Transcription-mediated amplification

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Performance Evaluation of the VERSANT HCV RNA Qualitative Assay by Using Transcription Mediated Amplification

Performance Evaluation of the VERSANT HCV RNA Qualitative Assay by Using Transcription Mediated Amplification

A preclinical evaluation of a qualitative assay for the detection of hepatitis C virus (HCV) RNA by transcription-mediated amplification (TMA) was conducted according to the guidelines of the National Com- mittee for Clinical Laboratory Standards and the U.S. Food and Drug Administration. Our results showed that this assay, HCV TMA, detected 95% of samples with HCV RNA concentrations of 5.3 IU/ml and 29 copies/ml. HCV TMA showed an overall specificity of 99.6% and was highly reproducible, detecting 99.3% of samples with HCV RNA concentrations of 50 copies/ml across seven different lots of reagents. Experiments with clinical samples showed that HCV TMA detected all HCV genotypes with similar efficiencies, detecting > 95% of samples at 50 HCV RNA copies/ml from patients infected with HCV genotypes 1a, 2b, 3a, 4a, 5a, and 6a. In experiments with RNA transcripts, HCV TMA detected > 96.6% of transcripts derived from HCV genotypes 1a, 1b, 2a, 2c, 3a, 4a, 5a, and 6a at 50 HCV RNA copies/ml. Detection of transcripts derived from HCV genotype 2b was slightly lower (88.4%) at 50 copies/ml but was 97.0% at 75 copies/ml. In addition, HCV TMA exhibited robust performance in detecting HCV RNA in samples subjected to various conditions commonly encountered in a clinical laboratory, including long-term storage, multiple freeze-thaw cycles, different collection tubes, and the presence of endogenous substances, commonly prescribed drugs, or other microorganisms and viruses. With its high sensitivity, specificity, reproducibility, and equivalent genotype reactivity, HCV TMA may provide an attractive alternative for routine qualitative HCV RNA testing in clinical laboratories.
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Female Epidemiology of Transcription Mediated Amplification Based Trichomonas vaginalis Detection in a Metropolitan Setting with a High Prevalence of Sexually Transmitted Infection

Female Epidemiology of Transcription Mediated Amplification Based Trichomonas vaginalis Detection in a Metropolitan Setting with a High Prevalence of Sexually Transmitted Infection

Recent literature has reported increased accuracy of Trichomonas vaginalis transcription-mediated amplification (TMA)-based analyte-specific reagent (ASR) testing in female populations. A retrospective investigation assessed 7,277 female first-void urine, cervical, or vaginal specimens submitted from a high-prevalence sexually transmitted infection (STI) community to characterize prevalence of disease etiologies. The most common STI phenotype reflected detection of solely T. vaginalis (54.2% of all health care encounters that resulted in STI detection). In females with detectable T. vaginalis, codetection of Chlamydia trachomatis and Neisseria gonorrhoeae occurred in 7.8% and 2.7% of health care encounters, respectively. The mean age of women with de- tectable T. vaginalis (30.6) was significantly higher than those for women with C. trachomatis or N. gonorrhoeae (22.3 and 21.6, respectively; P < 0.0001). T. vaginalis was the predominant sexually transmitted agent in women over the age of 20 (P < 0.0002). C. trachomatis was the most commonly detected agent in females under the age of 21, particularly from cervical specimens. However, first-void urine detection rates for T. vaginalis and C. trachomatis within this age demographic demonstrated no dif- ference (P ⴝ 0.92). While overall and cervical specimen-derived detection of T. vaginalis within African American majority geo- graphical locales outweighed that within majority Caucasian geographical regions (P < 0.004), this difference was not noted with first-void urine screening (P ⴝ 0.54). Health care professionals can consider TMA-based T. vaginalis screening for a wide age range of patients; incorporation of first-void urine specimens into screening algorithms can potentiate novel insight into the epidemiology of trichomoniasis.
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Urine Specimens from Pregnant and Nonpregnant Women Inhibitory to Amplification of Chlamydia trachomatis Nucleic Acid by PCR, Ligase Chain Reaction, and Transcription Mediated  Amplification: Identification of Urinary Substances  Associated with Inhibitio

Urine Specimens from Pregnant and Nonpregnant Women Inhibitory to Amplification of Chlamydia trachomatis Nucleic Acid by PCR, Ligase Chain Reaction, and Transcription Mediated Amplification: Identification of Urinary Substances Associated with Inhibition and Removal of Inhibitory Activity

The presence of endogenous amplification inhibitors in urine may produce false-negative results for the detection of Chlamydia trachomatis nucleic acids by tests such as PCR, ligase chain reaction (LCR), and transcription-mediated amplification (TMA). Consecutive urine specimens from 101 pregnant women and 287 nonpregnant women submitted for urinalysis were processed for C. trachomatis detection. Aliquots were spiked with the equivalent of one C. trachomatis elementary body and were tested by three commercial assays: AMPLICOR CT/NG, Chlamydia LCX, and Chlamydia TMA. The prevalence of inhibitors resulting in complete inhibition of amplification was 4.9% for PCR, 2.6% for LCR, and 7.5% for TMA. In addition, all three assays were partially inhibited by additional urine specimens. Only PCR was more often inhibited by urine from pregnant women than by urine from nonpregnant women (9.9 versus 3.1%; P 5 0.011). A complete urinalysis including dipstick and a microscopic examination was performed. Logistic regression analysis revealed that the following substances were associated with amplification inhibition: beta-human chorionic gonadotropin (odds ratio [OR], 3.3) and crystals (OR, 3.3) for PCR, nitrites for LCR (OR, 14.4), and hemoglobin (OR, 3.3), nitrites (OR, 3.3), and crystals (OR, 3.3) for TMA. Aliquots of each inhibitory urine specimen were stored at 4 and 2 70°C overnight or were extracted with phenol-chloroform and then retested at dilutions of 1:1, 1:4, and 1:10. Most inhibition was removed by storage overnight at 4 or 2 70°C and a dilution of 1:10 (84% for PCR, 100% for LCR, and 92% for TMA). Five urine specimens (three for PCR and two for TMA) required phenol-chloroform extraction to remove inhibitors. The results indicate that the prevalence of nucleic acid amplification inhibitors in female urine is different for each technology, that this prevalence may be predicted by the presence of urinary factors, and that storage and dilution remove most of the inhibitors.
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Quantitative Detection of Hepatitis B Virus by Transcription Mediated Amplification and Hybridization Protection Assay

Quantitative Detection of Hepatitis B Virus by Transcription Mediated Amplification and Hybridization Protection Assay

Several groups have reported the development of assay sys- tems for the detection of HBV DNA with or without DNA amplification (2, 4, 6–11, 14–21). The assay systems without amplification had lower detection sensitivities (from about 6.0 to 9.0 LGE/ml) and high quantitative accuracies, while the assay systems with amplification had higher sensitivities but lower quantitative accuracies. The detection ranges of both of these assay systems were apparently about 3 logs, regardless of whether amplification was used. As shown from the clinical performance of the assay in Fig. 3, HBV DNA amounts in patients with various disease conditions were widely distrib- uted over more than 5 logs. Furthermore, even HBV DNA levels in a single patient varied markedly (Fig. 4). Two clinical studies have suggested that HBV DNA amounts differ mark- edly in hepatitis B patients and carriers and that the detection range of some assays was apparently too narrow to monitor the amount of HBV DNA (11, 18). The results reported here suggest that diagnosis of HBV infection and monitoring of patients with HBV infection may require a test not only with adequate sensitivity but also one with a very wide detection range. The quantitative test for the detection of HBV that we have developed has adequate sensitivity and a broad dynamic range for monitoring the condition and prognosis of HBV patients, carriers, and especially, patients undergoing inter- feron therapy.
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Design and Validation of Transcription Mediated Amplification Nucleic Acid Amplification Tests for Mycoplasma genitalium

Design and Validation of Transcription Mediated Amplification Nucleic Acid Amplification Tests for Mycoplasma genitalium

prospectively enrolled cohort of patients seeking care. It is therefore possible that the specimens tested are not representative of a true intended use population, which could affect test clinical performance; for example, differences in infection prevalence or other comorbidities could affect the positive predictive value or sensitivity of the tests. An additional limitation is that, for the analytical studies, we used different quantitative DNA NAAT methods, target sequences, and calibration standards for the detection of genome equivalents of the eight strains used in the inclusivity panel (quantitative PCR [qPCR] of the MgPa gene with a whole-cell M. genitalium DNA calibrator) and the M30 strain included in the analytical sensitivity analysis (Invader chemistry-based assay of the 23S rRNA gene with an uncut plasmid DNA calibrator), possibly leading to the differences observed in the analytic sensitivity estimates from these two analyses. However, both analyses showed that the four TMA tests detected all nine strains at a titer at less than 1 GE/ml of specimen, indicating that the risk of a clinical false-negative result due to the analytical sensitivity capability of each test is likely to be small. We also did not have clinical penile meatal swab specimens, a potentially important new specimen type for STI testing (42, 43), to compare with test LOD capability in that specimen matrix. Finally, we did not compare test clinical performance to an absolute reference standard such as PCR Sanger sequencing for the identification of M. genita- lium infection. This concern is mitigated by previously published data showing the AMG IVD assay has 100% agreement with a validated reverse transcription-PCR/Sanger sequencing assay for M. genitalium 23S rRNA (19, 21).
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Very low sensitivity of wet mount microscopy compared to PCR against culture in the diagnosis of vaginal trichomoniasis in Uganda: a cross sectional study

Very low sensitivity of wet mount microscopy compared to PCR against culture in the diagnosis of vaginal trichomoniasis in Uganda: a cross sectional study

Recent advancement has led to the development of highly sensitive and specific polymerase chain reac- tion (PCR)/nucleic acid amplification techniques/tests (NAATs) that offer faster means to detection of TV infec- tion in endocervical, vaginal, or urine specimens from women. These include The APTIMA T. vaginalis assay (Hologic Gen-Probe, San Diego, CA), which detects RNA by transcription-mediated amplification with a sensitivity of 95.3–100% and specificity of 95.2–100% [16], the BD Probe Tec TV Qx Amplified DNA Assay (Becton–Dick- inson, Franklin Lakes, New Jersey), and the Affirm VP III (Becton–Dickinson, Sparks, MD), a DNA hybridiza- tion probe test that evaluates for T. vaginalis, G. vaginalis, and Candida albicans, in only 45 min with sensitivity and specificity of 63 and 99.9%, respectively [17]. Studies on the performance of these PCR assays have mainly been con- ducted in the developed countries such as USA, Europe, and Australia, and show PCR sensitivity to be 89–98% [18, 19]. These NAATs have become routine assays in developed countries however, in the resource-limited set- tings the high cost of these commercially available NAATs remains a prohibitive challenge to their use. The objective of this study was to evaluate the technical performance of
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Rapid Extraction Method of Mycobacterium ulcerans DNA from Clinical Samples of Suspected Buruli Ulcer Patients

Rapid Extraction Method of Mycobacterium ulcerans DNA from Clinical Samples of Suspected Buruli Ulcer Patients

The Mu DNA GenoLyse extraction protocol eliminates the need for high-speed centrifuges required for typical GenoLyse ® extraction procedures [22,23] or other M. ulcerans DNA extraction procedures (Table 4) with low-speed centrifuges which are applicable in a mobile suitcase laboratory under field conditions. Further, this protocol involved only two pipetting steps and the turnaround time from “samples in”, extraction, Mu RPA master mix preparation to amplification/detection was approximately 40 min. The centrifugation step introduced after cell lysis enables the partial purification of nucleic acid by the pelleting of cell debris and some inhibitors which might affect downstream applications. The cold chain independent nature of both GenoLyse ® and RPA reagents (i.e., reagents stable at room temperature for long period) provides an added advantage for the feasibility of this protocol and Mu RPA assay under field conditions. The field as used in this manuscript refers to Buruli ulcer treatment hospitals within endemic districts where patients receive care (point of need).
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Loop Mediated Isothermal Amplification (LAMP) Assay for GMO on: Recent Progresses and Future Perspectives

Loop Mediated Isothermal Amplification (LAMP) Assay for GMO on: Recent Progresses and Future Perspectives

As an alternative to normal PCR, the amplification of target DNAs with LAMP is obviously faster than con- ventional PCR-based technologies. It is less sensitive to inhibitors [18] [19], no need of complicate equipment, simple to operate, visual result, and easy to be developed into commercially available detection kits [20] [21]. All those advantageous characteristics make LAMP an ideal choice of on-site GMO detection method. However, why is it still in laboratory experiment stage? This review will focus first on its current applications, disadvan- taged features, and then future improvements needed for LAMP to be adopted for on-site GMO detection.
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Rapid detection of porcine kobuvirus in feces by reverse transcription loop mediated isothermal amplification

Rapid detection of porcine kobuvirus in feces by reverse transcription loop mediated isothermal amplification

As PCR requires an expensive thermal cycler and oper- ator skill, which are limited to the field, a novel technique, loop-mediated isothermal amplification (LAMP) was de- veloped by Notomi et al.[20] to overcome the difficulties of PCR-based techniques. RT-LAMP assays are more sen- sitive than conventional gel-based RT-PCR assays, fast and easy to perform since they require only a simple incubator, such as a heating block or a water bath to provide a

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Visual detection of H3 subtype avian influenza viruses by reverse transcription loop mediated isothermal amplification assay

Visual detection of H3 subtype avian influenza viruses by reverse transcription loop mediated isothermal amplification assay

The H3 subtype AIVs can provide genes for human influenza virus through gene reassortment, which raises great concerns in terms of its potential threat to human health [6]. Consequently, the development of a rapid, simple, sensitive detection method for H3 subtype AIVs is required. So far, there are several PCR-based methods being used to detect AIVs, but they all need precision instruments to amplify the nucleic acid of target sample; therefore, they can ’ t be applied in field conditions [24,25]. The loop-mediated isothermal amplification (LAMP) assay is a nucleic acid amplification method
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Development of a real time reverse transcription loop mediated isothermal amplification method for the rapid detection of porcine epidemic diarrhea virus

Development of a real time reverse transcription loop mediated isothermal amplification method for the rapid detection of porcine epidemic diarrhea virus

Amplification reactions were performed at 63 °C for 60 min using either a LA-320C Loopamp real-time tur- bidimeter (Teramecs, Japan) or in a water bath. The mixtures were heated at 80 °C for 10 min to terminate the reactions. The turbidity of the reaction was mea- sured in real time, and the result was indicated by the graph on the monitor of real-time turbidimeter, veri- fying the start of the amplification. LAMP products were then evaluated with a fluorescent detection re- agent (Eiken Chemical Co., Ltd., Japan). A negative control (a sample devoid of template) was included in each reaction.
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Reverse transcription, loop mediated isothermal amplification assay for the sensitive and rapid detection of H10 subtype avian influenza viruses

Reverse transcription, loop mediated isothermal amplification assay for the sensitive and rapid detection of H10 subtype avian influenza viruses

Specificity and sensitivity of the H10-RT-LAMP assay As expected, the turbidity value of the H10 subtype AIVs gradually increased at 20 min. We observed that the in- creased turbidity curve appeared only when H10 subtype AIVs was used as the template that indicated a positive re- sult, while straight lines were observed for the other avian pathogens (H1-H9 AIV, H11-H16 AIV, human influenza viruses, NDV, IBV and ILTV) indicating a lack of amplifi- cation (Fig. 1a). A green color was observed for the H10 subtype AIVs, and the other reactions remained the same orange color as before the incubation (Fig. 1b). The results of the detection assay for each strain are shown in Table 2. All the results suggest that this newly developed RT-LAMP assay only amplifies H10 subtype AIVs and shows no cross- reactivity with other avian pathogens and human influenza viruses (H1N1, H3N2 and influenza B virus), thus val- idating the high specificity of this H10-RT-LAMP assay. The sensitivity of the test was established by dilution series vitro-transcribed RNA standard sam- ples. The LOD for the RT-LAMP was 10 copies/μL (Fig. 2a and b). Direct visualization of the Calcein dye- aided color change not only determined amplification but also did not require expensive instruments, needing only a temperature-controlled water bath, and the calcein dye re- sults were consistent with the turbidity data.
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Rapid detection of wheat yellow mosaic virus by reverse transcription loop mediated isothermal amplification

Rapid detection of wheat yellow mosaic virus by reverse transcription loop mediated isothermal amplification

mM DTT, 4U RNase Inhibitor (TaKaRa, Biotechnology, Dalian, China), 200 U moloney murine leukemia virus (M-MLV) transcriptase or 1.25 U avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI, USA), 8 U Bacillus stearothermophilus (Bst) DNA polymerase (New England Biolabs, Ipswich, MA, USA), 1.0 μ l RNA extract and 4.0 μ l deionized distilled water. Four sets of primers (I-IV) located in the CP and the 72 kDa gene, were used to detect WYMV at 65°C for 25-80 min, and to compare RT-LAMP in the presence of M- MLV or AMV reverse transcriptase. The final products of RT-LAMP were a mixture of stem-loop DNAs with various stem lengths and cauliflower-like structures with multiple loops. For WYMV-positive sample, the linear- ized DNA form showed up in the lane by agarose gel analysis. Many pyrophosphate ions were produced dur- ing RT-LAMP, and the production of a white precipitate of magnesium pyrophosphate gave the tube a turbid appearance that could be observed directly. The positive tube was cloudy and the negative one was clear. With four sets of primers, the RT-LAMP reaction mixture was observed by the naked eye, and the amplification reactions were confirmed by complementary procedures, such as gel electrophoresis. Five microliters of amplified DNA fragments were electrophoresed in 1.5% agarose in TBE buffer.
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Development of a New Method for Diagnosis of Rubella Virus Infection by Reverse Transcription Loop Mediated Isothermal Amplification

Development of a New Method for Diagnosis of Rubella Virus Infection by Reverse Transcription Loop Mediated Isothermal Amplification

RT-LAMP for rubella would be expected to be a reliable and rapid diagnostic method in the clinical setting, because RT-LAMP showed an equivalent or higher detection rate com- pared with RT-PCR and virus isolation for nine samples ob- tained from clinically diagnosed patients. The RT-LAMP pro- cedure has clinical advantages of simplicity and rapidity, in comparison with virus isolation and RT-PCR. Virus isolation requires complex procedures for cell culture, is not always successful, and is not appropriate for a clinical laboratory di- agnostic tool. The genome amplification method always has the possibility of false positives due to cross-contamination. Since RT-LAMP is performed as a simple procedure in a single tube with sensitivity similar to that of nested PCR, cross- reaction would less likely occur in RT-LAMP than in RT-PCR. The detection limit of RT-PCR primers used in our study (30 PFU/ml) was similar to that in previous studies, and the RT-PCR primers were designed to have the same target as the RT-LAMP primers in order to compare the sensitivities of RT-LAMP and RT-PCR. In previous reports, RT-PCR for rubella targeting the E1 region detected up to 8 infectious units of WHO international standard/ml in amniotic fluid (8, 15) and a 10 ⫺6 dilution of culture fluid containing 10 6.8
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Development and Evaluation of a Reverse Transcription Loop Mediated Isothermal Amplification Assay for Rapid Detection of Enterovirus 71

Development and Evaluation of a Reverse Transcription Loop Mediated Isothermal Amplification Assay for Rapid Detection of Enterovirus 71

The loop-mediated isothermal amplification (LAMP) method is a cheap, rapid, and simple method that was first described in 2000 (24). This novel detection method works on the principle of a strand displacement reaction with the specific stem-loop structures, and the whole amplification reaction takes place continuously under isothermal conditions. Previ- ously, LAMP methods for detection of different viruses have been developed, including West Nile virus (27), dengue virus serotypes 1 to 4 (26), Japanese encephalitis virus (20, 28), avian H5 and H7 influenza viruses (29, 32), the 2009 pandemic H1N1 influenza virus (15, 21), Marburg virus (16), Ebola virus (17), foot-and-mouth disease virus (25), and herpes simplex virus 1 (30). All these assays showed high specificity, efficiency, and * Corresponding author. Mailing address: State Key Laboratory of
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Rapid and Real Time Detection of Chikungunya Virus by Reverse Transcription Loop Mediated Isothermal Amplification Assay

Rapid and Real Time Detection of Chikungunya Virus by Reverse Transcription Loop Mediated Isothermal Amplification Assay

A one-step, single-tube, real-time, accelerated, quantitative RT-LAMP assay was standardized for the rapid detection of CHIKV by targeting the highly conserved regions of the E1 gene based on multiple sequence alignments of all the circu- lating strains. The details of the each primer with regard to their positions in the genomic sequences are shown in Table 1. The detection of gene amplification is accomplished by real- time monitoring of turbidity at 63°C. The result indicated that the minimum time required for the initiation of amplification was 10 min with viral RNA preparations. It was also observed that there is continuous amplification of the target sequence as revealed through increased turbidity compared to the negative control having no template, wherein the turbidity got fixed around 0.01, well below the threshold value. None of the pos- itive samples tested over multiple times showed positivity in terms of increased turbidity after 45 min. Therefore, a sample with a Tp value of ⱕ 45 min and turbidity above the threshold value of ⱖ 0.1 was considered positive.
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Real Time Reverse Transcription Loop Mediated Isothermal Amplification for Rapid Detection of West Nile Virus

Real Time Reverse Transcription Loop Mediated Isothermal Amplification for Rapid Detection of West Nile Virus

phoresis with a 3% NuSieve 3:1 agarose gel (BMA) in Tris acetate-EDTA buffer (0.04 M Tris acetate, 1 mM EDTA), stained with ethidium bromide, and visu- alized on a UV transilluminator at 302 nm. Real-time monitoring of RT-LAMP amplification was done through spectrophotometric analysis by recording the optical density at 400 nm with a Loopamp real-time turbidimeter (LA-200; Teramecs) every 6 s. Positive real-time RT-LAMP assay results were determined by taking into account the time to positivity (Tp; in minutes), at which the turbidity increased above the threshold value fixed at 0.1, which is two times the average turbidity value of the negative controls of several replicates. None of the positive samples tested at multiple times showed positivity in terms of increased turbidity after 60 min. Therefore, a sample having Tp values of ⱕ60 min and turbidity above the threshold value of ⱖ0.1 was considered positive. The speci- ficity of the RT-LAMP-amplified product was analyzed by restriction digestion with the AluI enzyme, as well as by nucleotide sequencing of both digested and undigested products with two outer and two inner primers.
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Temporal Hierarchy of Gene Expression Mediated by Transcription Factor Binding Affinity and Activation Dynamics

Temporal Hierarchy of Gene Expression Mediated by Transcription Factor Binding Affinity and Activation Dynamics

FIG 3 Effects of binding affinities on gene expression of PhoB-regulated promoters. (A) Details of PhoB-regulated promoters. The promoters tested were from five operons: phoBR, phoE, ugpBAECQ, phoA, and phnCDEE=FGHIJKLMNOP. Boxes illustrate the positions of PhoB-binding sites, and positions are propor- tional to their relative distances from the transcription start (see Text S1 in the supplemental material for detailed sequences and the positioning of Pho boxes). The numbers at the top indicate information content (IC) calculated from the 22-bp PWM. All five promoters are fused to identical yfp genes for expression analyses. Alk., alkaline. (B) Dissociation of PhoB-DNA complexes measured by Bio-layer interferometry (BLI). Data are fitted using single exponential decay (solid lines), and the apparent off rates are listed in panel A. (C to E) Expression level and timing of the indicated promoters. Reporter activities (C and D) are measured in RU1616, RU1618, and RU1783 with the following reporter plasmids: pJZG202 (phoB), pRG347 (phoE), pRG346 (ugpB), pRG161 (phoA), and pRG162 (phnC). Error bars represent standard deviations (SDs) of the results from at least four independent experiments. The vertical black dotted line in panel D indicates the WT expression level of PhoB under P i -depleted conditions. OD normalized reporter activities (C) and activation onset times (E) were measured
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Three Isothermal Amplification Techniques for Rapid Identification of Cladophialophora carrionii, an Agent of Human Chromoblastomycosis

Three Isothermal Amplification Techniques for Rapid Identification of Cladophialophora carrionii, an Agent of Human Chromoblastomycosis

Innovations in molecular diagnostics are rapidly reaching rou- tine laboratories. Most molecular methods are PCR based, but during the past 2 decades, several isothermal amplification tech- niques, which amplify DNA by strand replacement without using a thermocycler, have become available. These non-PCR-based techniques have the advantages of simple application in the clin- ical lab and suitability for processing large numbers of specimens. In earlier studies on pathogenic fungi, Sun et al. (5, 6) developed loop-mediated isothermal amplification (LAMP) assays for the rapid diagnosis of Talaromyces marneffei and for Fonsecaea spe- cies. LAMP proved to be a fast and sensitive method for the rou- tine diagnosis of T. marneffei in the clinical lab, whereas the spec- ificity of the method was insufficient to distinguish between the different Fonsecaea species causing chromoblastomycosis. Najafzadeh et al. (7) introduced rolling-circle amplification (RCA) for use with Fonsecaea species, and this assay successfully distinguished the three species involved. Thus far, the multiplex
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Rapid detection of newly isolated Tembusu related Flavivirus by reverse transcription loop mediated isothermal amplification assay

Rapid detection of newly isolated Tembusu related Flavivirus by reverse transcription loop mediated isothermal amplification assay

Background: From April 2010 to January 2011, a severe new viral disease had devastated most duck-farming regions in China. This disease affected not only laying ducks but also meat ducks, causing huge economic losses for the poultry industry. The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of the new virus related to Tembusu-related Flavivirus. Results: The RT-LAMP assay is very simple and rapid, and the amplification can be completed within 50 min under isothermal conditions at 63°C by a set of 6 primers targeting the E gene based on the sequences analysis of the newly isolated viruses and other closely related Flavivirus.The monitoring of gene amplification can also be visualized by using SYBR green I fluorescent dye. In addition, the RT-LAMP assay for newly isolated Tembusu- related Flavivirus showed higher sensitivity with an RNA detection-limit of 2 copies/μL compared with 190 copies/ μL of the conventional RT-PCR method. The specificity was identified without cross reaction to other common avian pathogens. By screening a panel of clinical samples this method was more feasible in clinical settings and there was higher positive coincidence rate than conventional RT-PCR and virus isolation.
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