Direct assay for O6-methylguanine-acceptor protein in cell extracts

A direct in vitro assay for O⁶-methylguanine-acceptor protein in cell extracts that measures the transfer of radioactivity from labeled O⁶-methylguanine (O⁶MeGua) adducts in an exogenous DNA substrate to protein is described. The protein-bound radioactivity is released and separated from that remaining in the DNA by sequential digestion with protease K and aminopeptidase M, and appears in the alcohol-soluble fraction of the digest. Data obtained by the direct assay are similar to those obtained by an indirect assay that measures the amount of O⁶MeGua-acceptor protein as the loss of O⁶MeGua from the DNA. In addition to its accuracy, the direct assay is also simple and can measure the amount of O⁶MeGua-acceptor activity in cell extracts prepared from as few as 0.5–1.0 × 10⁶ mammalian cells.     

(Na+ + K+)-activated adenosinetriphosphatase: fluorimetric determination of the associated K+ -dependent 3-O-methylfluorescein phosphatase and its use for the assay of enzyme samples with low activities.

Data are presented which prove that 3-O-methylfluorescein phosphate is a substrate for the K⁺-dependent phosphatase that is associated with Na⁺,K⁺-ATPase. Conditions for the continuous fluorimetric assay of 3-O-methylfluorescein phosphatase are described. Enzyme preparations from three different tissues with widely different specific activities exhibit similar K_m values for 3-O-methylfluorescein phosphate. Correlation between Na⁺,K⁺-ATPase activity and K⁺-dependent 3-O-methylfluorescein phosphatase activity is demonstrated in several partially purified enzyme preparations and crude tissue fractions. When the K⁺-dependent 3-O-methylfluorescein phosphatase of a crude rat-brain homogenate is assayed, the activity is a linear function of the amount of homogenate added to the assay mixture. The equivalent of 10 μg of brain tissue may be assayed under the conditions used. The potential value of this highly sensitive fluorimetric method for the assay of enzyme in small samples of various tissues is suggested.     

Enzyme-Linked Immunosorbent Assays for the Specific and Sensitive Quantification of Methanosarcina mazei and Methanobacterium bryantii

Three microtitration plate enzyme-linked immunosorbent assays (ELISAs) have been developed: a competitive ELISA and a two-site (or indirect sandwich) ELISA for Methanosarcina mazei S6 and a two-site ELISA for Methanobacterium bryantii FR-2. The assays were sensitive, with limits of cell protein detection of 3 ng ml⁻¹, 5 ng ml⁻¹, and 50 ng ml⁻¹, respectively, and showed good precision. The M. mazei assays used monoclonal antibodies and were entirely species specific, showing no cross-reaction with methanogens of other genera or with other species of the same genus. The Methanobacterium bryantii assay, which used two polyclonal antisera, showed only a slight cross-reaction with one other Methanobacterium species but no cross-reaction with methanogens of other genera. The use of the ELISAs for quantitative analysis of mixed cultures and of sewage sludge samples was investigated. Sludge diluted at 1:10³ or more caused no significant interference in any of the three ELISAs. Various cultures of bacteria, methanogens, and nonmethanogens at a protein concentration of 50 μg ml⁻¹ showed no significant interference in the M. mazei competitive assay and the Methanobacterium bryantii two-site assay, although they did cause falsely low results in the M. mazei two-site assay.     

A simple real-time assay for in vitro translation

A high-throughput assay for real-time measurement of translation rates in cell-free protein synthesis (SNAP assay) is described. The SNAP assay enables quantitative, real-time measurement of overall translation rates in vitro via the synthesis of O⁶-alkylguanine DNA O⁶-alkyltransferase (SNAP). SNAP production is continuously detected by fluorescence produced by the reaction of SNAP with a range of quenched fluorogenic substrates. The capabilities of the assay are exemplified by measurements of the activities of Escherichia coli MRE600 ribosomes and fluorescently labeled E. coli mutant ribosomes in the PURExpress translation system and by determination of the 50% inhibitory concentrations (IC₅₀) of three common macrolide antibiotics.     

Quantitative and specific detection of the biocontrol agent, Serratia plymuthica, in plant extracts using a real-time TaqMan? assay

A Serratia plymuthica-specific TaqMan? assay was designed based on the consensus nucleotide sequence from the 3??- end of the luxS gene present in all S. plymuthica strains tested. The specificity of the assay was demonstrated by testing 21 Serratia spp. strains and 30 isolates belonging to various species that can potentially coexist with S. plymuthica in the same environment. Positive reactions in the TaqMan? assay were observed only for S. plymuthica isolates and not for other bacteria. The TaqMan? assay could detect down to 1.95 ng of S. plymuthica DNA, down to 5 bacterial cells per reaction (100?cfu ml^?1) in vitro, down to 50 bacterial cells per reaction (1,000?cfu ml^?1) in spiked potato root extracts and down to 5 bacterial cells per reaction (100?cfu ml^?1) in spiked potato haulm extracts. We used this assay to quantify S. plymuthica A30 cells in potato and tomato haulms and roots grown from S. plymuthica A30-inoculated potato seed tubers and tomato seeds. The results were comparable with the spread-plating of plant extracts on a newly developed S. plymuthica A30 selective medium (CVTR2Arif). The TaqMan? assay can be used to quantify S. plymuthica isolates in different ecosystems and in complex substrates.     

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity¹ and vibration². In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus³. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.EIW assays involve counting the number of eggs retained in the uterus of C. elegans⁴. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine^5-9.     

Rapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica

We have developed a rapid procedure for the detection of virulent Yersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5′ nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species of Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri, Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be ≥10² CFU/ml in pure cultures and ≥10³ CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked with Y. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocolitica with both 5′ nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.     

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock¹. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours^{2, 4}. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes⁵. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods⁶. This assay was based on a study in which PCR was used to measure the growth of bacteria⁷. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.     

Fractionation of simian virus 40 DNA fragments by RPC-5 column chromatography

This report describes several modifications of the original radioenzymatic assay for serotonin (4) which increase the sensitivity of the assay 10-fold as well as enhance its reliability. Serotonin is converted to [³H]melatonin, in two steps. First, serotonin is acetylated to N-acetylserotonin by acetic anhydride. The N-acetylserotonin is then incubated with hydroxyindole-O-methyltransferase and S-[methyl-³H]adenosyl methionine and is converted to [³H]melatonin. The radioactive melatonin is extracted with toluene-isoamyl alcohol (7:3), dried, reconstituted, isolated by one-dimensional, silica gel, thin-layer chromatography, and counted in a liquid scintillation counter. The assay is specific and sensitive to approximately 5 pg of serotonin and thus can be used to measure serotonin levels in single brain nuclei or microliter quantities of biological fluids. The assay can be easily adapted for the direct measurement of N-acetylserotonin. A large number of samples can be assayed in a single working day.     

A Real-Time PCR-Based Semi-Quantitative Breakpoint to Aid in Molecular Identification of Urinary Tract Infections

This study presents a novel approach to aid in diagnosis of urinary tract infections (UTIs). A real-time PCR assay was used to screen for culture-positive urinary specimens and to identify the causative uropathogen. Semi-quantitative breakpoints were used to screen for significant bacteriuria (presence of ≥10⁵ CFU/ml of uropathogens) or low-level bacteriuria (containing between 10³ and 10⁴ CFU/ml of uropathogens). The 16S rDNA-based assay could identify the most prevalent uropathogens using probes for Escherichia coli, Pseudomonas species, Pseudomonas aeruginosa, Staphylococcus species, Staphylococcus aureus, Enterococcus species and Streptococcus species. 330 urinary specimens were analysed and results were compared with conventional urine culture. Using a PCR Ct value of 25 as semi-quantitative breakpoint for significant bacteriuria resulted in a sensitivity and specificity of 97% and 80%, respectively. In 78% of the samples with monomicrobial infections the assay contained probes to detect the bacteria present in the urine specimens and 99% of these uropathogens was correctly identified. Concluding, this proof-of-concept approach demonstrates that the assay can distinguish bacteriuria from no bacteriuria as well as detect the involved uropathogen within 4 hours after sampling, allowing adequate therapy decisions within the same day as well as drastically reduce consequent urine culturing.     

Quantitative real-time PCR detection of Pseudomonas oleovorans subsp. lubricantis using TaqMan-MGB assay in contaminated metalworking fluids

Metalworking fluids (MWFs) are highly prone to microbial contamination, which leads to their degradation and biofouling. Pseudomonas oleovorans subsp. lubricantis, a newly described subspecies, was found to be important to MWF fouling. However, the actual distribution of P. oleovorans subsp. lubricantis in MWF is difficult to study using standard culturing techniques. To overcome this, a study was conducted to design a specific quantitative real-time PCR (qPCR) assay using TaqMan^®MGB (minor groove binding) probe for its identification and estimated quantification in contaminated MWFs. The gyrB housekeeping gene sequence was selected for designing a TaqMan^® MGB primer-probe pair using the Allele ID^® 5.0 probe design software for the assay. Whole-cell qPCR was performed with MWF spiked directly with P. oleovorans subsp. lubricantis (eliminating DNA extractions using commercial kit); the primer-probe pair’s sensitivity was 10¹ colony forming units (CFU) ml⁻¹. The assay provided no amplification with other closely related Pseudomonas species found in MWFs indicating its specificity. It was successful in identifying and enumerating P. oleovorans subsp. lubricantis from several used MWFs having between 10⁴ and 10⁶ CFU ml⁻¹. The designed TaqMan^® MGB probe thus can be successfully used for the subspecies-specific identification of P. oleovorans subsp. lubricantis and facilitates the study of its impact on MWFs.     

Development and validation of a new real-time assay for the quantification of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in the soil: a comparison with conventional soil plating

Verticillium wilt of olive, caused by the soil-borne fungus Verticillium dahliae, is one of the most serious diseases of olive tree. In this study, a SYBR Green-based quantitative polymerase chain reaction (Q-PCR) assay targeting the intergenic spacer (IGS) region of the ribosomal DNA (rDNA) was developed to quantify V. dahliae microsclerotia (MS) in soils cropped with olive tree. In order to make the assay quantitative, the number of rDNA units in the genome was estimated using Q-PCR and fixed at 25 copies/genome. The assay was highly specific for V. dahliae, with no cross-amplification with other soil-borne pathogens. The sensitivity analysis showed similar slopes and efficiency, from both fungal DNA (slope?=??3.405, r²?=?0.976, E?=?96.64 %) and the positive recombinant plasmid (y?=??3.36, r²?=?0.989, E = 98.43 %), thus indicating a high accuracy of the assay. The assay exhibits a high intra- and inter-run reproducibility at a very low concentration of 10² copies/μL (CV%?≈?1 %). When the real-time PCR assay was applied to quantify MS in five naturally infested soil samples, it was able to detect V. dahliae in as few as two MS g^?1 of soil. Q-PCR estimates of pathogen DNA were significantly correlated with disease severity (r²?=?0.944) and with the soil plating method (r²?=?0.845). This new assay will be a valuable tool and can be applied for disease risk prediction before installing new plantations, and provides a more complete and rapid examination for soils subjected to such a treatment program.     

Measurement of spermidine/spermine-N1-acetyltransferase activity by high-performance liquid chromatography with N1-dansylnorspermine as the substrate

We developed a nonradioactive assay to measure spermidine/spermine N¹-acetyltransferase (SSAT) activity by high-performance liquid chromatography (HPLC). N¹-dansylnorspermine was prepared and evaluated as a substrate of acetylation with acetyl-CoA by SSAT in rat hepatoma (HTC) cells. Kinetic studies revealed that the K_m values of N¹-dansylnorspermine and acetyl-CoA were approximately 11 and 13 μM, respectively. When the assay method was applied to HTC cell samples, the SSAT activity, even at the control level, could easily be detected in as few as 20 μg protein of cell extract corresponding to 1 × 10⁵ cells per determination, and 100 samples could be analyzed overnight. Thus, our HPLC method is a rapid and sensitive assay for the measurement of SSAT activity.     

Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily^1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya⁶. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila^7-14. An acetate kinase which can only utilize PP_i but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus^15,16.In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann^17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD⁺ by the enzymes pyruvate kinase and lactate dehydrogenase^21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine²³. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP⁺ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase²⁴.Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PP_i.     

A smiple specific assay for uracil tRNA methylases of enteric bacteria: use of ribothymidine-deficient tRNA

A simple quantitative assay that is about 95% specific for uracil tRNA methylases of E. coli and A. aerogenes has been developed. tRNA was isolated from a strain of E. coli carrying the trm^? mutation. These organisms have a low level of uracil methylase and consequently produce tRNA with a selective deficiency of ribothymidine. This RNA acted as a specific substrate for uracil tRNA methylases, when exposed to cell extracts from E. coli or A. aerogenes containing tRNA-methylating enzymes of multiple specificities. This assay can be used to screen organisms for trm^? mutations and for studies with inhibitors.     

Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling

To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10^-18 moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10^-18 moles of the p24/assay corresponds to ca. 10³ copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (10² copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.     

A novel assay for illegitimate recombination in Escherichia coli: Stimulation of λbio transducing phage formation by ultra-violet light and its independence from RecA function

We developed a novel assay system for illegitimate recombination, in which the frequency of the formation of λ Spi⁻ phages formed during prophage induction was measured with an E. coli P2 lysogen as the indicator bacteria. Since almost all of the λ Spi⁻ phages thus detected contain attR, they have essentially the same structures as λbio transducing phages, indicating that this assay system enables us to detect specialized transducing phages that produce heterogenote transductants, thus ignoring the occurrences of docL and docR particles which carry only one cohesive end. The following results on the formation of specialized transducing phages have been obtained by this assay system to date. (1) Irradiation with UV light greatly enhanced the formation of λ Spi⁻ phages. (2) Treatments with other DNA-damaging agents also enhanced the formation of λ Spi⁻ phages. (3) Illegitimate recombination during prophage induction does not require the RecA function, indicating that enhancement of λ Spi⁻ phage formation is not controlled by the SOS regulatory system. (4) Preliminary results suggested that DNA gyrase is involved in the formation of λ Spi⁻ phage during prophage induction. Since the above results were consistent with most of the previous observations on the illegitimate recombination in other systems, the Spi⁻ assay system can provide important clues to the mechanism of illegitimate recombination.     

Rapid Real-Time PCR Assay for Detection and Quantitation of Mycobacterium avium subsp. paratuberculosis DNA in Artificially Contaminated Milk

Using fluorescence resonance energy transfer technology and Lightcycler analysis, we developed a real-time PCR assay with primers and probes designed by using IS900 which allowed rapid detection of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Initially, the PCR parameters (including primer and probe levels, assay volume, Mg²⁺ concentration, and annealing temperature) were optimized. Subsequently, the quantitative ability of the assay was tested and was found to be accurate over a broad linear range (3 × 10⁶ to 3 × 10¹ copies). The assay sensitivity when purified DNA was used was determined to be as low as five copies, with excellent reproducibility. A range of DNA isolation strategies was developed for isolating M. avium subsp. paratuberculosis DNA from spiked milk, the most effective of which involved the use of 50 mM Tris HCl, 10 mM EDTA, 2% Triton X-100, 4 M guanidinium isothiocyante, and 0.3 M sodium acetate combined with boiling, physical grinding, and nucleic acid spin columns. When this technique was used in conjunction with the real-time PCR assay, it was possible to consistently detect <100 organisms per ml of milk (equivalent to 2,000 organisms per 25 ml). Furthermore, the entire procedure (extraction and PCR) was performed in less than 3 h and was successfully adapted to quantify M. avium subsp. paratuberculosis in spiked milk from heavily and mildly contaminated samples.     

Quantitative Detection of Listeria monocytogenes in Biofilms by Real-Time PCR

A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 × 10² CFU/cm².