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8/13/2019 J. Clin. Microbiol.-2013-Barletta-JCM.01397-13.pdf
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Title1
Multiplex real-time polymerase chain reaction for the diagnosis of Campylobacter , Salmonella ,2
and Shigella 3
Running Title4
Multiplex qPCR for Campylobacter , Salmonella , and Shigella 5
Authors6
Barletta F. 1 , Mercado EH 1 , Lluque A. 1, Ruiz J. 3,4, Cleary TG. 2, Ochoa TJ. 1,2 7
Institutions8
1Universidad Peruana Cayetano Heredia, Instituto de Medicina Tropical, Lima-Per.9
2Department of Epidemiology, University of Texas School of Public Health, Houston-USA.10
3Centre de Recerca en Salut Internacional de Barcelona, Hospital Clinic/Institut11
dInvestigacions Biomdiques August Pi i Sunyer, Universitat de Barcelona, Barcelona, Spain.12
4CIBERESP, Barcelona, Spain.13
14
These authors contributed equally to this work. 15
16
17
18
19
Corresponding Author20
Theresa J. Ochoa, MD21
Instituto de Medicina Tropical Alexander von Humboldt22
Universidad Peruana Cayetano Heredia23
Av. Honorio Delgado 43024
San Martin de Porras, Lima 33, Per25
Phone 51-1-482-391026
Fax: 51-1-482-340427
E-mail: [email protected] 28
Copyright 2013, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.01397-13JCM Accepts, published online ahead of print on 12 June 2013
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Abstract 29
Infectious diarrhea can be classified based on its clinical presentation into non-inflammatory30
and inflammatory. In developing countries, among inflammatory diarrhea, Shigella is the most31
common cause, followed by Campylobacter and Salmonella . Because the time frame in which32
treatment choices must be made is short, and the conventional stool cultures lack good33
sensitivity there is a need for a rapid, sensitive, and inexpensive detection technique. The34
purpose of our study was to develop a multiplex real-time PCR procedure to simultaneously35
identify Campylobacter spp ., Salmonella spp ., and Shigella spp . Primers were designed to36
amplify invA, ipaH , and 16SrRNA simultaneously in a single reaction to detect Salmonella ,37
Shigella , and Campylobacter , respectively. One hundred two of one hundred three strains of the38
targeted enteropathogens, and 34/34 of other pathogens were correctly identified using this39
approach. The melting temperatures for invA was 82.96C 0.05, for ipaH was 85.56 C 0.2840
and for 16SrRNA was 89.21C 0.24. The limit of accurately quantification of the assay in41
stool samples was 10 4 CFU g -1 however the limit of detection was 10 3 CFU g -1. This assay is a42
simple, rapid, inexpensive, and reliable system for the practical detection of these three43
enteropathogens in clinical specimens.44
45
46
47
48
49
KEY WORDS:50
Multiplex real time PCR, Salmonella spp ., Shigella spp ., Campylobacter spp . 51
52
53
54
55
56
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Background57
Infectious diarrhea is a global health problem still responsible for thousands of deaths58
worldwide, especially in children 4. It can be classified, based on its clinical presentation, into59
two syndromesnoninflammatory and inflammatory diarrhea 14. Among cases of inflammatory60
diarrhea Shigella is the most common cause, followed by Campylobacter and Salmonella 18.61
These invasive organisms primarily target the lower bowel; they invade the intestinal mucosa to62
induce an acute inflammatory reaction and activate cytokines and inflammatory mediators 16.63
Because the time frame in which treatment choices must be made is short and the conventional64
stool cultures lack good sensitivity, there is a need for a rapid, sensitive, and inexpensive65
detection technique. We searched for DNA sequences that were highly conserved between the66
different species of each genera; and we selected the following genes as targets: invA (invasion67
A gene) for Salmonella spp ., ipaH (invasion plasmid antigen H) for Shigella spp ., and 16SrRNA 68
(16S ribosomal RNA) for Campylobacter spp ., to develop a fluorescence-based real-time PCR69
procedure to simultaneously identify these enteropathogens. In this analysis the post-PCR70
products are identified based on melting-point curve analysis. We have also standardized the71
technique to quantify these bacteria directly from stool samples.72
73
Methods 74
Bacterial Strains75
One hundred forty seven enteropathogenic strains (Table 1) were analyzed, including clinical76
isolates representative of the species of Salmonella spp ., Shigella spp ., and Campylobacter spp .,77
as well as other enteropathogens. These clinical strains had previously been identified based78
on serology, biochemical assays and real time PCR for the Diarrhoeagenic E. coli 8 . In addition79
we used Samonella enteritidis ATCC 13076, Shigella flexneri ATCC 12022, and80
Campylobacter jejuni subspp . jejuni ATCC 33560 as positive controls.81
DNA isolation from pure culture82
Strains were sub-cultured from frozen or peptone stocks onto MacConkey (Merck, Darmstadt,83
Alemania) for Salmonella and Shigella , and Chocolate Agar (TSA; Oxoid, Basingstoke,84
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Hampshire, Englandcon + 5% sheep blood) for Campylobacter using quadrant streaking85
methods and incubated at 37C to produce isolated colonies. After overnight incubation (48 86
72 hours for Campylobacter cultures) a bacterial suspension was carefully prepared (0.5 Mac87
Farland scale), avoiding agar contamination, an important cause of erratic amplification. Crude88
lysates were prepared and used directly as templates for the PCR reaction. DNA was extracted89
by boiling the bacterial suspension in 500uL of PCR or molecular grade water for 5 minutes90
followed by room temperature incubation for 10 min, a centrifugation at 12,000 rpm for 1091
minutes. 2 l of this crude lysate was used as template with 18 l PCR master mix to make a 20 l92
total reaction volume.93
94
Primer Design95
The primers were designed to detect three different virulence genes simultaneously in a single96
reaction (Table 2). Primers were designed so that amplicons would be produced having melting97
temperatures (Tm) ranging from 77-95 C with greater than 1 C between each peak. We98
targeted the amplicon Tm as the first parameter seeking appropriate primer sequences to99
amplify unique regions in a given virulence gene that would result in an amplicon of the desired100
Tm. Sequences of each gene were examined for features such as areas of high or low GC101
content, size, and identity among reported BLAST sequences for the target gene. These areas102
were analyzed by an oligonucleotide properties calculator (PrimerPremier 5.0) which uses the103
nearest neighbor method to predict the amplicon Tm. After selecting areas likely to produce104
amplicons with the desired Tm, primers were designed using the Primer3 program105
(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and were synthesized by Belomed106
S.R.L. (Lima, Peru). These primers were then sequentially added to the mix to determine their107
actual Tm as well as to determine whether non specific primer amplification occurred in the108
presence of other oligonucleotide primers. Primer concentrations were then optimized to109
produce melting curves of similar peak height (area under the curve) between products and110
across a series of dilutions to simulate the varying concentration of template DNA extracted111
from the crude lysate preparation.112
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PCR Conditions113
Initially we evaluated previously reported multiplex assays 6,7,9,10,12,19 to determine whether they114
would work well in a real time PCR. Non specific amplification or interference with new115
primers made most of the primers in these assays problematic. We sequentially eliminated116
primers and eventually were able to use several previously described primers in our system117
(Table 2). PCR was performed using a LightCycler 480 Real-Time PCR System (Roche118
Applied Science). Each multiplex PCR assay was performed in a final reaction volume of 20 l119
containing 0.5U Phusion polymerase (Finnzyme OY, Espoo, Finland) in High Fidelity Phusion120
buffer with a final concentration of 200 M dNTPs and 3mM MgCl 2. The primers were used at a121
final concentration of 0.2 to 0.4 M (Table 2). SYBR Green I (Cambrex Bio Science, Rockland,122
ME) was diluted as recommended by the manufacturer. The hot-start technique was used to123
prevent nonspecific amplification. The amplification cycles consisted of incubation at 98C for124
30sec, 65C for 30 sec, 72C for 30 sec, and 72C for 10 sec. After 30 cycles a melting curve125
using fluorescence of SYBR Green was determined with a ramp speed of 0.2C/sec between126
72C and 98C with a reading every 0.2C. Melting peaks were automatically calculated by the127
software LightCycler 480 SW 1.5 (Roche Diagnostics) which after subtracting background128
fluorescence from a set of water blanks, plotted the negative derivative of fluorescence with129
respect to temperature (-d(F)/dT versus T). Representative strains of each species were analyzed130
by agarose gel electrophoresis (2.0% agarose gels) to ensure that no unwanted bands were seen131
and that the predicted product size was found. 132
133
Limit of detection and amplification effiency in spiked stool134
We reactivated one strain of each pathogen onto MacConkey (Merck, Darmstadt, Alemania),135
(Salmonella and Shigella ), and chocolate agar (TSA; Oxoid, Basingstoke, Hampshire,136
Englandcon + 5% sheep blood) ( Campylobacter ) plate. After 37C/18h ( Salmonella and137
Shigella ) and 42C/48h ( Campylobacter ) we made 10-fold serial dilution (equivalent to 10 0 to138
107 CFU) and spiked 100 mg of stool sample from a healthy volunteer. The number of CFU139
was determined by plating culture dilutions on MacConckey ( Salmonella and Shigella ) and140
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Chocolate agar ( Campylobacter ). Bacterial DNA was extracted from the stool samples with the141
Roche Kit (High Pure PCR Template Preparation Kit) and resuspended into 200 uL of Buffer142
TE (Tris EDTA). Amplification efficiency (E) was estimated by using the slope of the standard143
curve and the formula E = (10 1/slope) - 1. A reaction with 100% efficiency will generate a slope144
of -3.32.145
146
Results147
We evaluated three enzymes: (i) Phusion hot-start DNA polymerase (Finnzymes, Finland), a148
Pyrococcus- like enzyme with a processivity-enhancing domain; (ii) Roche (FastStart Taq DNA149
polymerase), a chemically modified form of thermostable recombinant Taq, and (iii) Promega150
(GoTaq DNA polymerase). Of these, Phusion was the only enzyme that gave reliably151
reproducible amplication. The average melting temperature (Tm) for the invA amplicon was152
82.96C 0.05; 85.56C 0.28 for ipaH; and 89.21C 0.24 for 16SrRNA (Table 2). The spacing153
between peaks for each gene is shown in Fig. 1A. Remarkably little variation in intensity and154
Tm was observed among the different strains tested. On an individual-gene basis, all genes155
tested were reliably amplified except for 16SrRNA , which was detected in 39/40 strains156
expected to be positive. Among the other enteropathogens tested for cross reaction, one strain157
could be considered false positive (1/34), although as an Enteroinvasive E. coli (EIEC) strain it158
is expected to have the ipaH gene. The amplitudes of the melting curves were quite similar for159
all strains in each category as well. The individual peaks were symmetric, as shown in Fig. 1B,160
demonstrating the overlap and reproducibility of the assay for multiple strains. Analysis using161
agarose gel confirmed that the amplicons represented on the melting-curve graph were indeed of162
the correct molecular weights expected based on the primer sequences (Figure 1C).163
Mix infections are common, especially in diarrheal disease. For this reason we simulated164
possible coinfections between these pathogens. We have not observed competition between the165
different set of primers used to detect the genes involved in the virulence of these pathogens166
(Figure 2).167
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The ability to detect Salmonella , Shigella and Campylobacter in stool samples was tested by168
spiking serial dilutions of each enteropathogen into 100mg of sample. The assay in stool169
sample detected the presence of these pathogens in the range of 10 3 107 CFU g -1 (Figure 3A,170
3B, 3C), but could only accurately quantify from 10 4 to 10 7 CFU g -1. The standard plot showed171
that the regression coefficient was linear (R 2 = 0.99, 0.98 and 0.99, respectively) over a 5 log172
dilution range and the reaction efficiencies were 100.3%, 91.7% and 99.0% for the invA, ipaH ,173
and 16SrRNA genes, respectively, (4A, 4B, 4C).174
The total cost starting from the stool sample, including the DNA extraction and qPCR analysis175
was higher than the cost starting from culture (US$ 6.0 vs. US$ 4.8) however the time for176
obtaining a confirmed result was much less (4 hours vs. 24-72 hours, depending of the177
pathogen).178
179
Discussion180
Real-time PCR assays have been developed independently for the detection and quantification181
of some enteropathogens; Helicobacter pylor 20, Clostridium difficile 20 , Campylobacter 17 , 182
Cryptosporidium 15 , Salmonella 19 and enteropathogenic Escherichia coli 2,enterohemorrhagic183
Escherichia coli 3. Although the PCR conditions were standardized, when we tested our184
conventional PCR in the real time thermo cycler we saw that the melting temperature (Tm) of185
the products of Campylobacter spp . and Shigella spp . were similar and the peaks overlapped.186
We examined the sequence of the ipaH gene for the most conserved region for all the strains of187
Shigella spp with the BLAST software and analyzed the new set of primers with the188
PrimerPremier 5.0. The best target region was located between nucleotides 16 and 123, but the189
Tm for the PCR product overlapped with the invA gene amplified for Salmonella spp . We kept190
this region and solved the problem of the overlapping with the CG tails strategy 22, adding 3191
nucleotides (CGC) in front of the forward primer for Shigella spp . Comparing to previous192
studies 13,21,23 this Multiplex have shown a higher analytical sensitivity to detect these three193
enteropathogens in stool samples. The effenciencies (90% to 110%) and the regression194
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coefficient (almost 1.00) obtained for all the targets were in agreement with the MIQE195
Guidelines 5.196
In this study we first standardized a conventional PCR using primers that were previously197
reported against 16SrRNA (Salmonella spp . and Campylobacter spp .)11-13 and ipaH (Shigella198
spp .)7. Unfortunately when we put all the primers in a single PCR reaction we obtained cross199
reaction. Conventional PCR requires amplification in a thermocycler followed by product200
separation by gel electrophoresis. The cost, and the time delay required to do gel analysis of201
PCR products, and the inability to analyze large numbers of strains, are major impediments to202
this approach. In addition, while ethidium bromide is a relatively inexpensive reagent for DNA203
gel staining, it has human and environmental safety concerns. For these reasons, real-time204
fluorescence-based multiplex PCR has become an attractive technique. It offers the advantage205
of being a faster and more robust assay because it does not require post-PCR procedures to206
detect amplification products.207
Our study has some limitations. First, although the sequences used as target in this multiplex208
PCR are from highly conserved regions of the genes, a weakness of this or any multiplex assay209
is that new variants of virulence genes could fail to amplify with the primers described. Second,210
this assay also identifies Enteroinvansive E. coli (EIEC) since both Shigella spp . and EIEC have211
the ipaH gene. Third, it was not possible to compare the performance of the assay between212
cultures and stool samples because these last had been stored for long time and DNA might be213
degraded.214
Nevertheless, this assay represents a simple, rapid, sensitive, and inexpensive system for the215
practical presumptive detection of these three enteropathogens. An advantage of this technique216
is that there is no need to run an electrophoresis gel to determine the presence of the amplicons217
because each has a characteristic melting temperature (Tm) that is detected in the denaturation218
curve. Indeed, this approach also allows identification of possible mixed infections involving219
several of these pathogens. The cost of materials was under $6.00 per sample analyzed. Thus,220
the new real-time multiplex PCR provides reliable results within a short time and might be221
useful as an additional diagnostic tool whenever time is important in the diagnosis of222
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enteropathogenic bacteria. Further studies should focus on the comparison between culture and223
qPCR from stool samples to calculate the real sensitivity and specificity of this assay.224
225
Acknowledgements 226
This work was supported by a Public Health Service award (grants 1K01TW007405 to T.J.O.227
and R01-HD051716 to T.G.C.) from the National Institute of Health; and by Agencia Espaola228
de Cooperacin Internacional para el Desarrollo (AECID), Spain, Programa de Cooperacin229
Interuniversitaria e Investigacin Cientfica con Iberoamrica (D/019499/08 and D/024648/09)230
(J.R and T.J.O).231
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Legends334
335
Figure 1A:336
Real-time PCR simultaneously detects three different genes. Data from individual tubes, each337
containing the ATCC strains: S. enteritidis 13076, S. flexneri 12022 and C. jejuni subspp. jejuni338
33560, are shown in a single gure so that the separation between individual amplicon melting339
curves is illustrated (from left to right: invA, ipaH , and 16SrRNA ). The y axis (uorescence)340
represents the negative derivative of uorescence over temperature versus temperature.341
342
Figure 1B:343
Melting curves of DNA isolated from pure cultures of (a) Salmonella (n=26), (b) Shigella 344
(n=49) and (c) Campylobacter (n=41). Curves are superimposed showing the reproducibility345
within specie. The y axis (uorescence) represents the negative derivative of uorescence over346
temperature versus temperature.347
348
Figure 1C:349
Agarose gel (2%) of amplicons of representative strains from the multiplex real-time PCR.350
From left to right: (1) 100bp molecular weight ladder, (2) C. coli , (3) C. jejuni , (4) S. enteritidis ,351
(5) S. infantis , (6) Salmonella spp. , (7) S. boydii , (8) S. dysenteriae , (9) S. flexneri , (10) S.352
sonnei , (11) C. jejuni subspp . jejuni ATCC 33560, (12) S. enteritidis ATCC 13076, (13) S.353
flexneri ATCC 12022, (14) Diffusely adherence E. coli , (15) Enteroaggregative E. coli , (16)354
Enteropathogenic E. coli , (17) Enterotoxigenic E. coli , (18) Enteroinvasive E. coli , (19)355
Shigatoxin-like producere E. coli , (20) E. coli K12, (21) Pseudomonas aeruginosa , (22)356
Klebsiella pneumonia , (23) Proteus mirabilis . 357
358
359
360
361
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Figure 2:362
Mixed infection detected in a pool of colonies corresponding to S. enteritidis ATCC 13076363
[invA], S. flexneri ATCC 12022 [ ipaH ] and C. jejuni subspp . jejuni ATCC 33560 [ 16SrRNA ]364
(A), and another corresponding to S. enteritidis ATCC 13076 [ invA] and S. flexneri ATCC365
12022 [ ipaH ].366
367
Figure 3:368
The qPCR detected serial dilutions from 10 7 to 10 3 CFUg-1 of Salmonella (A), Shigella (B) and369
Campylobacter (C) in stool samples.370
371
Figure 4:372
In stool samples, the qPCR showed an efficiency of 100.3%, 91.7% and 99.0% for the invA (A),373
ipaH (B), and 16SrRNA (C) genes, respectively, with correlation coefficients of 0.99, 0.98, and374
0.99, respectively.375
376
377
378
379
380
381
382
383
384
385
386
387
388
390
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Table 1: Clinical enteropathogenic strains analyzed in the real-time PCR assay system
Species NSalmonella spp.
S. enteritidis ATCC 13076
S. enteritidis
S. infantis
Salmonella spp.
26
1
6
2
17
Shigella spp .
S. flexneri ATCC 12022
S. boydii
S. dysenteriae
S. flexneri
S. sonnei
Shigella spp.
49
1
6
3
5
6
28
Campylobacter spp .
C. jejuni subspp . jejuni ATCC 33560
C. coli
C. jejuni
Campylobacter spp.
41
1
10
10
20
Negative control: other enteropathogens
Diffusely adherent E. coli
Enteroaggregative E. coli
Enteroinvasive E. coli
Enteropathogenic E. coli
Enterotoxigenic E. coli
Shiga toxin-producing E. coli
E. coli K12
Pseudomonas aeruginosa
Klebsiella pneumonia
Proteus mirabilis
34
6
6
1
6
6
5
1
1
1
1
TOTAL 147*
*Without including the 3 ATCC strains as positive control
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Table 2: Primers for Multiplex real-time PCR
Pathogen Gen O 1 Primer
(5 3)
Final Concen
(uM)
Amplicon
Size (bp)
Amplicon Tm
(mean SD)Ref
Salmonella spp. invA
F
R
CATTTCTATGTTCGTCATTCCATTA
CC
AGGAAACGTTGAAAAACTGAGGA
TTCT
0.40 132 82.96 0.05Pusterla
(2009)
Shigella spp. ipaH
F
R
CGCGACGGACAACAGAATACACT
CCATC
ATGTTCAAAAGCATGCCATATCTG
TG
0.20 108 85.56 0.28This
study
Campylobacter spp. 16SrRNAF
R
GGATGACACTTTTCGGAGC
CATTGTAGCACGTGTGTC0.40 812 89.21 0.24
Logan
(2001)
1 O = Orientation, F = Forward, R = Reverse
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FIGURES:1
Figure 12
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B
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9
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C
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Figure 226
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A
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Figure 339
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A
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C
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Figure 447
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B
A
C