Evaluation of the Virus Counter® for rapid baculovirus quantitation.

The utility of a new instrument for rapid virus quantitation, the Virus Counter, was evaluated in a blind study conducted at three sites. This instrument is a substantially improved version of the original academic research instrument described previously by Stoffel and Rowlen (2005a). The addition of hydrodynamic focusing, a self-contained fluidics system and customized software for system control and data analysis has resulted in a commercially viable and available design. Baculovirus samples were provided by Protein Sciences Corporation and blinded to InDevR and Baylor College of Medicine. Protein Sciences Corporation and Baylor College of Medicine analyzed the samples by plaque assay and InDevR analyzed the samples using the Virus Counter. Serial dilution of stock viruses into growth media and buffer allowed for comparison of measured versus intended concentrations. Direct log-scale comparison between pooled Virus Counter results and pooled plaque assay results indicated a linear relationship (slope=1.1±0.2, R(2)=0.86) with statistically significant Pearson correlation (r=0.93, p<0.001).

Fingerprinting of single viral genomes.

We demonstrate the use of technology developed for optical mapping to acquire DNA fingerprints from single genomes for the purpose of discrimination and identification of bacteria and viruses. Single genome fingerprinting (SGF) provides not only the size but also the order of the restriction fragments, which adds another dimension to the information that can be used for discrimination. Analysis of single organisms may eliminate the need to culture cells and thereby significantly reduce analysis time. In addition, samples containing mixtures of several organisms can be analyzed. For analysis, cells are embedded in an agarose matrix, lysed, and processed to yield intact DNA. The DNA is then deposited on a derivatized glass substrate. The elongated genome is digested with a restriction enzyme and stained with the intercalating dye YOYO-1. DNA is then quantitatively imaged with a fluorescence microscope and the fragments are sized to an accuracy >or=90% by their fluorescence intensity and contour length. Single genome fingerprints were obtained from pure samples of adenovirus, from bacteriophages lambda and T4 GT7, and from a mixture of the three viral genomes. SGF will enable the fingerprinting of uncultured and unamplified samples and allow rapid identification of microorganisms with applications in forensics, medicine, public health, and environmental microbiology.

Statistics of single-molecule measurements: applications in flow-cytometry sizing of DNA fragments.

BACKGROUND: The measurement of physical properties from single molecules has been demonstrated. However, the majority of single-molecule studies report values based on relatively large data sets (e.g., N > 50). While there are studies that report physical quantities based on small sample sets, there has not been a detailed statistical analysis relating sample size to the reliability of derived parameters. METHODS: Monte Carlo simulations and multinomial analysis, dependent on quantifiable experimental parameters, were used to determine the minimum number of single-molecule measurements required to produce an accurate estimate of a population mean. Simulation results were applied to the fluorescence-based sizing of DNA fragments by ultrasensitive flow cytometry (FCM). RESULTS: Our simulations show, for an analytical technique with a 10% CV, that the average of as few as five single-molecule measurements would provide a mean value within one SD of the population mean. Additional simulations determined the number of measurements required to obtain the desired number of replicates for each subpopulation within a mixture. Application of these results to flow cytometry data for lambda/HindIII and S. aureus Mu50/SmaI DNA digests produced accurate DNA fingerprints from as few as 98 single-molecule measurements. CONCLUSIONS: A surprisingly small number of single-molecule measurements are required to obtain a mean measurement descriptive of a normally-distributed parent population.

Performance assessment of DNA fragment sizing by high-sensitivity flow cytometry and pulsed-field gel electrophoresis.

The sizing of restriction fragments is the chief analytical technique utilized in the production of DNA fingerprints. Few techniques have been able to compete with pulsed-field gel electrophoresis (PFGE), which is capable of discriminating among bacteria at species and strain levels by resolving restriction fragments. However, an ultrasensitive flow cytometer (FCM) developed in our lab has also demonstrated the ability to discriminate bacteria at species and strain levels. The abilities of FCM warrant a quantitative parallel comparison with PFGE to assess and evaluate the accuracy and precision of DNA fragment sizing by both techniques. Replicate samples of Staphylococcus aureus Mu50 were analyzed along with two clinical S. aureus isolates. The absolute fragment sizing accuracy was determined for PFGE (5% +/- 2%) and FCM (4% +/- 4%), with sequence-predicted Mu50 SmaI fragment sizes used as a reference. Precision was determined by simple arithmetic methods (relative standard deviation for PFGE [RSD(PFGE) ] = 3% +/- 2% and RSD(FCM) = 1.2% +/- 0.8%) as well as by the use of dendrograms derived from Dice coefficient-unweighted pair group method with arithmetic averages (UPGMA) and Pearson-UPGMA analyses. All quantitative measures of PFGE and FCM precision were equivalent, within error. The precision of both methods was not limited by any single sample preparation or analysis step that was tracked in this study. Additionally, we determined that the curve-based clustering of fingerprint data provided a more informative and useful assessment than did traditional band-based methods.

Quantitative intercomparison of transmission electron microscopy, flow cytometry, and epifluorescence microscopy for nanometric particle analysis.

Nanometric biological particles such as viruses have received increased attention in a wide range of scientific fields. Evaluation of viral contributions to environmental processes and the use of viruses in medical applications such as gene therapy require viruses to be routinely and accurately enumerated. There are a variety of existing techniques for counting viruses, namely, plaque assays, transmission electron microscopy (TEM), epifluorescence microscopy (EFM), and flow cytometry (FCM); each has advantages and disadvantages. While there have been attempts to intercompare some of these techniques to determine the most effective means to count viruses, no previous study used a technique-independent standard for quantitative comparison of collection efficiency, accuracy, and precision. In this work, polystyrene nanospheres were used as standards for the intercomparison of performance characteristics for TEM, EFM, FCM, as well as a custom-built flow cytometer (the Single Nanometric Particle Enumerator, SNaPE). EFM and SNaPE exhibited the highest degree of accuracy and precision, with particle concentrations deviating < or =5% from true and relative errors less than half that of TEM, EFM and SNaPE are also significantly more time and cost efficient than TEM.

Rapid enumeration of respiratory viruses.

Virus detection and enumeration has become increasingly important in fields ranging from medicine and biotechnology to environmental science. Although there are a wide variety of techniques that can be used to count viruses, there is demand for a rapid and more accurate means for virus enumeration. In this work, the performance of a flow cytometer that was designed and custom-built specifically for rapid detection of single viruses was evaluated. The instrument, designated a single nanometric particle enumerator (SNaPE), was characterized and calibrated using fluorescent polystyrene nanospheres. The reliability of the instrument with respect to virus enumeration was demonstrated for three medically relevant viruses, adenovirus-5, respiratory syncytial virus, and influenza A, treated with a fluorescent nucleotide stain. In each case, the SNaPE yielded a virus particle concentration consistent with, but slightly lower than, transmission electron microscopy (TEM) results, as expected. In addition, on the basis of calibration of signal intensity, the average peak height for a given virus was correlated with genome size, as expected. In contrast to time-consuming analyses such as TEM and plaque titers, SNaPE analysis of pure virus samples (including sample handling, data collection, and data processing) can be completed within 1 h.