Técnica de PCR: Fundamentos y Aplicaciones

Vista previa del material en texto

Taller 1 Biología Celular. 
Departamento de Biología Celular e Histología 
1° Unidad Académica, FMED, UBA. 
1 
 
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. 
Taller 1 Biología Celular. 
Departamento de Biología Celular e Histología 
1° Unidad Académica, FMED, UBA. 
2 
 
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. 
 
Taller 1 Biología Celular. 
Departamento de Biología Celular e Histología 
1° Unidad Académica, FMED, UBA. 
3 
 
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). 
 
 
 
 
 
 
 
 
 
 
 
 
 
2
Kamal, 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) 
 
Taller 1 Biología Celular. 
Departamento de Biología Celular e Histología 
1° Unidad Académica, FMED, UBA. 
4 
 
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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Taller 1 Biología Celular. 
Departamento de Biología Celular e Histología 
1° Unidad Académica, FMED, UBA. 
5 
 
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 
Taller 1 Biología Celular. 
Departamento de Biología Celular e Histología 
1° Unidad Académica, FMED, UBA. 
6 
 
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 human
brucellosis in endemic areas
Ibrahim 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 a
persistent health problem in many developing countries around the world. It is one of the most frequently
reported diseases in Saudi Arabia and incidence is particularly high in the Central region, and around the city of
Riyadh. The aim of this study was to evaluate a two-stage PCR assay for detection of human brucellosis particularly
in endemic areas.
Methods: A total of 101 serum samples were collected from patients with acute febrile illness (AFI) of unknown
cause from two different locations in the Western region of Saudi Arabia. The first location (Northern) is
characterized by a nomadic rural population while the second (Central) is a modern urban city. All samples were
subjected 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 samples
collected from the Central region proved to be Brucella negative. Samples positive for Brucella were subjected to
multiplex species-specific Brucella amplification. B. abortus was detected in 10% and B. melitensis in 8% of the
samples, while the majority (82%) of samples showed both B. abortus and B. melitensis. As expected, B. suis was not
detected in any of the samples.
Conclusions: This study concluded that a two-stage PCR assay could be useful as a rapid diagnostic tool to allow
the consideration of brucellosis as a possible cause of AFI, particularly in non-urban locations. It also recommends
the collection of epidemiological data for such patients to obtain further information that may help in rapid
diagnosis.
Background
The etiology and incidence of acute febrile illness (AFI)
represents a major public health problem because clin-
ical diagnosis is usually unreliable, and diagnostic tests
are often not available in disease endemic areas [1]. Sur-
veillance based on symptoms alone frequently results in
classification errors, because febrile illnesses resulting
from 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 the
causes of febrile illnesses in a population. Brucellosis is a
severe acute febrile disease causedby Gram-negative
bacteria of the genus Brucella. It is the cause of a wide
range of significant veterinary and public health prob-
lems, and economic loss [2]. The eradication of human
brucellosis is difficult and the disease has a serious med-
ical impact worldwide [3]. The acute febrile clinical
symptoms of brucellosis always overlap with those of
other 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 the
Central region and around the city of Riyadh [4-10].
Since brucellosis is a zoonotic disease, it is transmitted
from animals to humans by direct contact with infected
* Correspondence: imbc57@yahoo.com
1Biochemistry Department, Faculty of Science, King Abdulaziz University,
Jeddah, Saudi Arabia
2Biochemistry Department, Faculty of Science, Ain Shams University, Cairo,
Egypt
Full 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 Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Kamal et al. BMC Infectious Diseases 2013, 13:145
http://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 as
unpasteurized milk or cheese. Direct contact with infected
animals, their secretions or their carcasses can lead to in-
fection through inhalation or accidental skin and mucous
membrane penetration [11,12]. In Saudi Arabia, brucel-
losis has been recognized as a major health problem, and
measures to control the disease were implemented as
early 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 (from
pigs), and B. canis (from dogs) [14]. Queipo-Ortuno and
coworkers [15] found 100% sensitivity and 98.3% specifi-
city using the B4/B5 primer pair amplifying a 223 bp
fragment of the bcsp31 gene, compared with 70% sensi-
tivity for diagnosis by blood culture. PCR identification
of Brucella strains at the species or biovar level has been
described 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 target
the specific integration of IS711 elements within the
genome 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 as
bacterial culturing and serological tests, even though
thermal cyclers may be available. In this study, we took
the initiative to evaluate a two-stage PCR assay as a
rapid sensitive diagnostic tool for diagnosis of human
brucellosis, to highlight the need to consider brucellosis
in the differential diagnosis of AFI, particularly in non-
urban areas where patients are known to have a risk of
exposure 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 unpasteurized
dairy products and ingest fresh camel, goat and sheep
milk. The Central location was in a modern urban city
where the Bedouin population is low and the chances of
using unpasteurized dairy products or direct exposure to
animals are limited. These two locations were selected
to test the likelihood of Brucella infection being a major
cause of AFI especially in rural locations, where the life-
style of the population allows contact with Brucella-
infected animals.
Methods
Subjects
A total of 101 serum samples were collected from the
two selected locations in the Western region of Saudi
Arabia. In the Northern region, samples were from the
Armed Forces hospital of Tabuk during the period June
2009 to January 2011, and in the Central region samples
were 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 older
suffering from unknown fever of more than 2 days’ dur-
ation who sought medical help in hospital. AFI was
defined as a body temperature ≥ 38°C at the time of collec-
tion, or fever of more than 2 days, and no other identified
cause of fever such as diarrhea, hepatitis or any respiratory
tract infections. Control serum samples were collected
from 20 healthy volunteers from the same locations. No
family history or any occupational exposure to Brucella
infection was recorded for the healthy controls. All partic-
ipants meeting inclusion criteria were asked to participate
in this study. Informed written consent was obtained from
adult 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/ml
of proteinase K were added to 300 ul of serum and incu-
bated for 2 h at 37°C. Proteinase K in the digest was
inactivated by heating at 90–95°C for 10–l5 min. After
phenol-chloroform-isoamyl alcohol extraction and etha-
nol precipitation, DNA was dissolved in 50 ul of
nuclease-free water.
Brucella genus-specific DNA amplification
To diagnose the Brucella positive samples, the first PCR
amplification was carried out using primers designed to
target a 223 bp fragment of the bcsp31 gene. This se-
quence encodes an immunogenic membrane protein of a
31 kDa antigen of B. abortus and is conserved in all
Brucella 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 obtained
from Bioline, Inc., (Taunton, MA, USA), as described by
Baily et al. [19]. PCR was performed in a 25 ul mixture
containing template DNA; PCR buffer (10 mM Tris HCl
[pH 8.4], 50 mM KCl, 1.5 mM MgCl2); 10 pmol of each
primer; 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 preheating
step at 95°C for 5 min followed by 35 cycles of 90°C for
1 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 on
DNA from a B. abortus reference strain was included in
all tests, as well as a negative control containing all of the
components of the reaction mixture except DNA. 20% of
each PCR product was visualized on a 1% agarose gel
stained with 2 ug/ml of ethidium bromide.
Multiplex species-specific Brucella DNA amplification
All samples positive using the B4/B5 primers were
subjected to multiplex PCR to determine which Brucella
Kamal et al. BMC Infectious Diseases 2013, 13:145 Page 2 of 5
http://www.biomedcentral.com/1471-2334/13/145
ANEXO, TALLER 1 BIOLOGÍA CELULAR Departamento de Biología Celular e Histología 
1° Unidad Académica, FMED, UBA
species might be causing the infection. Species-specific
DNA segments of B. abortus, B. melitensis and B. suis
were targeted for amplification using specific primers
derived from the IS711 element [20]. The forward pri-
mer (50 CAT GCG CTA TGT CTG GTT AC 30) spans
803 to 823 nt of IS711 and generates a 113 bp PCR
product with B. abortus reverse primer (50 GGC TTT
TCT ATC ACG GTA TTC 30), 252 bp PCR product with
B. melitensis reverse primer (50 AGT GTT TCG GCT
CAG AAT AAT C 30), and 170 bp product with B. suis
reverse primer (50 ACC GGA ACA TGC AAA TGA C
30). Amplification conditions were the same as for the
first PCR, except for the use of an annealing temperature
of 58°C. Positiveand negative PCR controls were used in
all tests. B. suis primers were used as an internal nega-
tive PCR control. B. suis is pathogenic to pigs, which are
not found in Saudi Arabia. PCR products were visualized
on a 1% agarose gel as previously described.
Results
A total of 101 AFI patients were enrolled in this study in
the Western region of Saudi Arabia; 61 and 40 from the
Northern and Central locations, respectively. Their char-
acteristics are presented in Table 1. Forty-four samples
(72%) from the Northern location were serologically
positive 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 of
Brucella 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. The
PCR control of the Brucella reference strain amplified a
product of a similar size. No amplification was detected
in the negative PCR control, or in the negative control
subjects. Conventional PCR confirmed that 50 samples
(81.9%) were diagnosed as Brucella positive out of the
61 samples collected from the Northern location, leaving
11 (18%) patients whose AFI was of non-Brucella origin.
No human brucellosis was detected in the 40 samples
collected 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, which
illustrates the presence of 113 bp and 252 bp bands spe-
cific for B. abortus and B. melitensis, respectively. Among
the 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 of
both B. abortus and B. melitensis (Table 2). The B. suis
amplification product (170 bp) was not detected in any of
the samples. These results confirmed the specificity and
sensitivity of these primers for the targeted region in
Brucella DNA.
Discussion
AFI still represents a common clinical syndrome among
patients seeking hospital care. Brucella is one of a num-
ber of pathogens causing febrile illness, and is a serious
public health problem in many developing countries,
including Saudi Arabia; where many people, by their
traditional lifestyle, consume raw milk or have close ani-
mal contact [21]. The true human brucellosis incidence
has been estimated to be between 10 and 25 times
higher than the number of annual reported cases [22].
Diagnosis of human brucellosis in Saudi Arabia cur-
rently depends mainly on culture [23] and serological
tests [24]. Brucella is a highly virulent bacterium and may
constitute exposure hazards for laboratory personnel. Fur-
thermore, its culture is time consuming and the isolation
rate is low, which may cause critical diagnostic delays [25].
At the early stage of infection, the sensitivity of serologic
tests 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 technique
has proved to be a very useful, simple, quick, sensitive, spe-
cific and relatively inexpensive technique that merits its
adoption in clinical laboratories. Several articles have been
published dealing with various PCR-based methods for
Brucella detection.
In this study, a two-stage PCR assay was tested. The
genus-specific PCR assay, which targets the 223 bp
sequence of the gene encoding a 31 kDa Brucella
abortus antigen [19], and the multiplex amplification for
the 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
Kamal et al. BMC Infectious Diseases 2013, 13:145 Page 3 of 5
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ANEXO, TALLER 1 BIOLOGÍA CELULAR Departamento de Biología Celular e Histología 
1° Unidad Académica, FMED, UBA
AMOS PCR for B. abortus, B. melitensis, B. ovis and B.
suis [20], were carried out on 110 Saudi Arabian serum
samples from patients with AFI. Brucella genus-specific
B4/B5 primers detected the presence of the predicted
223 bp fragment in 81.9% of serum samples collected
from the Northern location, but did not detect any
Brucella cases out of the 40 samples collected from
Central location. These results indicated that using
serum as a clinical sample and the two PCR sequential
assays provided a sensitive assay for diagnosis of human
brucellosis. Similar results were reported by Elfaki and
coworkers [27], who reported the presence of the same
PCR fragment (223 bp) in 96% of the sera samples from
25 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. abortus
and B. melitensis), which represented the majority of
cases (81%). B. suis primers failed, as predicted, since
swine are not domestic in Saudi Arabia. Similar results
were previously recorded in Saudi Arabia [27].
Our results support other studies [27,28], which
recommended the use of PCR as the diagnostic tool of
choice for human brucellosis. Since the samples in this
study were collected randomly with very limited case
histories, 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 free
DNA of one or both species in the tested samples. The
free DNA in serum may reflect the degradation of
Brucella cells during the bacteremic phase of infection
[27]. Double infection may be attributed to keeping live-
stock of different species. The incidence of animal
brucellosis in the Saudi Arabia Makkah region was pre-
viously found to be 0.8% in goats, 0.5% in sheep, 2.8% in
camels and 3.6% in cows [29]. Ten years later in the Asir
region, 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, where
the non-urban area (Northern location) showed a high
incidence of human brucellosis among the AFI patients,
which could be due to probable exposure to infected
dairy products or direct contact with infected animals
common in the nomadic Bedouin population lifestyle in
such rural locations. On the other hand, our assay failed
to detect any brucellosis in samples collected from the
urban 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 socioeconomic
factors, with brucellosis affecting poor, marginalized
communities who often do not have the means to seek
treatment. A study conducted in rural Tanzania revealed
that 1 in 5 patients did not present to a health center for
assessment 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 their
first visit. These cases were treated for other diseases such
as malaria, which is much more common in the rural area
than brucellosis, and the brucellosis remained untreated
[32]. Given the high proportion of brucellosis cases with
fever, brucellosis should be considered as a differential
diagnosis for fevers of unknownorigin. Many patients from
non-urban areas do not report to healthcare facilities.
Conclusions
This study concluded that a two-stage PCR assay could
be useful as a rapid diagnostic tool to highlight the need
to 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 also
shortens the diagnostic time. It may also be considered
as 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-specific
multiplex 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
Kamal et al. BMC Infectious Diseases 2013, 13:145 Page 4 of 5
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ANEXO, TALLER 1 BIOLOGÍA CELULAR Departamento de Biología Celular e Histología 
1° Unidad Académica, FMED, UBA
tracing Brucella spp. transmission and identification of
infection sources.
The study recommends that healthcare authorities de-
termine the patient’s geographic location, lifestyle, age,
gender, occupational exposure, food consumption, other
health conditions (antibiotic treatment) and family his-
tory. This information plus species-specific diagnosis is
useful for improving the diagnostic capacity, reducing
the diagnostic delay, introducing new treatment regi-
mens and providing strategies to effectively cure even
the most complex cases of brucellosis often seen in en-
demic areas.
Consent statement
Institutional ethical approval for the study was obtained
from the Ethical Committee of King Abdulaziz University.
Competing interests
The authors declared that they have no competing interests.
Authors’ contributions
IHK designed and performed the study experiments and was responsible for
writing the manuscript. BAG participated in the laboratory experiments. SSM,
TAK and KOA were responsible for data management, and revised the draft
carefully for important intellectual content. All authors read and approved
the final version of this manuscript.
Acknowledgements
This project was funded by the deanship of Scientific Research (DSR), King
Abdulaziz University, Jeddah, under grant no. 03/079/429. The authors,
therefore, acknowledge with thanks DSR technical and financial support.
Author details
1Biochemistry Department, Faculty of Science, King Abdulaziz University,
Jeddah, Saudi Arabia. 2Biochemistry Department, Faculty of Science, Ain
Shams University, Cairo, Egypt. 3Experimental Biochemistry Unit, King Fahd
Medical Research Center (KFMRC), King Abdulaziz University, Jeddah, Saudi
Arabia.
Received: 8 October 2012 Accepted: 15 March 2013
Published: 21 March 2013
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doi:10.1186/1471-2334-13-145
Cite this article as: Kamal et al.: Two-stage PCR assay for detection of
human brucellosis in endemic areas. BMC Infectious Diseases 2013 13:145.
Kamal et al. BMC Infectious Diseases 2013, 13:145 Page 5 of 5
http://www.biomedcentral.com/1471-2334/13/145
ANEXO, TALLER 1 BIOLOGÍA CELULAR Departamento de Biología Celular e Histología 
1° Unidad Académica, FMED, UBA
	TALLER 1 BC 2018 nueva version
	Brucelosis diagnostic PCR
	Abstract
	Background
	Methods
	Results
	Conclusions
	Background
	Methods
	Subjects
	Brucella genus-specific DNA amplification
	Multiplex species-specific Brucella DNA amplification
	Results
	Discussion
	Conclusions
	Consent statement
	Competing interests
	Authors’ contributions
	Acknowledgements
	Author details
	References