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#!/usr/bin/perl
use strict;
use warnings;
use Test::More tests => 1;
use Lingua::EN::Tokenizer::Offsets qw/get_tokens/;
use Test::Differences;
use utf8::all;



my ($original,$expected) = &load_strings;
my $tokens = get_tokens($original);
my $got = join "\n",@$tokens;

eq_or_diff "$got\n",  $expected,   "testing strings";


sub load_strings {
my $original = <<'END'
I'm testing'this.
Real-time PCR assays using TaqMan or Molecular
Beacon probes were developed and optimized for the
quantification of total bacteria, the nitrite-oxidizing bacteria
Nitrospira, and Nitrosomonas oligotropha-like ammonia
oxidizing bacteria (AOB) in mixed liquor suspended solids
(MLSS) from a municipal wastewater treatment plant
(WWTP) using a single-sludge nitrification process. The
targets for the real-time PCR assays were the 16S rRNA
genes (16S rDNA) for bacteria and Nitrospira spp. and
the amoA gene for N. oligotropha. A previously reported
assay for AOB 16S rDNA was also tested for its application
to activated sludge. The Nitrospira 16S rDNA, AOB 16S
rDNA, and N. oligotropha-like amoA assays were loglinear over 6 orders of magnitude and the bacterial 16S
rDNA real-time PCR assay was log-linear over 4 orders
of magnitude with DNA standards. When these real-time
PCR assays were applied to DNA extracted from MLSS,
dilution of the DNA extracts was necessary to prevent
PCR inhibition. The optimal DNA dilution range was
broad for the bacterial 16S rDNA (1000-fold) and
Nitrospira 16S rDNA assays (2500-fold) but narrow for
the AOB 16S rDNA assay (10-fold) and N. oligotrophalike amoA real-time PCR assay (5-fold). In twelve MLSS
samples collected over one year, mean cell per L
values were 4.3 ( 2.0 × 1011 for bacteria, 3.7 ( 3.2 ×
1010 for Nitrospira, 1.2 ( 0.9 × 1010 for all AOB, and
7.5 ( 6.0 × 109 for N. oligotropha-like AOB. The percent
of the nitrifying population was 1.7% N. oligotropha-like
AOB based on the N. oligotropha amoA assay, 2.9% total
AOB based on the AOB 16S rDNA assay, and 8.6% nitriteoxidizing bacteria based on the Nitrospira 16S rDNA assay.
Ammonia-oxidizing bacteria in the wastewater treatment
plant were estimated to oxidize 7.7 ( 6.8 fmol/hr/cell based
on the AOB 16S rDNA assay and 12.4 ( 7.3 fmol/hr/cell
based on the N. oligotropha amoA assay.
* Corresponding author phone: (865)974-8080; fax: (865)974-8086;
e-mail: sayler@utk.edu.
† Department of Microbiology.
‡ Department of Civil and Environmental Engineering.
§ Center for Environmental Biotechnology.
10.1021/es0257164 CCC: $25.00
Published on Web 12/04/2002

© 2003 American Chemical Society

1. Introduction
END
;