Revista Argentina de Microbiología (2010) 42: 4-10
Revista Argentina de Microbiología (2010) 42: 4-10
Preparation of a working seed lot of BCG and quality
control by PCR genotyping
A. TROVERO1*, C. ARGÜELLES1, A. CATALDI2
1Instituto Nacional de Producción de Biológicos (INPB), ANLIS “Dr. Carlos G. Malbrán”. Av. Vélez Sarsfield 563, (1281) Ciudad
Autónoma de Buenos Aires; 2Instituto de Biotecnología, CNIA-INTA Castelar, Prov. de Buenos Aires, Argentina.
*Correspondence. E-mail: firstname.lastname@example.org
The bacillus Calmette-Guérin (BCG) was obtained in 1920 after successive passages leading to the attenuation of a
Mycobacterium bovis strain. For the following 40 years, BCG had been replicated, resulting in substrains with genotypic
and phenotypic differences. Several genomic studies have compared two BCG strains, M. bovis and Mycobacterium
tuberculosis, and observed that deleted regions in the different strains could be related to differences in antigenic
properties. In this work, a working seed lot was obtained from a lyophilized secondary seed lot from the BCG Pasteur
strain 1173 P2 and genetically characterized. The genome was analyzed by PCR directed to five regions (RD1, RD2,
RD14, RD15, DU2), using the seed lot and different available strains as templates. No genetic differences were found
in the fragments studied as compared to the Pasteur strain. A total of 20 passages were carried out and no differences
were found in the size of the fragments amplified by PCR. In conclusion, this method allows to control a working seed
lot genotypically and to assess the stability of the BCG genome.
Key words: BCG strains, genome, working seed lot, PCR, quality control
Preparación de un lote semilla de trabajo de BCG y control de calidad por genotipificación por PCR. El bacilo
de Calmette-Guérin (BCG) se obtuvo en 1920, después de sucesivos pasajes que llevaron a la atenuación de una
cepa de Mycobacterium bovis. A lo largo de los 40 años subsiguientes la cepa BCG fue replicada y surgieron sub-
cepas con diferencias fenotípicas y genotípicas. Se realizaron varios estudios de comparación genómica de diferentes
cepas de BCG, M. bovis y Mycobacterium tuberculosis, y se observó que las deleciones de regiones en las diferentes
cepas podrían estar relacionadas con diferencias en las propiedades antigénicas. En este trabajo se describe la
preparación y caracterización genética de un lote semilla de trabajo obtenido a partir de un lote semilla secundaria
liofilizado de la cepa BCG Pasteur 1173 P2. Se analizaron por PCR cinco regiones (RD1, RD2, RD14, RD15, DU2) en
el lote semilla de trabajo utilizando como control las diferentes cepas disponibles. No se hallaron diferencias genéticas
en los fragmentos estudiados al comparar el lote semilla de trabajo con la cepa BCG Pasteur 1173 P2. Asimismo, se
efectuaron hasta 20 pasajes y no se encontraron diferencias en el tamaño de los fragmentos amplificados por PCR.
En conclusión, se ha puesto a punto un método que permite controlar el genotipo de un lote semilla de trabajo y
evaluar la estabilidad del genoma del BCG.
Palabras clave: cepas BCG, genoma, lote semilla de trabajo, PCR, control de calidad
The history of the bacillus Calmette-Guérin (BCG)
started in 1908 when Calmette and Guérin began their
work from a virulent strain of Mycobacterium bovis by
performing serial passages in a bile-containing medium.
For the following thirteen years, a total of 230 passages
were performed until the M. bovis strain lost its virulence
in animals. The original BCG strain had then been main-
tained and distributed worldwide, since 1921 for over 40
years (24). It was noted that several BCG strains main-
tained by continuous subcultures suffered phenotypic
changes (14, 28). In 1950, the World Health Organiza-
tion (WHO) gave the first recommendation for the pro-
duction and control of BCG vaccines. Since 1960, the
Pasteur strain has been freeze-dried, keeping the form of
a primary seed lot. There are heterogeneous phenotypic
characteristics in vitro and in vivo among the different
strains. Behr et al. (5) collected data on the distribution of
different strains. According to historical records, the first
distribution of a documented daughter strain is the BCG
Russia obtained in 1924. From then on, until its lyophili-
zation in 1961, BCG-Pasteur daughter strains were ob-
tained after 1173 passages, either directly or indirectly at
Quality control of BCG by PCR genotyping5
irregular times. After BCG Russia, the following strains were
obtained: Moreau (1925), Japan (1925), Sweden (1926)
and Park (1926 given to Phipps 1928) and Denmark (1931),
Tice (1934), Frappier (1937), Birkhaug (1946), Connaught
(1948 from Frappier), Prague (1947 from Denmark), Glaxo
(1954 from Denmark) and Pasteur (lyophilized in 1961 af-
ter 1173 serial passages) (5, 24).
Much later, the genome sequence of M. tuberculosis
(H37Rv and CDC1551) (13, 16) and the sequence of two
M. bovis strains (BCG Pasteur and AF2122/97) became
available (17). Since then, these strains have been used
as references in comparative genomic studies. The com-
parison of genomes between different strains and/or spe-
cies of mycobacteria has led to the identification of ge-
nomic differences that may explain the observed
phenotypic differences, such as host range, virulence and
pathogenesis. In addition, the analysis of deletions by
differential hybridization has allowed the construction of
a phylogenetic tree of the species of the M. tuberculosis
complex (10). The different BCG strains have been stud-
ied by comparative genomics, subtractive genomic hy-
bridization (11, 18, 22), or DNA microarray technologies
(6), generating deletions and insertions known as regions
of difference (RD). Although the BCG genome has un-
dergone a number of deletions, it is 30 kb longer than the
wild type M. bovis AF2122/97, resulting from two tandem
duplications, DU1 and DU2. The comparison of the ge-
nome sequences of M. tuberculosis with those of M. bovis
and BCG Pasteur led to find 42 RD. These comprise 170
genes, of which BCG Pasteur has lost 133 (9).
Although new tuberculosis vaccines are currently un-
der investigation (1), BCG is the cornerstone of the strat-
egy to fight tuberculosis. The genetic differences between
BCG vaccine strains have renewed the interest in the in-
fluence of the vaccine strain on the protective efficacy
against tuberculosis. Although there is good evidence to
support the notion that the induced immune response and
protection afforded against tuberculosis differs between
BCG vaccine strains, there are currently insufficient data
to favour or recommend one particular strain (2, 4, 7, 8,
12, 20, 23, 26).
In our laboratory, the production of an intravesical BCG
for cancer has been attained from a lyophilized second-
ary seed lot Pasteur strain. The seed is cultivated in syn-
thetic liquid culture media (Sauton medium). The produc-
tion is performed according to the recommendations of
the WHO, with the “Seed Lot System”. To avoid the oc-
currence of mutants and possible changes in immuno-
genicity, it is recommended that no more than 12 pas-
sages should be performed in culture medium from the
primary seed lot. According to the WHO´s recommenda-
tions, the controls that should be performed are the mi-
croscopic observation of bacilli, Ziehl-Neelsen staining,
the determination of the aspect of the colonies, the exclu-
sion of bacterial or fungal contamination, and the absence
of virulent mycobacteria (30).
The aim of this study was to obtain a working seed lot of
BCG from a Pasteur 1173P2 secondary seed lot and to
perform a molecular characterization of the batch obtained,
since it may serve as a genetic quality control for future lots.
MATERIAL AND METHODS
Production of a working seed lot
The working seed lot was obtained by conventional cultivation
as a film surface in Sauton media, from strain BCG1173P2, with
only two passages, according to the recommendation of the WHO
(27, 30). The cultures were separated from their media by filtration
in a Birkhaug funnel and rinsed with sterile sodium glutamate
1.5% w/v. A total of 20 g of bacteria was transferred to another
flask with stainless steel balls to disperse the bacilli by rotation
of the flask for 25 minutes at 40 RPM. The bacillary mass was
then resuspended in 1.5% sodium glutamate at a concentration
of 20 mg/ml. These preparations were lyophilized and frozen at
-25 °C at vacuum. After 12 h, the secondary drying started at a
temperature of 18 °C, in a vacuum chamber and stored at 4 °C.
Connaught (Pasteur Merieux Connaught-Canada), Danish
(Staten Serum Institut-Copenhague), Pasteur 1173 P2 (Pasteur
Institute), Russia (BB-NCPID Ltd. Bulgaria), Glaxo (Glaxo
Laboratories), and M. bovis AN5.
Purification of genomic DNA
DNA was purified from BCG colonies on solid Lowenstein-
Jensen according to the method described by van Soolingen et
PCR was performed on fragments related to regions RD1,
RD2, RD14, RD15, duplications DU2-I, DU2-III and DU2-IV, and
the internal deletion of DU2, to distinguish different strains of
BCG. Primers (See Table 1) were used for the internal and
flanking regions of RD2 and RD14. For duplications, internal
primers with opposite direction were used. The nomenclature of
RD and duplicated regions was used as by Brosch et al. (9). For
preliminary genome comparisons between M. tuberculosis and
M. bovis BCG, web sites http://genolist.pasteur.fr/TubercuList/
and http://genolist.pasteur.fr/BCGList were used. Primers for RD
15 were designed using the Primer3 web site http://www-
genome.wi.mit.edu/cgi-bin/primer/primer3. A PCR was performed
with the different BCG strains and the working lot, and with
successive passages in Sauton liquid medium. The PCR reaction
was performed in a final volume of 50 µl with 1.25 U of Promega
Taq polymerase, 2 mM MgCl2, 0.05 mM dNTP and 50 nM of
each primer. The PCR conditions were 1 cycle of 96 °C (4 min),
29 cycles of 96 °C (1 min), between 50-60 °C [depending on the
annealing temperature (Ta) for each pair of primers, Table 1] for
1 min, 72 °C (2 min) and 1 cycle of 72 °C (10 min). The reaction
was carried out in a Perkin Elmer thermocycler. The PCR products
were analyzed by electrophoresis in horizontal 1.2% agarose
gels, with ethidium bromide.
Controls were performed according to the WHO stand-
ards: in the seed lot and other control BCG strains, acid-
fast bacilli in Ziehl-Neelsen staining with small clumps,
which are typical of BCG, were observed. In Lowenstein-
Jensen, the bacilli presented themselves as expanded
colonies without pigment and with slow growth. In the test
6Revista Argentina de Microbiología (2010) 42: 4-10
of purity in trypticase soy broth and thioglycolate broth,
there was no development of bacteria or fungi.
There was a survival of 60% of guinea pigs after in-
oculation with the BCG seed lot. The average initial weight
of the surviving guinea pigs was 398 g and the average
final weight 763 g. The average weight gain in six months
was 365 g. The guinea pigs were killed after six months
and no signs of tuberculosis were found. According to
the WHO recommendations the result was satisfactory.
RD1 region: all BCG strains studied gave an amplifica-
tion product of approximately 500 bp, with primers flanking
the RD1 region, indicating a deletion of RD1, whereas the
DNA of M. bovis wild type was not amplified (Figure 1).
RD2 region: only the Russia strain produced an am-
plification product of approximately 500 bp with primers
internal to the region, while the other strains did not show
such product, because they have a deletion of that re-
gion. With primers flanking the region, a band of approxi-
mately 1 kb confirmed the existence of a deletion of ap-
proximately 10 kb region of RD2 in all the strains, except
for the Russia strain, where the 1 kb band was not ob-
served (Figure 2).
RD14 region: the Pasteur strain and the working lot
gave a 500 bp fragment in the amplification with primers
flanking RD14, but gave no amplification with the internal
primers. Therefore, these strains have a deletion of the
RD14 region. The other strains gave a 470 bp fragment
with the internal primers, and no amplification with the
external ones. As previously described, these strains have
the RD14 region (Figure 3).
RD15 region: this region is present in all BCG strains
tested, except for the Connaught strain. These data are
Figure 1. PCR corresponding to the RD1 region. Lanes: 1- DNA
ladder, 2- BCG Connaugt, 3- BCG Danish, 4- BCG Pasteur,
5- BCG Glaxo, 6- BCG Russia, 7- Working seed lot, 8- M. bovis AN5.
Table 1. List of primers
RegionName Sequence Genomic
RD14 internal left
RD14 internal right CGTCGAATACGAGTCGAACA
RD14 left flank TTGATTCGCCAACAACTGAA
RD14 right flankGGGCTGGTTAGTGTCGATTC
RD15 internal leftCAGTTGGTGGGGTTGCTTG
RD15 internal right CGAGTGTGGAACGAAAGACG
57 Behr et al. (6)
RD253 Brosch et al. (10)
RD257 Brosch et al. (10)
RD1453 Brosch et al. (10)
RD1451 Brosch et al. (10)
RD15 59 Our design
DU2-I 51Brosch et al. (9)
DU2-III54Brosch et al. (9)
DU2-IV 54Brosch et al. (9)
A Int-DU2 54Brosch et al. (9)
(1) All coordinates relative to M. tuberculosis H37Rv
Quality control of BCG by PCR genotyping7
consistent with previous results (5, 6, 9), since there was
an amplification product of approximately 500 bp with
primers internal to the region in all the strains except for
Connaught (Figure 4).
DU2 group I duplication: the amplification with the Rus-
sia strain gave a 500 bp fragment. This coincides with
the existence of the duplication described by Brosch et
al. (9) in that strain. In the other strains, no band was
observed, thus indicating that there is no such duplica-
tion in these strains, including the working lot (Figure 5).
DU2 group III duplication: this amplification was ob-
served with the Danish and Glaxo strains, yielding a frag-
ment slightly smaller than 500 bp which corresponds to
the duplication described by Brosch et al. (9). There was
no amplification band in the working lot, the Pasteur, the
Figure 2. Left: PCR corresponding to the RD2 internal region. Right: PCR corresponding to the RD2 flanking region. Lanes:
1- BCG Connaught, 2- BCG Danish, 3- BCG Pasteur, 4- BCG Glaxo, 5- BCG Russia, 6- Working seed lot, 7- negative control,
8- DNA ladder.
Figure 3. Left: PCR corresponding to the RD14 internal region. Lanes: 1- BCG Connaught, 2- BCG Danish, 3- BCG Glaxo, 4- BCG
Pasteur, 5- BCG Russia, 6-Working seed lot, 7- negative control, 8- DNA ladder. Right: PCR corresponding to the RD14 flanking
region: Lanes: 1- BCG Connaught, 2- BCG Danish, 3- BCG Glaxo, 4- BCG Russia, 5- BCG Pasteur, 6- Working seed lot, 7- negative
control, 8- DNA ladder.
Figure 4. PCR corresponding to the RD15 region. Lanes:
1- BCG Connaught, 2- BCG Danish, 3- BCG Pasteur, 4- BCG
Glaxo, 5- BCG Russia, 6- Working seed lot, 7- DNA ladder;
8- negative control.
8Revista Argentina de Microbiología (2010) 42: 4-10
Connaught and the Russia strains, thus concluding that
there is no such duplication in these strains (Figure 6).
DU2 group IV duplication: in the amplification with the
Connaught and Pasteur strains, there is a 500 bp frag-
ment that corresponds to the duplication of the DU2 group
IV described by Brosch et al. (9). In the working seed lot
and the Pasteur strain, the band was observed, thus con-
cluding that the DU2 duplication is present in these strains
Internal deletion of DU2 duplication: with the primers
that flank the deletion, a fragment shorter than 500 bp
was amplified in all strains except in the Russia strain,
thus suggesting that in this region there is a deletion of
about 60 kb, as described by Brosch et al. (9) (Figure 8).
Figure 5. PCR corresponding to the DU2-group 1 duplication.
Lanes: 1- DNA ladder, 2- BCG Connaught, 3- BCG Danish,
4- BCG Russia, 5- BCG Glaxo, 6- BCG Pasteur 1173P2,
7- Working seed lot, 8- negative control.
Figure 6. PCR corresponding to the DU2-Group III duplication.
Lanes: 1- DNA ladder, 2- BCG Connaught, 3- BCG Danish,
4- BCG Russia, 5- BCG Glaxo, 6- BCG Pasteur 1173P2,
7- Working seed lot.
Figure 7. PCR corresponding to the DU2-Group IV duplication.
Lanes: 1- BCG Connaught, 2- BCG Danish, 3- BCG Russia,
4- BCG Glaxo, 5- BCG Pasteur 1173P2, 6- Working seed lot,
7- negative control, 8- DNA ladder.
Figure 8. PCR corresponding to internal deletion of DU2. Lanes:
1- DNA ladder, 2- BCG Connaught, 3- BCG Danish, 4- BCG
Russia, 5- BCG Glaxo, 6- BCG Pasteur 1173P2, 7- Working seed
lot, 8- negative control.
The results are summarized in Table 2.
Genomic stability after serial passages: PCR reactions
were tested with the working seed lot alter 3, 6, 10, 15
and 20 passages in Sauton medium and no differences
were observed when compared to the initial working seed
Phenotypic characterization: enzymatic biochemical
characterization was performed in the Pasteur BCG strain
and the seed lot, and both gave similar results. Catalase
at 25 °C and urease were positive, and the following tests
were negative: catalase at 68 °C, nitrate reductase, hy-
drolysis of Tween 80, β-glucosidase, β-galactosidase,
pyrazinamidase, arylsulphatase, and iron reduction. Sus-
ceptibility tests to antibiotics were also performed to the
Quality control of BCG by PCR genotyping9
Pasteur BCG and the seed lot strains by the proportion
method on Löwenstein Jensen medium. Both gave simi-
lar results, they were susceptible to: hydrazide of
phenocarboxylic acid 2 µg/ml, aminosalicylic acid 0.5 µg/
ml, isoniazid 0.2 µg/ml, streptomycin 4 µg/ml, ethambutol
2 µg/ml, and rifampicin 40 µg/ml; they were resistant to
cycloserine 30 µg/ml.
In this work, we produced and genetically character-
ized a working seed lot of BCG, which may be used in
the production of an intravesical BCG or intradermal vac-
cine. This methodology allows to standardize the pro-
duction process, with a limited number of passages. We
established a procedure with only two passages from
the seed working lot, and therefore, only four from the
Pasteur 1173P2 secondary seed lot, used to start the
The importance of reducing the number of passages
is that upon subculturing, the bacteria can undergo ge-
netic changes and, depending on the culture conditions,
mutants may be selected. In fact, this happened with the
original BCG strain that originated the different substrains
that are available at present. Although the WHO recom-
mends to limit the number of passages to 12 (30), it is
preferable to reduce them to 3 (31-33). In the laboratory
production of BCG during subcultures, changes, such as
deletions or duplications, may occur. These mutations can
alter the strain phenotypically and may be involved in the
virulence, survival, and induction of immune responses.
The attenuation is generated by deletions of virulence
genes (19, 21, 25). Reversing the mutation is impossible,
but mutations would further attenuate the strain and gen-
erate a decrease in protective efficacy. In order to avoid
the occurrence of mutants during the production process
and to reduce the number of passages, we produced a
seed working lot, which will be involved in the production
process with only two passages.
On the other hand, the seed working lot was geneti-
cally characterized, to assess genomic stability. We
analyzed regions that characterize the different strains
available by PCR. Five regions (RD1, RD2, RD14, RD15,
DU2) were analyzed and primers directed outward from
the DU2 duplicated region were used to find out whether
or not there was a duplication.
No changes were found in the working seed lot as
compared to the Pasteur strain in the regions studied or
after twenty successive passages.
We herein describe a PCR technique that is simple
and economical, and that can be performed in any qual-
ity control laboratory of biologics. A more thorough analy-
sis of the genome can be carried out with other molecular
biology techniques, such as microarray hybridization or
pulsed field electrophoresis (34). A multiplex PCR method
that analyzed six regions, but did not include the tandem
duplications, has been previously described (3). The
methodology presented here uses internal and flanking
primers, and allows to detect the presence of mixed cul-
tures with and without deletions.
Another advantage of PCR in this work is the discrimi-
nation of BCG strains. If we take into account that differ-
ent strains are available in the country for the BCG vac-
cine and bladder cancer treatment, it is important to have
a potential method for the identification and characteriza-
tion of BCG. In the case of any complication such as sep-
sis or infection in vaccinated patients or in patients with
bladder cancer treatment, the causative organism can be
identified because the method can distinguish different
BCG strains as well as M. bovis from M. tuberculosis.
In summary, a working seed lot was prepared and
genetically characterized, following the controls recom-
mended by the WHO (30) and the Argentine Pharmaco-
poeia (15), with satisfactory results.
Table 2. Results of the PCR amplification with the different BCG strains
PCRConnaught DanishRussiaGlaxo PasteurSeed lot
DU2- Internal deletion
(-) no amplification
10Revista Argentina de Microbiología (2010) 42: 4-10 Download full-text
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Recibido: 21/07/09 – Aceptado: 24/11/09