TALLER 1 DE BIOLOGÍA CELULAR Guía de actividades Introducción · Taller 1 Biología Celular. Departamento de Biología Celular e Histología 1° Unidad Académica, FMED, UBA. 2

Taller 1 Biología Celular. Departamento de Biología Celular e Histología

1° Unidad Académica, FMED, UBA.

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TALLER 1 DE BIOLOGÍA CELULAR

Guía de actividades

Introducción:

El conocimiento científico acerca de los procesos moleculares que subyacen al mantenimiento

del genoma y que permiten simultáneamente su variabilidad, han permitido comprender los

fenómenos biológicos de replicación, reparación, mutación y reorganización del ADN.

Pero además, este conocimiento ha permitido desarrollar procedimientos tecnológicos

innovadores que poseen múltiples aplicaciones en la clínica y en la investigación básica. En

este taller analizaremos una de estas técnicas: PCR (Reacción en cadena de la polimerasa),

cuyo desarrollo está basado en los procesos de replicación de los ácidos nucleicos.

PARTE 1: Técnica de PCR (Reacción en cadena de la polimerasa). Fundamentos . Semejanzas

y diferencias con la replicación celular del ADN1.

La técnica de reacción en cadena de la polimerasa (PCR, Polymearse Chain Reaction) permite la

amplificación de un fragmento de ADN en una reacción in vitro. Como consecuencia de la

aplicación de esta técnica pueden generarse millones de copias del fragmento de ADN

amplificado a partir de un número muy bajo de copias iniciales. Esta técnica fue desarrollada

en 1983 por el bioquímico estadounidense Kary Mullis por la que recibió el premio Nobel de

Química en 1993.

1.1 ¿Qué elementos son imprescindibles en esta reacción?

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1.2.¿Qué principios/propiedades básicas del ADN y de la enzima ADN polimerasa son

fundamento de la PCR?

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1 Bibliografía recomendada:

Cooper “La célula” 7ma ed. Ed. Marbán.

Alberts “Introducción a la Biología Celular” 3ra ed. Ed.Panamericana.

Para contestar las siguientes preguntas, le sugerimos que lea previamente

los fundamentos de las técnicas de PCR y electroforesis de ácidos nucleicos

de la bibliografía recomendada1 en la materia.



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1.3. Compare las fases-procesos de desnaturalización, hibridación, y extensión de la PCR con la

replicación in vivo en eucariotas ¿Qué principales diferencias observa?

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1.4. ¿Puede haber errores de copiado durante la PCR? Explique brevemente.

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1.5. Volviendo al proceso de replicación del ADN in vivo en eucariotas, durante el mismo

pueden ocurrir errores de copiado, ¿hay mecanismos que pueden subsanar este fenómeno?

¿Cuáles?

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1.6. La electroforesis de ácidos nucleicos es una técnica complementaria que se utiliza para

visualizar los productos de amplificación de una PCR. Explique los fundamentos de esta

técnica.y la información que puede obtener de la misma.

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PARTE 2: Técnica de PCR (Reacción en cadena de la polimerasa).

Aplicaciones en medicina I: Diagnóstico de enfermedades infecciosas.

Una aplicación de rutina de la técnica de PCR en el ámbito clínico es en el diagnóstico de

enfermedades infecciosas, mediante la detección de ADN específico del agente patógeno

(bacterias, virus, hongos, etc) en muestras de pacientes.

El siguiente ejercicio está basado en un trabajo de investigación (ver

anexo). Los trabajos de investigación, usualmente denominados

informalmente con de la palabra inglesa ‘papers’, son el vehículo de

comunicación de resultados que utiliza la comunidad científica.

Si bien la resolución del ejercicio no requiere de la información del

trabajo original, recomendamos su lectura. Si le resultara muy difícil,

puede concentrarse en la lectura e interpretación del Abstract

(Resumen) al inicio del trabajo.



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Un ejemplo de esta aplicación se describe en un trabajo de investigación publicado en el año

20132 (ver anexo), en el que se utiliza una reacción de PCR para la detección de brucelosis

humana en áreas endémicas.

La brucelosis es una enfermedad infecciosa de distribución mundial, frecuente en zonas

rurales, que constituye un problema persistente en muchos países en desarrollo. Esta

enfermedad es producida por bacterias del género Brucella, que suelen transmitirse de

animales utilizados como ganado a los seres humanos (zoonosis), en los que produce un

síndrome febril agudo severo.

Los síntomas producidos por la brucelosis suelen solaparse con aquellos producidos por otros

agentes etiológicos, por lo que es alta la probabilidad de diagnóstico incorrecto basado

únicamente en la sintomatología, lo que puede conducir a la administración de tratamientos

ineficaces a los pacientes.

El trabajo de investigación mencionado propone un método de diagnóstico de la brucelosis

que consiste en los siguientes pasos:

1°-Toma de muestras de sangre de pacientes con síntomas de síndrome febril agudo.

2°-Extracción de ADN del suero de las muestras.

3°-Utilización del ADN extraído en el paso anterior como molde en una reacción de PCR con

primers diseñados para amplificar un fragmento de 223 pb del gen bcsp31, específico de

bacterias del género Brucella.

2.1 Represente en el siguiente esquema de un gel de agarosa el patrón de bandas esperado al

realizar la detección por PCR de brucelosis. Considere que las muestras de los pacientes A

y C resultaron positivas para la presencia de Brucella y que la muestra del paciente B

resultó negativa. (M: marcador de peso molecular).

2Kamal, I. H., Gashgari, B. Al, Moselhy, S. S. & Abdullah, T. "Two-stage PCR assay for detection of human

brucellosis in endemic areas". BMC Infecttious Dis. 13, 145 (2013)



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2.2 ¿Qué tipo de muestra podría utilizarse como control positivo? ¿Y como control

negativo?¿Cuál es la utilidad de incluir controles positivos y negativos en la reacción de

PCR?

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2.3 Explique cuál es el fundamento de la especificidad de la técnica. ¿Podría esta técnica dar

un falso positivo si hubiera ADN del paciente en la muestra de suero?

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Cuatro especies de bacterias del género Brucella son patogénicas para humanos, siendo las

más prevalentes B.meltiensis (transmitida por ganado caprino y ovino) y B.abortus (transmitida

por bovinos).

Determinar cuál es la especie de Brucella responsable de la enfermedad de cada uno de los

pacientes puede brindar información epidemiológica que permita elaborar estrategias de

prevención primaria. Con ese objetivo, los autores del trabajo de investigación mencionado,

desarrollaron una segunda reacción de PCR para realizar con aquellas muestras que hubieran

resultado positivas para la presencia de Brucella. Para ello, realizaron una PCR multiplex: una

variante de la técnica que PCR que utiliza varios pares de primers simultáneamente en una

misma reacción. En este caso, se utilizaron primers para amplificar un producto de 113 pb,

específico de B.abortus y primers diferentes para amplificar un producto de 252 pb específico

de B.melitensis.

2.4 Interprete la siguiente figura extraída del trabajo de investigación y determine para cada

uno de los pacientes (del 1 al 9) la(s) especie(s) de Brucella encontradas en sus muestras.

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PARTE 3: Técnica de PCR (Reacción en cadena de la polimerasa).

Aplicaciones en medicina II: Detección de mutaciones.

La distrofia muscular de Duchenne (DMD) es una enfermedad genética provocada por

mutaciones en el gen de la distrofina, localizado en el cromosoma X. El curso de la enfermedad

–que afecta frecuentemente a varones- se caracteriza por atrofia y debilidad musculares

progresivas. En muchos pacientes las mutaciones encontradas son deleciones de exones

completos del gen de distrofina. Poblacionalmente ha podido determinarse que el exón 49 –

que posee una longitud de 87 pb- está delecionado en un elevado número de pacientes.

Considere la estrategia de detectar la presencia o ausencia de la deleción del exón 49 del gen

de distrofina mediante la técnica de PCR. Para ello, analice el siguiente esquema de la región

de interés del gen de distrofina en el que se indican los sitios de hibridación de los primers (P1

y P2). Los exones se representan mediante bloques numerados y los intrones con líneas

delgadas:

3.1 Un niño de 5 años es diagnosticado con DMD y se ha determinado que posee una deleción

del exón 49 en el gen de distrofina. Para determinar la presencia o ausencia de esa

mutación en otros miembros de la familia se realiza una PCR con la estrategia mencionada

anteriormente, a partir de ADN extraído de muestras de sangre periférica. Interprete los

resultados obtenidos para cada miembro de la familia y dibuje el patrón esperado de

bandas obtenidas para el paciente diagnosticado con DMD.

(NOTA: Considere que los individuos de sexo masculino poseen un único cromosoma X

en su genoma, mientras que los individuos de sexo femenino poseen dos)

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Durante la cursada de Genética se analizarán otras aplicaciones de la PCR

en el diagnóstico de mutaciones



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Para seguir practicando:

A continuación se muestran ejemplos de ejercicios tomados en parciales de años anteriores

respecto de los contenidos abordados en el Seminario de Integración I y el Taller I.

Indique la respuesta correcta (sólo una opción es correcta):

1- En el proceso de replicación de ADN eucariota se observa que: A: Puede iniciarse en cualquier posición de las secuencias de ADN B: Se caracteriza por ser un proceso mayormente dispersivo C: Oligonucleótidos de ARN actúan como cebadores (primers) en ambas cadenas D: Los telómeros son replicados usando como molde una secuencia de ADN conservada 2- Las ADN polimerasas δ y ε que participan en el proceso de replicación del ADN: A: Añaden desoxinucleótidos solamente en el extremo 3´ B: Inician la polimerización independientemente de la presencia de cebador (primer) en la cadena adelantada C: La polimerización del ADN procede en dirección 3´-5´ mediante uniones fosfodiéster D: Carecen de actividad 3´exonucleasa 3- La reacción en cadena de la polimerasa (PCR) se diferencia de la replicación in vivo de ADN de

eucariotas en que en la PCR: A: Se utilizan cebadores (primers) que son oligonucleótidos de ARN B: La enzima Taq polimerasa no introduce errores en el copiado C: El incremento de temperatura permite la desnaturalización de las hebras de ADN D: Requiere de una helicasa y ligasa distinta a la replicación in vivo

RESEARCH ARTICLE Open Access

Two-stage PCR assay for detection of humanbrucellosis in endemic areasIbrahim Hassan Kamal1,2*, Basim Al Gashgari1, Said Salama Moselhy1,2, Taha Abdullah Kumosani1,3

and Khalid Omar Abulnaja1

Abstract

Background: Brucellosis is a common zoonosis that can cause a severe febrile illness in humans. It constitutes apersistent health problem in many developing countries around the world. It is one of the most frequentlyreported diseases in Saudi Arabia and incidence is particularly high in the Central region, and around the city ofRiyadh. The aim of this study was to evaluate a two-stage PCR assay for detection of human brucellosis particularlyin endemic areas.

Methods: A total of 101 serum samples were collected from patients with acute febrile illness (AFI) of unknowncause from two different locations in the Western region of Saudi Arabia. The first location (Northern) ischaracterized by a nomadic rural population while the second (Central) is a modern urban city. All samples weresubjected to DNA extraction and Brucella genus-specific PCR amplification using B4/B5 primers of the bcsp31 gene.Positive B4/B5 samples were subjected to multiplex species-specific Brucella PCR amplification.

Results: In the Northern location, 81.9% of the AFI samples were confirmed Brucella positive, while all the samplescollected from the Central region proved to be Brucella negative. Samples positive for Brucella were subjected tomultiplex species-specific Brucella amplification. B. abortus was detected in 10% and B. melitensis in 8% of thesamples, while the majority (82%) of samples showed both B. abortus and B. melitensis. As expected, B. suis was notdetected in any of the samples.

Conclusions: This study concluded that a two-stage PCR assay could be useful as a rapid diagnostic tool to allowthe consideration of brucellosis as a possible cause of AFI, particularly in non-urban locations. It also recommendsthe collection of epidemiological data for such patients to obtain further information that may help in rapiddiagnosis.

BackgroundThe etiology and incidence of acute febrile illness (AFI)represents a major public health problem because clin-ical diagnosis is usually unreliable, and diagnostic testsare often not available in disease endemic areas [1]. Sur-veillance based on symptoms alone frequently results inclassification errors, because febrile illnesses resultingfrom different pathogens may be clinically indistinguish-able. Ideally a good surveillance system should be sup-ported by modern molecular diagnostic tests and be

sensitive and specific enough to accurately reflect thecauses of febrile illnesses in a population. Brucellosis is asevere acute febrile disease caused by Gram-negativebacteria of the genus Brucella. It is the cause of a widerange of significant veterinary and public health prob-lems, and economic loss [2]. The eradication of humanbrucellosis is difficult and the disease has a serious med-ical impact worldwide [3]. The acute febrile clinicalsymptoms of brucellosis always overlap with those ofother etiological pathogens, and this may lead to misdiag-nosis as well as improper antibiotic treatment regimes.Human brucellosis is one of the most frequently

reported diseases in Saudi Arabia, particularly in theCentral region and around the city of Riyadh [4-10].Since brucellosis is a zoonotic disease, it is transmittedfrom animals to humans by direct contact with infected

* Correspondence: [email protected] Department, Faculty of Science, King Abdulaziz University,Jeddah, Saudi Arabia2Biochemistry Department, Faculty of Science, Ain Shams University, Cairo,EgyptFull list of author information is available at the end of the article

© 2013 Kamal et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

Kamal et al. BMC Infectious Diseases 2013, 13:145http://www.biomedcentral.com/1471-2334/13/145

ANEXO, TALLER 1 BIOLOGÍA CELULAR Departamento de Biología Celular e Histología

1° Unidad Académica, FMED, UBA

animals or consumption of raw animal products such asunpasteurized milk or cheese. Direct contact with infectedanimals, their secretions or their carcasses can lead to in-fection through inhalation or accidental skin and mucousmembrane penetration [11,12]. In Saudi Arabia, brucel-losis has been recognized as a major health problem, andmeasures to control the disease were implemented asearly as 1983 [13].Four species of the genus Brucella are pathogenic for

humans, namely B. melitensis (from sheep and goats), B.abortus (from cattle and other bovidae), B. suis (frompigs), and B. canis (from dogs) [14]. Queipo-Ortuno andcoworkers [15] found 100% sensitivity and 98.3% specifi-city using the B4/B5 primer pair amplifying a 223 bpfragment of the bcsp31 gene, compared with 70% sensi-tivity for diagnosis by blood culture. PCR identificationof Brucella strains at the species or biovar level has beendescribed by Redkar et al. [16], who developed a real-time PCR assay for the detection of B. abortus, B.melitensis, and B. suis biovar 1. These PCR assays targetthe specific integration of IS711 elements within thegenome of the respective Brucella species or biovar.In most developing countries, especially in non-urban

areas, real-time PCR facilities are not available as a diag-nostic tool for human brucellosis. Most diagnostic la-boratories still rely on routine laboratory tests such asbacterial culturing and serological tests, even thoughthermal cyclers may be available. In this study, we tookthe initiative to evaluate a two-stage PCR assay as arapid sensitive diagnostic tool for diagnosis of humanbrucellosis, to highlight the need to consider brucellosisin the differential diagnosis of AFI, particularly in non-urban areas where patients are known to have a risk ofexposure and Brucella incidence is expected to be high.Two different locations in the Western region of Saudi

Arabia were selected to test the two-stage PCR strategy.The Northern location is characterized by a nomadic,mostly Bedouin population who consume unpasteurizeddairy products and ingest fresh camel, goat and sheepmilk. The Central location was in a modern urban citywhere the Bedouin population is low and the chances ofusing unpasteurized dairy products or direct exposure toanimals are limited. These two locations were selectedto test the likelihood of Brucella infection being a majorcause of AFI especially in rural locations, where the life-style of the population allows contact with Brucella-infected animals.

MethodsSubjectsA total of 101 serum samples were collected from thetwo selected locations in the Western region of SaudiArabia. In the Northern region, samples were from theArmed Forces hospital of Tabuk during the period June

2009 to January 2011, and in the Central region sampleswere collected at King Abdulaziz Hospital of Jeddah dur-ing the period November 2009 to November 2011.Serum was obtained from patients aged 1 year or oldersuffering from unknown fever of more than 2 days’ dur-ation who sought medical help in hospital. AFI wasdefined as a body temperature ≥ 38°C at the time of collec-tion, or fever of more than 2 days, and no other identifiedcause of fever such as diarrhea, hepatitis or any respiratorytract infections. Control serum samples were collectedfrom 20 healthy volunteers from the same locations. Nofamily history or any occupational exposure to Brucellainfection was recorded for the healthy controls. All partic-ipants meeting inclusion criteria were asked to participatein this study. Informed written consent was obtained fromadult participants and parents of minors.All samples were subjected to DNA extraction as pre-

viously described [17], with minor modifications as fol-lows: 1% of sodium dodecyl sulfate (SDS) and 10 mg/mlof proteinase K were added to 300 ul of serum and incu-bated for 2 h at 37°C. Proteinase K in the digest wasinactivated by heating at 90–95°C for 10–l5 min. Afterphenol-chloroform-isoamyl alcohol extraction and etha-nol precipitation, DNA was dissolved in 50 ul ofnuclease-free water.

Brucella genus-specific DNA amplificationTo diagnose the Brucella positive samples, the first PCRamplification was carried out using primers designed totarget a 223 bp fragment of the bcsp31 gene. This se-quence encodes an immunogenic membrane protein of a31 kDa antigen of B. abortus and is conserved in allBrucella biovars [18]. A pair of 21-nucleotide primers, B4(50 TGG CTC GGT TGC CAA TAT CAA 30) and B5 (50

CGC GCT TGC CTT TCA GGT CTG 30), were obtainedfrom Bioline, Inc., (Taunton, MA, USA), as described byBaily et al. [19]. PCR was performed in a 25 ul mixturecontaining template DNA; PCR buffer (10 mM Tris HCl[pH 8.4], 50 mM KCl, 1.5 mM MgCl2); 10 pmol of eachprimer; 200 uM (each) of dATP, dCTP, dTTP and dGTP(Bioline, Inc.), and 1.25 U of Taq polymerase (Qiagen,Chatsworth, NJ, USA). The cycle consisted of a preheatingstep at 95°C for 5 min followed by 35 cycles of 90°C for1 min, 60°C for 30 s, and 72°C for 1 min with a final incu-bation at 72°C for 10 min. A positive control based onDNA from a B. abortus reference strain was included inall tests, as well as a negative control containing all of thecomponents of the reaction mixture except DNA. 20% ofeach PCR product was visualized on a 1% agarose gelstained with 2 ug/ml of ethidium bromide.

Multiplex species-specific Brucella DNA amplificationAll samples positive using the B4/B5 primers weresubjected to multiplex PCR to determine which Brucella

Kamal et al. BMC Infectious Diseases 2013, 13:145 Page 2 of 5http://www.biomedcentral.com/1471-2334/13/145



species might be causing the infection. Species-specificDNA segments of B. abortus, B. melitensis and B. suiswere targeted for amplification using specific primersderived from the IS711 element [20]. The forward pri-mer (50 CAT GCG CTA TGT CTG GTT AC 30) spans803 to 823 nt of IS711 and generates a 113 bp PCRproduct with B. abortus reverse primer (50 GGC TTTTCT ATC ACG GTA TTC 30), 252 bp PCR product withB. melitensis reverse primer (50 AGT GTT TCG GCTCAG AAT AAT C 30), and 170 bp product with B. suisreverse primer (50 ACC GGA ACA TGC AAA TGA C30). Amplification conditions were the same as for thefirst PCR, except for the use of an annealing temperatureof 58°C. Positive and negative PCR controls were used inall tests. B. suis primers were used as an internal nega-tive PCR control. B. suis is pathogenic to pigs, which arenot found in Saudi Arabia. PCR products were visualizedon a 1% agarose gel as previously described.

ResultsA total of 101 AFI patients were enrolled in this study inthe Western region of Saudi Arabia; 61 and 40 from theNorthern and Central locations, respectively. Their char-acteristics are presented in Table 1. Forty-four samples(72%) from the Northern location were serologicallypositive for Brucella with varying titers (data not shown).All samples were subjected to Brucella genus amplifica-tion using B4/B5 primers that amplify a conserved re-gion in all Brucella species to detect the presence ofBrucella DNA as one of the possible causes of the AFI.Agarose gel electrophoresis of the B4/B5 conventional

PCR amplification gave a product size of 223 bp, indicat-ing the presence of Brucella genus in these patients. ThePCR control of the Brucella reference strain amplified aproduct of a similar size. No amplification was detectedin the negative PCR control, or in the negative controlsubjects. Conventional PCR confirmed that 50 samples(81.9%) were diagnosed as Brucella positive out of the61 samples collected from the Northern location, leaving11 (18%) patients whose AFI was of non-Brucella origin.No human brucellosis was detected in the 40 samplescollected from the Central location.DNA from the 50 Brucella positive patients were

subjected to the species-specific multiplex PCR. Multiplex

PCR electrophoresis results are shown in Figure 1, whichillustrates the presence of 113 bp and 252 bp bands spe-cific for B. abortus and B. melitensis, respectively. Amongthe 50 samples, B. abortus alone was evident in five sam-ples (10%) and B. melitensis alone in four samples (8%),while the rest of the samples (82%) showed products ofboth B. abortus and B. melitensis (Table 2). The B. suisamplification product (170 bp) was not detected in any ofthe samples. These results confirmed the specificity andsensitivity of these primers for the targeted region inBrucella DNA.

DiscussionAFI still represents a common clinical syndrome amongpatients seeking hospital care. Brucella is one of a num-ber of pathogens causing febrile illness, and is a seriouspublic health problem in many developing countries,including Saudi Arabia; where many people, by theirtraditional lifestyle, consume raw milk or have close ani-mal contact [21]. The true human brucellosis incidencehas been estimated to be between 10 and 25 timeshigher than the number of annual reported cases [22].Diagnosis of human brucellosis in Saudi Arabia cur-

rently depends mainly on culture [23] and serologicaltests [24]. Brucella is a highly virulent bacterium and mayconstitute exposure hazards for laboratory personnel. Fur-thermore, its culture is time consuming and the isolationrate is low, which may cause critical diagnostic delays [25].At the early stage of infection, the sensitivity of serologictests is low and false-negative or only weak-positive reac-tions may occur [26]. Because of limitations of culture tech-niques and serological tests, various molecular methods,particularly PCR, have been developed for rapid identifica-tion of organisms in clinical samples. The PCR techniquehas proved to be a very useful, simple, quick, sensitive, spe-cific and relatively inexpensive technique that merits itsadoption in clinical laboratories. Several articles have beenpublished dealing with various PCR-based methods forBrucella detection.In this study, a two-stage PCR assay was tested. The

genus-specific PCR assay, which targets the 223 bpsequence of the gene encoding a 31 kDa Brucellaabortus antigen [19], and the multiplex amplification forthe identification of Brucella to the species level called

Table 1 Characteristic features of patients with AFI

Characteristic Values

Locality at Western region

Northern Central

No. of AFI samples collected 61 40

No. of Brucella seropositive samples (data not shown) 44/61 00/40

No. of samples subjected to conventional PCR (B4/B5) amplification 61/61 40/40

No. of samples subjected to species-specific PCR 50/61 00/40




AMOS PCR for B. abortus, B. melitensis, B. ovis and B.suis [20], were carried out on 110 Saudi Arabian serumsamples from patients with AFI. Brucella genus-specificB4/B5 primers detected the presence of the predicted223 bp fragment in 81.9% of serum samples collectedfrom the Northern location, but did not detect anyBrucella cases out of the 40 samples collected fromCentral location. These results indicated that usingserum as a clinical sample and the two PCR sequentialassays provided a sensitive assay for diagnosis of humanbrucellosis. Similar results were reported by Elfaki andcoworkers [27], who reported the presence of the samePCR fragment (223 bp) in 96% of the sera samples from25 patients with symptoms of brucellosis from two refer-ence hospitals in Central Saudi Arabia.The Brucella species-specific multiplex PCR classified

the Brucella genus positive samples into single B. abor-tus or B. melitensis or double infection (both B. abortusand B. melitensis), which represented the majority ofcases (81%). B. suis primers failed, as predicted, sinceswine are not domestic in Saudi Arabia. Similar resultswere previously recorded in Saudi Arabia [27].Our results support other studies [27,28], which

recommended the use of PCR as the diagnostic tool ofchoice for human brucellosis. Since the samples in thisstudy were collected randomly with very limited casehistories, we could not classify the brucellosis as acute,chronic or relapsing cases.The existence of the double product may be attributed

to active double infection or to the coexistence of freeDNA of one or both species in the tested samples. The

free DNA in serum may reflect the degradation ofBrucella cells during the bacteremic phase of infection[27]. Double infection may be attributed to keeping live-stock of different species. The incidence of animalbrucellosis in the Saudi Arabia Makkah region was pre-viously found to be 0.8% in goats, 0.5% in sheep, 2.8% incamels and 3.6% in cows [29]. Ten years later in the Asirregion, it had risen to 18.2% in goats, 12.3% in sheep,22.6% in camels and 15.5% in cows [30].Our results succeeded in highlighting the differences

between the two selected locations in this study, wherethe non-urban area (Northern location) showed a highincidence of human brucellosis among the AFI patients,which could be due to probable exposure to infecteddairy products or direct contact with infected animalscommon in the nomadic Bedouin population lifestyle insuch rural locations. On the other hand, our assay failedto detect any brucellosis in samples collected from theurban city (Central location), where the Bedouin popula-tion is limited and exposure risk is low.As mentioned by Dean and co-authors [31], health

service inadequacies are compounded by socioeconomicfactors, with brucellosis affecting poor, marginalizedcommunities who often do not have the means to seektreatment. A study conducted in rural Tanzania revealedthat 1 in 5 patients did not present to a health center forassessment until more than 1 year after the onset of ill-ness. As a result of false-negative results, 44.8% brucel-losis cases were not diagnosed at the hospitals on theirfirst visit. These cases were treated for other diseases suchas malaria, which is much more common in the rural areathan brucellosis, and the brucellosis remained untreated[32]. Given the high proportion of brucellosis cases withfever, brucellosis should be considered as a differentialdiagnosis for fevers of unknown origin. Many patients fromnon-urban areas do not report to healthcare facilities.

ConclusionsThis study concluded that a two-stage PCR assay couldbe useful as a rapid diagnostic tool to highlight the needto consider brucellosis as a possible cause of AFI, par-ticularly in non-urban locations.The two-stage PCR assay minimizes exposure risks to

laboratory personnel for this virulent bacterium and alsoshortens the diagnostic time. It may also be consideredas an epidemiological tool for disease confirmation,

1 2 3 4 5 6 7 8 9 M +ve -ve

Figure 1 Agarose gel electrophoresis of species-specificmultiplex assay products. Lane #1 B. melitensis only (252 bp).Lanes 5 and 6, B. abortus only (113 bp); lanes 2, 3, 4, 7, 8, and 9,both of B. abortus and B. melitensis (113 bp and 252 bp respectively).Lane (+ve) and (−ve) contain positive and negative PCR controls.Lane (M) contains a 100-bp ladder (HyperLadder, Bioline, Inc.,MA, USA).

Table 2 Brucella DNA amplification using B4/B5 and species-specific multiplex PCR

(AFI) Samples locality Brucella genus B4/B5 conventional PCR Brucella species-specific multiplex

(Western region)(number)

-ve amplification +ve amplification Double product Single product Single product

Non- Brucella patients Brucella patients B. abortus & B. melitensis B. abortus B. melitensis

(Northern region) (61) 11 50 41 (82%) 5 (10%) 4 (8%)

(Central region) (40) 40 ————— Not applicable




tracing Brucella spp. transmission and identification ofinfection sources.The study recommends that healthcare authorities de-

termine the patient’s geographic location, lifestyle, age,gender, occupational exposure, food consumption, otherhealth conditions (antibiotic treatment) and family his-tory. This information plus species-specific diagnosis isuseful for improving the diagnostic capacity, reducingthe diagnostic delay, introducing new treatment regi-mens and providing strategies to effectively cure eventhe most complex cases of brucellosis often seen in en-demic areas.

Consent statementInstitutional ethical approval for the study was obtainedfrom the Ethical Committee of King Abdulaziz University.

Competing interestsThe authors declared that they have no competing interests.

Authors’ contributionsIHK designed and performed the study experiments and was responsible forwriting the manuscript. BAG participated in the laboratory experiments. SSM,TAK and KOA were responsible for data management, and revised the draftcarefully for important intellectual content. All authors read and approvedthe final version of this manuscript.

AcknowledgementsThis project was funded by the deanship of Scientific Research (DSR), KingAbdulaziz University, Jeddah, under grant no. 03/079/429. The authors,therefore, acknowledge with thanks DSR technical and financial support.

Author details1Biochemistry Department, Faculty of Science, King Abdulaziz University,Jeddah, Saudi Arabia. 2Biochemistry Department, Faculty of Science, AinShams University, Cairo, Egypt. 3Experimental Biochemistry Unit, King FahdMedical Research Center (KFMRC), King Abdulaziz University, Jeddah, SaudiArabia.

Received: 8 October 2012 Accepted: 15 March 2013Published: 21 March 2013

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doi:10.1186/1471-2334-13-145Cite this article as: Kamal et al.: Two-stage PCR assay for detection ofhuman brucellosis in endemic areas. BMC Infectious Diseases 2013 13:145.




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