J. Clin. Microbiol.-2013-Barletta-JCM.01397-13.pdf

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

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382

383

384

385

386

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388

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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

3

4

5

6

A

B


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7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

C


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Figure 226

27

28

29

30

31

32

33

34

35

36

37

38

B

A


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Figure 339

40

41

42

43

44

45

46

A

B

C


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Figure 447

48

49

50

51

B

A

C

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J. Clin. Microbiol.-2013-Barletta-JCM.01397-13.pdf