An important consideration in the design of oligonucleotide probes for homogeneous hybridization assays is efficiency energy transfer between fluorophore and quencher used to label probes. We have determined a large number combinations commonly fluorophores quenchers. also measured quenching effect nucleotides on fluorescence each fluorophore. Quenching efficiencies were both resonance static modes quenching. found that, addition their photochemical characteristics, tendency bind other has...

We describe a multiplex nucleic acid assay that identifies and determines the abundance of four different pathogenic retroviruses (HIV-1, HIV-2, human T-lymphotrophic virus types I II). Retroviral DNA sequences are amplified in single, sealed tube by simultaneous PCR assays, resulting amplicons detected real time hybridization differently colored, amplicon-specific molecular beacons. The color fluorescence generated course amplification which present, number thermal cycles required for...

In this work, we characterize genes in Mycobacterium tuberculosis that are regulated by IdeR (iron-dependent regulator), an iron-responsive DNA-binding protein of the DtxR family has been shown to regulate iron acquisition smegmatis. To identify some constitute regulon, searched M. genome for promoter regions containing consensus IdeR/DxR binding sequence. Genes preceded boxes included a set encoding proteins necessary acquisition, such as biosynthesis siderophores (mbtA, mbtB, mbtI),...

A collection of Aspergillus fumigatus mutants highly resistant to itraconazole (RIT) at 100 micro g ml(-1) were selected in vitro (following UV irradiation as a preliminary step) investigate mechanisms drug resistance this clinically important pathogen. Eight the RIT found have mutation Gly54 (G54E, -K, or -R) azole target gene CYP51A. Primers designed for conserved regions multidrug (MDR) pumps used reverse transcriptase PCR amplification reactions identify novel genes encoding potential...

The mechanism of transport mRNA-protein (mRNP) complexes from transcription sites to nuclear pores has been the subject many studies. Using molecular beacons track single mRNA molecules in living cells, we have characterized diffusion mRNP nucleus. move freely by Brownian at a rate that assures their dispersion throughout nucleus before they exit into cytoplasm, even when site is located near periphery. restricted extranucleolar, interchromatin spaces. When wander dense chromatin, tend...

Variants of concern (VOC) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including alpha, beta, gamma, delta, and omicron, threaten to prolong the pandemic, leading more global morbidity mortality. Genome sequencing is mainstay tracking evolution virus, but costly, slow, not easily accessible. Multiplex quantitative RT-PCR assays for SARS-CoV-2 have been developed that identify all VOCs as well other mutations interest in viral genome, nine total, using single-nucleotide...

OBJECTIVE: The N-methyl-d-aspartic acid (NMDA) class of glutamate receptors has received attention in the pathophysiology schizophrenia because similarity between some schizophrenic symptoms and caused by NMDA antagonists. To determine if receptor abnormalities were present at mRNA level, expression (NR) subunits NR1, NR2A, NR2B was measured specimens from dorsolateral prefrontal cortex occipital elderly patients with normal subjects. METHOD: Postmortem antemortem assessed diagnosed (N=26)...

ABSTRACT Current clinical assays for determining antibiotic susceptibility in Mycobacterium tuberculosis require many weeks to complete due the slow growth of bacilli. Here we demonstrate an extremely sensitive single-tube PCR assay that takes less than 3 h and reliably identifies rifampin-resistant M. DNA extracted directly from sputum. Ninety-five percent mutations associated with rifampin resistance occur 81-bp core region bacterial RNA polymerase gene, rpoB . All within this result...

Mycobacterium tuberculosis possesses a diversity of potential virulence factors including complex branched lipids such as the phenolic glycolipid PGL-tb. PGL-tb expression by clinical M. isolate HN878 has been associated with less efficient Th1 response and increased in mice rabbits. It suggested that W-Beijing family is only group strains an intact pks1-15 gene, required for synthesis capable producing We have found some do not produce while others may variant examined early host cytokine...

Amplification of signals by the hybridization chain reaction (HCR) is a powerful approach for increasing signal strength in single-molecule fluorescence situ hybridization, but probes tagged with an HCR initiator sequence are prone to producing false signals. Here we describe system interacting hairpin binary which conditionally sequestered. The binding these perfectly complementary target unmasks initiator, enabling generation amplified signal. This probe can distinguish single-nucleotide...

ABSTRACT An automated PCR with fluorescent probes (molecular beacons) detected Mycobacterium avium subsp. paratuberculosis in bovine feces. When the was compared culture testing 41 fecal samples, kappa scores of 0.94 to 0.96, a sensitivity 93 96%, and specificity 92% were obtained. Results quantitated by using standard curve derived from plasmid containing IS 900 . A minimum quantity 1.7 × 10 −4 pg DNA, correlating 1 8 CFU, detected.

ABSTRACT Candida dubliniensis is an opportunistic fungal pathogen that has been linked to oral candidiasis in AIDS patients, although it recently isolated from other body sites. DNA sequence analysis of the internal transcribed spacer 2 (ITS2) region rRNA genes reference strains was used develop molecular beacon probes for rapid, high-fidelity identification C. as well albicans . Molecular beacons are small nucleic acid hairpin brightly fluoresce when they bound their targets and have a...

Novel amino-reactive derivatives of lanthanide-based luminescent labels enhanced brightness and metal retention were synthesized used for the detection cDNA oligonucleotides by molecular beacons. Time-resolved acquisition signal that occurs upon hybridization probe to target enabled avoidance short-lived background fluorescence, markedly enhancing sensitivity detection, which was less than 1 pM. This value is about 50 100 times more sensitive level achieved with conventional...

New approaches for imaging dynamic processes involving RNAs in living cells are continuously being developed and optimized. The use of molecular beacons synthesized from 2'-O-methylribonucleotides (which resistant to cellular nucleases) is an established approach visualizing native mRNAs real time. In order spatially temporally resolve steps RNA cells, need efficiently hybridize their targets. To expand the repertoire target sites accessible beacons, we decreased length probe sequences...

The infection with Borrelia burgdorferi can result in acute to chronic Lyme disease. In addition, coinfection tick-borne pathogens, Babesia species and Anaplasma phagocytophilum has been increasing endemic regions of the USA Europe. currently used serological diagnostic tests are often difficult interpret and, moreover, antibodies against pathogens persist for a long time making it confirm cure these cannot be diagnosis early disease state before adaptive immune response is established....

During the last few years, several innovative technologies have become available for performing sensitive and accurate genetic analyses. These techniques use fluorescent detection strategies in combination with nucleic acid amplification protocols. Most commonly used is real-time polymerase chain reaction (PCR). To achieve maximum potential of a realtime PCR assay, parameters must be evaluated optimized independently. This chapter describes different steps necessary establishing molecular...

Kaposi's sarcoma-associated herpesvirus (KSHV) lytic switch protein, Rta, is a ligand-independent inducer of the Notch signal transduction pathway, and KSHV cannot reactivate from latency in cells null for target protein RBP-Jk. Here we show that Rta promotes DNA binding RBP-Jk, mechanism fundamentally different established RBP-Jk-activating proteins, intracellular domain (NICD) Epstein-Barr virus EBNA2. Although constitutively active RBP-Jk NICD do not transactivate promoters independently,...

A real-time PCR assay with the ability to rapidly identify all pathogenic bacteria would have widespread medical utility. Current technologies cannot accomplish this task due severe limitations in multiplexing ability. To end, we developed a new system which supports very high degrees of multiplexing. We class mismatch-tolerant "sloppy" molecular beacons, modified them provide an extended hybridization range, and multiprobe, multimelting temperature (T(m)) signature approach bacterial...

We report here the use of novel "sloppy" molecular beacon probes in homogeneous PCR screening assays which thermal denaturation resulting probe-amplicon hybrids provides a characteristic set amplicon melting temperature (T(m)) values that identify species is present sample. Sloppy beacons possess relatively long probe sequences, enabling them to form with amplicons from many different despite presence mismatched base pairs. By using four sloppy beacons, each possessing sequence and labeled...