAEBV. This microarray result was verified using a real-time reverse-transcriptase polymerase chain reaction assay in a larger group of patients with CAEBV. Eventually, 3 genes were found to be significantly upregulated: guanylate binding protein 1, tumor necrosis factor-induced protein 6, and guanylate binding protein 5. These genes may be associated with the inflammatory reaction or with cell proliferation.

High-throughput real-time quantitative reverse transcription PCR.

PubMed

Bookout, Angie L; Cummins, Carolyn L; Mangelsdorf, David J; Pesola, Jean M; Kramer, Martha F

2006-02-01

Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.

Armored RNA as Virus Surrogate in a Real-Time Reverse Transcriptase PCR Assay Proficiency Panel

PubMed Central

Hietala, S. K.; Crossley, B. M.

2006-01-01

In recent years testing responsibilities for high-consequence pathogens have been expanded from national reference laboratories into networks of local and regional laboratories in order to support enhanced disease surveillance and to test for surge capacity. This movement of testing of select agents and high-consequence pathogens beyond reference laboratories introduces a critical need for standardized, noninfectious surrogates of disease agents for use as training and proficiency test samples. In this study, reverse transcription-PCR assay RNA targets were developed and packaged as armored RNA for use as a noninfectious, quantifiable synthetic substitute for four high-consequence animal pathogens: classical swine fever virus; foot-and-mouth disease virus; vesicular stomatitis virus, New Jersey serogroup; and vesicular stomatitis virus, Indiana serogroup. Armored RNA spiked into oral swab fluid specimens mimicked virus-positive clinical material through all stages of the reverse transcription-PCR testing process, including RNA recovery by four different commercial extraction procedures, reverse transcription, PCR amplification, and real-time detection at target concentrations consistent with the dynamic ranges of the existing real-time PCR assays. The armored RNA concentrations spiked into the oral swab fluid specimens were stable under storage conditions selected to approximate the extremes of time and temperature expected for shipping and handling of proficiency panel samples, including 24 h at 37°C and 2 weeks at temperatures ranging from ambient room temperature to −70°C. The analytic test performance, including the reproducibility over the dynamic range of the assays, indicates that armored RNA can provide a noninfectious, quantifiable, and stable virus surrogate for specific assay training and proficiency test purposes. PMID:16390950

Human Coronavirus NL63 Molecular Epidemiology and Evolutionary Patterns in Rural Coastal Kenya.

PubMed

Kiyuka, Patience K; Agoti, Charles N; Munywoki, Patrick K; Njeru, Regina; Bett, Anne; Otieno, James R; Otieno, Grieven P; Kamau, Everlyn; Clark, Taane G; van der Hoek, Lia; Kellam, Paul; Nokes, D James; Cotten, Matthew

2018-05-05

Human coronavirus NL63 (HCoV-NL63) is a globally endemic pathogen causing mild and severe respiratory tract infections with reinfections occurring repeatedly throughout a lifetime. Nasal samples were collected in coastal Kenya through community-based and hospital-based surveillance. HCoV-NL63 was detected with multiplex real-time reverse transcription PCR, and positive samples were targeted for nucleotide sequencing of the spike (S) protein. Additionally, paired samples from 25 individuals with evidence of repeat HCoV-NL63 infection were selected for whole-genome virus sequencing. HCoV-NL63 was detected in 1.3% (75/5573) of child pneumonia admissions. Two HCoV-NL63 genotypes circulated in Kilifi between 2008 and 2014. Full genome sequences formed a monophyletic clade closely related to contemporary HCoV-NL63 from other global locations. An unexpected pattern of repeat infections was observed with some individuals showing higher viral titers during their second infection. Similar patterns for 2 other endemic coronaviruses, HCoV-229E and HCoV-OC43, were observed. Repeat infections by HCoV-NL63 were not accompanied by detectable genotype switching. In this coastal Kenya setting, HCoV-NL63 exhibited low prevalence in hospital pediatric pneumonia admissions. Clade persistence with low genetic diversity suggest limited immune selection, and absence of detectable clade switching in reinfections indicates initial exposure was insufficient to elicit a protective immune response.

Tamm-Horsfall Protein Regulates Granulopoiesis and Systemic Neutrophil Homeostasis

PubMed Central

Micanovic, Radmila; Chitteti, Brahmananda R.; Dagher, Pierre C.; Srour, Edward F.; Khan, Shehnaz; Hato, Takashi; Lyle, Allison; Tong, Yan; Wu, Xue-Ru

2015-01-01

Tamm-Horsfall protein (THP) is a glycoprotein uniquely expressed in the kidney. We recently showed an important role for THP in mediating tubular cross-talk in the outer medulla and in suppressing neutrophil infiltration after kidney injury. However, it remains unclear whether THP has a broader role in neutrophil homeostasis. In this study, we show that THP deficiency in mice increases the number of neutrophils, not only in the kidney but also in the circulation and in the liver, through enhanced granulopoiesis in the bone marrow. Using multiplex ELISA, we identified IL-17 as a key granulopoietic cytokine specifically upregulated in the kidneys but not in the liver of THP−/− mice. Indeed, neutralization of IL-17 in THP−/− mice completely reversed the systemic neutrophilia. Furthermore, IL-23 was also elevated in THP−/− kidneys. We performed real-time PCR on laser microdissected tubular segments and FACS-sorted renal immune cells and identified the S3 proximal segments, but not renal macrophages, as a major source of increased IL-23 synthesis. In conclusion, we show that THP deficiency stimulates proximal epithelial activation of the IL-23/IL-17 axis and systemic neutrophilia. Our findings provide evidence that the kidney epithelium in the outer medulla can regulate granulopoiesis. When this novel function is added to its known role in erythropoiesis, the kidney emerges as an important regulator of the hematopoietic system. PMID:25556169

Molecular detection of viral causes of encephalitis and meningitis in New York State.

PubMed

Dupuis, Michelle; Hull, Rene; Wang, Heng; Nattanmai, Seela; Glasheen, Bernadette; Fusco, Heather; Dzigua, Lela; Markey, Katie; Tavakoli, Norma P

2011-12-01

The etiology of encephalitis and meningitis, serious diseases of the central nervous system (CNS), in most cases remains unknown. The importance of establishing a diagnosis however, becomes even more important as advances are made in effective therapy. Molecular methods of detection, in particular, PCR, are being used routinely and have established a place in the arsenal of tools for diagnosis of CNS infections. In this study a viral etiological agent was detected by PCR in 340 of the total 2,357 specimens from patients who exhibited symptoms of encephalitis or meningitis. The detection rate increased from 8.9% during the first year of the study to 14.8% during the second year of the study with improved methodology and an expanded panel of viral agents. Methods were enhanced by developing real-time PCR assays (some multiplexed), using increased automation, superior nucleic acid extraction, and reverse transcription (RT) methods, and incorporation of an internal extraction control. Additionally, adenovirus and human herpes virus 6 (HHV-6) were added to the original panel of 10 viruses that included enteroviruses, herpesviruses, and arboviruses. The most common viruses detected were enteroviruses (129; 5.5%), Epstein-Barr virus (EBV) (85; 3.6%), herpes simplex viruses (HSVs) 1 and 2 (67; 2.8%), and varicella zoster virus (VZV) (44; 1.9%). Copyright © 2011 Wiley Periodicals, Inc.

Real-Time PCR Identification of Six Malassezia Species.

PubMed

Ilahi, Amin; Hadrich, Inès; Neji, Sourour; Trabelsi, Houaida; Makni, Fattouma; Ayadi, Ali

2017-06-01

Lipophilic yeast Malassezia species is widely found on the skin surface of humans and other animals. This fungus can cause pityriasis versicolor, Malassezia folliculitis, and seborrheic dermatitis. Still now, there is a problem with species identification of Malassezia with conventional methods. We developed a real-time polymerase chain reaction (PCR) assay with multiple hybridization probes for detecting M. globosa, M. furfur, M. restricta, M. sympodialis, M. slooffiae, and M. pachydermatis. The amplification curves and specific melting peaks of the probes hybridized with real-time PCR product were used for species identifications. The assay was further evaluated on 120 samples which were performed by swabbing from 60 domestic animals (23 goats, 10 dogs, 15 cows, 3 cats, 8 rabbits, and 1 donkey) and in 70 human samples (28 patients with pityriasis versicolor, 17 breeders, and 25 control group). Fifteen M. pachydermatis were identified from animals. From human, 61 isolates were identified as M. globosa (28), M. furfur (15), M. restricta (6), M. sympodialis (8), M. slooffiae (2), and M. pachydermatis (2). Eight cases of co-detection from 6 patients and 2 breeders were revealed. Our findings show that the assay was highly effective in identifying Malassezia species. The application of multiplex real-time PCR provides a sensitive and rapid identification system for Malassezia species, which may be applied in further epidemiological surveys from clinical samples.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Fan; Wang, Yuanqing, E-mail: yqwang@nju.edu.cn; Li, Fenfang

The avalanche-photodiode-array (APD-array) laser detection and ranging (LADAR) system has been continually developed owing to its superiority of nonscanning, large field of view, high sensitivity, and high precision. However, how to achieve higher-efficient detection and better integration of the LADAR system for real-time three-dimensional (3D) imaging continues to be a problem. In this study, a novel LADAR system using four linear mode APDs (LmAPDs) is developed for high-efficient detection by adopting a modulation and multiplexing technique. Furthermore, an automatic control system for the array LADAR system is proposed and designed by applying the virtual instrumentation technique. The control system aimsmore » to achieve four functions: synchronization of laser emission and rotating platform, multi-channel synchronous data acquisition, real-time Ethernet upper monitoring, and real-time signal processing and 3D visualization. The structure and principle of the complete system are described in the paper. The experimental results demonstrate that the LADAR system is capable of achieving real-time 3D imaging on an omnidirectional rotating platform under the control of the virtual instrumentation system. The automatic imaging LADAR system utilized only 4 LmAPDs to achieve 256-pixel-per-frame detection with by employing 64-bit demodulator. Moreover, the lateral resolution is ∼15 cm and range accuracy is ∼4 cm root-mean-square error at a distance of ∼40 m.« less

Reconfigurable Carbon Nanotube Multiplexed Sensing Devices.

PubMed

Xu, Xinzhao; Clément, Pierrick; Eklöf-Österberg, Johnas; Kelley-Loughnane, Nancy; Moth-Poulsen, Kasper; Chávez, Jorge L; Palma, Matteo

2018-06-26

Here we report on the fabrication of reconfigurable and solution processable nanoscale biosensors with multisensing capability, based on single-walled carbon nanotubes (SWCNTs). Distinct DNA-wrapped (hence water-soluble) CNTs were immobilized from solution onto different prepatterned electrodes on the same chip, via a low-cost dielectrophoresis (DEP) methodology. The CNTs were functionalized with specific, and different, aptamer sequences that were employed as selective recognition elements for biomarkers indicative of stress and neuro-trauma conditions. Multiplexed detection of three different biomarkers was successfully performed, and real-time detection was achieved in serum down to physiologically relevant concentrations of 50 nM, 10 nM, and 500 pM for cortisol, dehydroepiandrosterone-sulfate (DHEAS), and neuropeptide Y (NPY), respectively. Additionally, the fabricated nanoscale devices were shown to be reconfigurable and reusable via a simple cleaning procedure. The general applicability of the strategy presented, and the facile device fabrication from aqueous solution, hold great potential for the development of the next generation of low power consumption portable diagnostic assays for the simultaneous monitoring of different health parameters.

Coherent ultra dense wavelength division multiplexing passive optical networks

NASA Astrophysics Data System (ADS)

Shahpari, Ali; Ferreira, Ricardo; Ribeiro, Vitor; Sousa, Artur; Ziaie, Somayeh; Tavares, Ana; Vujicic, Zoran; Guiomar, Fernando P.; Reis, Jacklyn D.; Pinto, Armando N.; Teixeira, António

2015-12-01

In this paper, we firstly review the progress in ultra-dense wavelength division multiplexing passive optical network (UDWDM-PON), by making use of the key attributes of this technology in the context of optical access and metro networks. Besides the inherit properties of coherent technology, we explore different modulation formats and pulse shaping. The performance is experimentally demonstrated through a 12 × 10 Gb/s bidirectional UDWDM-PON over hybrid 80 km standard single mode fiber (SSMF) and optical wireless link. High density, 6.25 GHz grid, Nyquist shaped 16-ary quadrature amplitude modulation (16QAM) and digital frequency shifting are some of the properties exploited together in the tests. Also, bidirectional transmission in fiber, relevant in the context, is analyzed in terms of nonlinear and back-reflection effects on receiver sensitivity. In addition, as a basis for the discussion on market readiness, we experimentally demonstrate real-time detection of a Nyquist-shaped quaternary phase-shift keying (QPSK) signal using simple 8-bit digital signal processing (DSP) on a field-programmable gate array (FPGA).

Distributed rendering for multiview parallax displays

NASA Astrophysics Data System (ADS)

Annen, T.; Matusik, W.; Pfister, H.; Seidel, H.-P.; Zwicker, M.

2006-02-01

3D display technology holds great promise for the future of television, virtual reality, entertainment, and visualization. Multiview parallax displays deliver stereoscopic views without glasses to arbitrary positions within the viewing zone. These systems must include a high-performance and scalable 3D rendering subsystem in order to generate multiple views at real-time frame rates. This paper describes a distributed rendering system for large-scale multiview parallax displays built with a network of PCs, commodity graphics accelerators, multiple projectors, and multiview screens. The main challenge is to render various perspective views of the scene and assign rendering tasks effectively. In this paper we investigate two different approaches: Optical multiplexing for lenticular screens and software multiplexing for parallax-barrier displays. We describe the construction of large-scale multi-projector 3D display systems using lenticular and parallax-barrier technology. We have developed different distributed rendering algorithms using the Chromium stream-processing framework and evaluate the trade-offs and performance bottlenecks. Our results show that Chromium is well suited for interactive rendering on multiview parallax displays.

Simultaneous Identification of Four "Legal High" Plant Species in a Multiplex PCR High-Resolution Melt Assay.

PubMed

Elkins, Kelly M; Perez, Anjelica C U; Quinn, Alicia A

2017-05-01

The international prevalence of "legal high" drugs necessitates the development of a method for their detection and identification. Herein, we describe the development and validation of a tetraplex multiplex real-time polymerase chain reaction (PCR) assay used to simultaneously identify morning glory, jimson weed, Hawaiian woodrose, and marijuana detected by high-resolution melt using LCGreen Plus ® . The PCR assay was evaluated based on the following: (i) specificity and selectivity-primers were tested on DNA extracted from 30 species and simulated forensic samples, (ii) sensitivity-serial dilutions of the target DNA were prepared, and (iii) reproducibility and reliability-sample replicates were tested and remelted on different days. The assay is ideal for cases in which inexpensive assays are needed to quickly detect and identify trace biological material present on drug paraphernalia that is too compromised for botanical microscopic identification and for which analysts are unfamiliar with the morphology of the emerging "legal high" species. © 2016 American Academy of Forensic Sciences.

The use of reverse iontophoresis based surface plasmon resonance for the development of a noninvasive real time transdermal biomarker sensor

NASA Astrophysics Data System (ADS)

Gupta, Niraj K.; Hwang, Yongsoon; Cameron, Brent D.

2016-03-01

Recent developments in the identification of biomarkers offer a potential means to facilitate early disease detection, gauge treatment in drug therapy clinical trials, and to assess the impact of fatigue and/or stress as related to human physical and cognitive performance. For practical implementation, however, real-time sensing and quantification of such physiological biomarkers is preferred. Some key aspects in this process are continuous sample collection and real time detection. Traditionally, blood is considered the gold standard for samples but frequent phlebotomy is painful and inconvenient. Other sources like saliva and passive sweat cannot be precisely controlled and are affected by other limitations. Some of these can be addressed by reverse iontophoresis which is a noninvasive technique capable of facilitating controlled transport of biomolecules up to 20kDa in size across the skin barrier by passing a low level current between two dermal electrodes. The samples collected at the electrode site can then be monitored at site or transported via a microfluidic channel towards a sensor. In the case reported here, the sensor is based on surface plasmon resonance (SPR), which is a label free, real time, and highly sensitive optical sensing technique. The real time SPR detection of targeted biomarkers is then achieved through the use of aptamer surface modification. In this experiment, extraction and detection of orexin A, a stress related biomarker, is used for demonstration purposes.

Dynamical Interplay between Awareness and Epidemic Spreading in Multiplex Networks

NASA Astrophysics Data System (ADS)

Granell, Clara; Gómez, Sergio; Arenas, Alex

2013-09-01

We present the analysis of the interrelation between two processes accounting for the spreading of an epidemic, and the information awareness to prevent its infection, on top of multiplex networks. This scenario is representative of an epidemic process spreading on a network of persistent real contacts, and a cyclic information awareness process diffusing in the network of virtual social contacts between the same individuals. The topology corresponds to a multiplex network where two diffusive processes are interacting affecting each other. The analysis using a microscopic Markov chain approach reveals the phase diagram of the incidence of the epidemics and allows us to capture the evolution of the epidemic threshold depending on the topological structure of the multiplex and the interrelation with the awareness process. Interestingly, the critical point for the onset of the epidemics has a critical value (metacritical point) defined by the awareness dynamics and the topology of the virtual network, from which the onset increases and the epidemics incidence decreases.