TITLE

Collaborative Study to Evaluate a Rapid Total Bacteria Count Assay using

Quantitative Real-Time PCR.

AUTHORS

Thomas Romick. Industrial Microbial Testing. Newport Coast, CA

Rafael Marfil. Grupo Sinergia. Tlalnepantla Edo. de Mexico

Eduardo Lopez. Grupo Sinergia. Monterey, Mexico

CORRESPONDENCE

thom.romick@imt-pcr.com

POTENTIAL REVIEWERS

Frank Busta
National Center for Food Protection and Defense
University of Minnesota
fbusta@umn.edu

Sharon Brunelle
AOAC research institute
sbrunelle@aoac.org
ABSTRACT

A collaborative study was undertaken to demonstrate the precision and accuracy of a total
bacteria count (TBC) assay developed to run with commercially available PCR reagents and
a rapid thermocycling platform. The TBC assay can be completed and reported for quality
assurance action within 1 hour after sample preparation. A collaborator kit was supplied to
each lab with all the necessary reagents and consumables that were detailed on a chain of
custody document. Collaborating laboratories were diversified among government,
academic and industrial. Results from the 7 collaborative labs were analyzed using the
Taguchi Method to demonstrate robust design performance. The β value indicated how
close the average result was to the target and ranged from 0.83 to 1.07 (1.00 target). The
signal to noise ratio which includes ANOVA and the β factor ranged from 14.13 to 23.19
(20.00 minimum target).

KEY WORDS

Quantitative PCR, Total Bacteria Count, HACCP.

INTRODUCTION

A quantitative real-time PCR was evaluated by 7 collaborating laboratories for accuracy and
precision. The TBC (Total Bacterial Count) assay uses universal primers that flank a highly
conserved DNA segment of the 23s ribosomal gene in prokaryotes. The amplicon is
detected by Temperature Dependent Fluorescence (TDF). The primer concentration and
fluorescent dye have been optimized to detect the slightest amount of target DNA and so
can be used as a sterility test and quantitative bacteria load test. Real-time PCR tests assays
have been developed for many applications targeting the ribosomal operon (Kabir et al.
2003; Khan, et al. 2007; Lee and Levin 2007) and also using TDF for confirmation of
amplicon production on automated PCR platforms (Rawsthorne and Phister 2006). Bacteria
counts, whether specific or total, can be correlated easily to standard methods for routine use
in the laboratory per sample type (Bach, et al. 2002; Nadkarni, et al. 2002; Takahashi et al.
2006). The TBC assay gives quantitative results in 30 minutes after sample preparation
compared to compendial standard plate count methods that require 48 hours. The TBC
assay was used in this collaborative study to demonstrate a rapid method for quantification
and ease-of-use in diverse laboratory environments as measured by Taguchi robust design
statistical analysis (Taguchi 1987).

MATERIALS AND METHODS

Protocol

Each laboratory was provided with a kit of reagents and consumables that were documented
on a chain of custody form. The kit contained the TBC assay (Industrial Microbial Testing,
IMT lot #03359, Newport Coast, CA), Cepheid Smarmix® master mix beads lot #009-04,
Cepheid DNA beads lot #00901, and Cepheid reaction tubes lot # 722C7-7157 (Cepheid,
Sunnyvail, CA), pipette barrier tips VWR lot #0706080202 (VWR International USA), and
PCR grade water lot #W334507L601(Teknova, Hollister, CA) Each laboratory was
provided with a protocol to conduct the study that included production of a standard curve
in triplicate followed by a series of unknowns in triplicate. The standard curve was made
using the Cepheid DNA bead standard with serial 10-fold dilutions and the unknowns were
provided by IMT in a dried format. The testing was performed on the Cepheid
SamrtCycler® platform using the TBC protocol and data transmission of results done via
Email in an Excel format. Statistical analysis was done using the Taguchi method for robust
design determination (Taguchi 1987; Taguchi and Wu 1990).

Collaborators

Brigham Young University, Department of Microbiology and Molecular Biology, Provo,

UT.
California Department of Public Health, Food & Drug Laboratory Branch, Richmond, CA.
NASA Ames Research Center, Moffett Field, CA.
NASA Jet Propulsion Laboratory, Biotechnology and Planetary Protection Group, Pasadena,
CA.
 Nestle USA Quality Assurance Center, Dublin, OH.
North Carolina State University, Department of Food Bioprocessing and Nutrition, Raleigh,
NC.
San Francisco Public Utilities Commission, Water Quality Division, Millbrae, CA

Laboratories are listed in alphabetical order and do not represent the same order data are
presented in tables and graphs for discretion.

RESULTS

Figure 1 shows a typical standard curve generated by each laboratory. Correlation

coefficients (r-square values) ranged from 0.9835 to 0.9993 for the 7 collaborating
laboratories. The standard curve for each laboratory was used to calculate the unknown
samples. The results were transmitted electronically in an Excel spreadsheet format.

Lab B

6
5
log standards

4
3
y = -0.2901x + 10.899
2
R2 = 0.9993
1
0
0 5 10 15 20 25 30 35
Ct values

Figure 1. A typical standard curve generated by each collaborating laboratory. Ct values are determined as the
cycle needed to cross a threshold value during real-time PCR.

Table 1 shows the resultant values of each collaborating lab against the expected unknown
values based on the standard curve generated by each lab:

R esults (log)
Expected Expected
Sam ple ID value value (log) 1 2 3 4 5 6 7
1-R 1 5000 3.69897 2.71770 3.29175 3.62876 3.54594 3.15925 3.64032 3.62920
1-R 2 5000 3.69897 3.08612 3.35173 3.70587 3.60877 3.78136 3.41096 3.67400
1-R 3 5000 3.69897 3.04614 3.20036 3.75442 3.59163 3.66388 3.76074 3.75520
U 1-R 1 500 2.69897 2.16078 2.82051 3.17751 2.25788 2.36626 2.34731 2.67160
U 1-R 2 500 2.69897 2.21504 2.88906 3.16609 2.25788 2.65195 2.33297 2.63800
U 1-R 3 500 2.69897 2.10937 2.54062 3.01186 2.33785 2.41165 2.50499 2.61560
U 2-R 1 100 2.00000 1.84090 2.38354 2.36070 1.55816 2.10727 1.71370 1.80360
U 2-R 2 100 2.00000 1.88089 2.33785 2.37498 1.59529 2.29417 1.81691 1.61880
U 2-R 3 100 2.00000 1.76950 2.14078 2.25502 1.71238 2.18737 1.81691 1.73360
10-R1 50 1.69897 1.44392 1.91230 1.65526 1.59814 1.85896 1.66496 1.53200
10-R2 50 1.69897 1.55530 1.67811 1.83234 1.48390 1.72546 1.43560 1.50120
10-R3 50 1.69897 1.66098 1.77807 1.82948 1.63527 1.86697 1.29512 1.77000
Table 1. Results from each laboratory for unknown samples relative to expected values for statistical analyses.
Figure 2 shows a beta curve function according to Taguchi theory :

y = BM
x
y x
x
(obs.)
x
x
x

x
x
x

Standards
(
M1 M2 ............ (Signals)
Mk

Figure 2. The β calculation used for Taguchi statistical analysis where Yi is the average value of the three
observations made on the standards, Mi.

β̃ = M1Y1 + M2Y2 + ………. + MkYk

k

∑ Mi2
i =1

Figure 3 shows typical results from a collaborating lab for the β calculation :

Individual Value Plot of lab 2 vs Signal

3.5

3.0
lab 2

2.5

2.0

1.5
1.69897 2.00000 2.69897 3.69897
Signal

Figure 3. A typical β curve from a collaborating laboratory.

Table 2 summarizes the Taguchi statistics for β, ANOVA (σ2 ) and Signal-to-Noise ratio (η)
for Robust Design determination :

Laboratory β σ2 η
1 0.827 0.01465 16.69
2 0.976 0.01758 17.34
3 1.069 0.00679 22.26
4 0.910 0.00397 23.19
5 0.978 0.03698 14.13
6 0.928 0.01977 16.39
7 0.967 0.00880 20.26
Global 0.951 0.07443 18.61
Table 2. Data from all collaborating laboratories summarizing the β value, σ2, and η (signal to noise ratio).

DISCUSSION

The 23s region of the prokaryotic ribosome was targeted for known sequence conservation
due to environmental pressure to preclude significant mutation that would be lethal. The
16s region has also been a target for stable and variable regions and both genes have been
used in environmental and food applications for real-time PCR ( Chotar et al. 2006; Kabir et
al. 2003; Khan et al. 2007; Lee and Levin 2007; Takahashi et al. 2006). The 23s sequence
target produces a 200 base pair amplicon that is an optimal size for temperature dependent
fluorescence detection using commercially available PCR thermocycling equipment (Fykse
et al. 2003; Khan et al. 2007; Rawsthorne and Phister 2006). The platform used in this
study was the Cepheid SmartCycler®. Quantitative data can be easily determined after a 30
minute reaction time. The TBC assay was designed to perform on the platform with the
Cepheid SmartMix® master mix bead format. The master mix beads are rehydrated with
the TBC primers and intercalating dye in one step then aliquoted into the reaction tubes with
the sample in a simplified 3-step process. This automation greatly reduces the chance of
contamination and pipetting errors as shown by the collaboration data.

It is suggested that robust design statistics are essential to describe the usefulness of a
method of testing in an industrial setting (Phadke 1989; Taguchi 1987). According to the
Taguchi method, measurement systems need to have 3 attributes; precision, accuracy
(exactitude) and robustness.

Precision is evaluated by matching average measurements against specific standards.

Accuracy is estimated using ANOVA and it is usually referred as measurement error.
However, robustness is what is known as the “optimization statistic” and it is measured
through the “Signal–to-Noise” ratio (Taguchi and Wu 1990).

Robust design is defined to be the application of something (process, product, methodology)

obtaining consistent results under a wide variety of extreme conditions. This means that a
measurement system has a robust design if precision and accuracy are simultaneously steady.
That is what signal-to-noise ratio attempts to evaluate.

Signal-to-Noise ratio is not new but has different expressions according to the application.
The signal-to-noise formula was originally defined by taking the square root of the
“measure of noncentrality” and established criteria for goodness-of-fit while modelling
behaviour of observed data (Box and Draper 1987). Another signal-to-noise ratio comes
from taking the square root of the quotient σ2p/ σ2m to determine the suitability of a
measurement system to control a process (Wheeler and Lyday 1989). In this case, σ2p is the
variance of the manufacturing process and σ2m is the variance of the measurement
instrument. In both cases, the proposed ratio is a maximization statistic.

Taguchi developed a signal-to-noise ratio that is appropriate for robust design. It analyzes a
signal that travels through a noisy channel. By using a formula that involves precision and
accuracy in a logarithmic expression, the resulting statistic turns out to be a maximization
technique which demands a value approaching 20, in order to assure robustness. That is
equivalent to say that the standard error of measurement will not exceed 10% over its target.

With respect to the 7 laboratories that used the PCR method to measure several standards,
the fact that 3 of them achieved a signal-to-noise ratio value of more than 20 indicates good
solid design. Care should be exercised when training technicians to avoid loss of accuracy
which seemed to be the largest source of error. Technicians were not formally trained on
the equipment for the TBC assay. Each lab was sent a protocol to follow and reagent
package to perform the study.

The results show that all collaborators successfully used the TBC assay to quantify bacterial
load accurately. The TBC assay can be used for a rapid quantitative measure of bacteria in
many diverse applications and support HACCP for real-time statistical process control.
Robust design statistical analysis demonstrates that this system can be applied in the
industrial environment.

This study was designed to show how diverse laboratories can use the TBC assay in their
labs for specific applications. Once a standard curve is constructed for a sample type, it can
be used time again for quantifying unknown samples. Quantitative real-time PCR has been
shown successful for food, environmental and clinical applications (Bach et al. 2002;
Nadkarni et al. 2002; Takahashi et al. 2006).
REFERENCES

BACH, H.J., TOMANOVA, J., SCHLOTER, M. and MUNCH, J.C. 2002. Enumeration of
total bacteria and bacteria with genes for proteolytic activity in pure cultures and in
environmental samples by quantitative PCR mediated amplification. J. Microbial Methods.
49, 235-245.

BOX, G.E.P. and DRAPER, N.R. 1987. Empirical Model Building and Response Surfaces.
John Wiley and Sons, Hoboken, NJ.

CHOTAR, M. VIDOVA, B. and GODANY, A. 2006. Development of specific and rapid

detection of bacterial pathogens in dairy products by PCR. Folia Microbiol. (Praha). 51,
639-646.

FYKSE, E.M., OLSEN, J.S. and SKOGAN, G. 2003. Application of sonication to release
DNA from Bacillus cereus for quantitative detection by real-time PCR. J. Microbiol.
Methods. 55, 1-10.

GOTO, M., TAKAHASHI, H., SEGAWA, Y., HAYASHIDANI, H., TAKATORI, K. and
HARA-KUDO, Y. 2007. Real-time PCR method for quantification of Stapylococcus
aureus in milk. J. Food Prot. 70, 90-96.

KABIR, S., RAJENDRAN, N., AMEMIYA, T., and ITOH, K. 2003. Real-time
quantitative PCR assay on bacterial DNA: In a model soil system and environmental
samples. J. Gen. Appl. Microbiol. 49, 101-109.

KHAN, U.H., GANNON, V., KENT, R., WENDELL, K., LAPEN, D.R., MILLER, J.,
NEUMANN, N., PHILLIPS, R., ROBRERTSON, W., TOPP, E., van BOCHOVE, E. and
EDGE, T.A. 2007. Development of a rapid quantitative PCR assay for direct detection and
quantification of culturable and non-culturable Escherichia coli from agriculture watersheds.
J. Microbial Methods. 69, 480-488.

LEE, J.L., and LEVIN, R. 2007. Quantification of total viable bacteria on fish fillets by
using ethidium bromide monoazide real-time polymerase chain reaction. Int. J. Food
Microbiol. 118, 312-317.

LIMING, S.H. and BHAGWAY, A.A. 2004. Application of a molecular beacon-real-time

PCR technology to detect Salmonella species contaminating fruits and vegetables. Int. J.
Food Microbiol. 95, 177-187.

NADKARNI, M.A., MARTIN, F.E., JACQUES, N.A., HUNTER, N. 2002. Determination

of bacterial load by real-time PCR using broad-range (universal) probe and primers set.
Microbiology. 148, 257-266.
PHADKE, M.S. 1989. Quality Engineering Using Robust Design. Prentice Hall,
Englewood Cliff, NJ.

RAWSTHORNE, H., and PHISTER, T.G. 2006. A real-time PCR assay for the
enumeration and detection of Zygoscccharomyces bailii from wine and fruit juices. Int. J.
Food Microbiol. 112, 1-7.

TAGUCHI, G. 1987. System of Experimental Design. UNIPUB/Krass International

Publications, New York.

TAGUCHI, G. and WU, Y. 1990. Quality Engineering: Dynamic Characteristics and

Measurement Engineering. Implementation Manual, American Supplier Institute
International. Detroit, MI.

TAKAHASHI, H., KONUMA, H. and HARA-KUDO, Y. 2006. Development of a

quantitative real-time PCR method to enumerate total bacteria counts in ready-to-eat fruits
and vegetables. J. Food Prot. 69, 2504-2508
.
WHEELER, D.J. and LYDAY, R.W. 1989. Evaluating the Measurement Process.
SPC Press, Inc. Knoxville, TN.