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Journal of Clinical Microbiology, May 1998, p. 1428-1429, Vol. 36, No. 5
Menzies School of Health Research, Darwin,
Australia
Received 25 November 1997/Returned for modification 22 January
1998/Accepted 4 February 1998
We describe a simplification of a highly discriminatory molecular
typing method, called Vir typing, for Streptococcus
pyogenes (D. Gardiner, J. Hartas, B. Currie, J. D. Mathews,
D. J. Kemp, and K. S. Sriprakash, PCR Methods Appl.
4:288-293, 1995). The procedure can be completed within a day, is
reproducible, and can be applied directly to colonies growing on
primary culture plates, allowing rapid establishment of strain identity
in an outbreak.
Streptococcus pyogenes
(group A streptococcus [GAS]) is historically typed based on the
antiphagocytic M protein. There are nearly 74 recognized M types
(11). However, a great majority of isolates collected from
regions of GAS endemicity, such as northern Australia, Malaysia, and
Thailand, could not be typed by the available sera (10, 12,
14) despite the presence of M protein in these nontypeable
strains (17). Several molecular typing methods for GAS have
been reported (3, 4, 7, 8, 13, 15). One of these, Vir typing
(8), is based on the restriction fragment length
polymorphism (RFLP) of a PCR-amplified region representing the
mga regulon of GAS. This locus encodes M and M-related
proteins, their positive regulator (the mga product), and
C5a peptidase (5, 6). This method is applicable to all isolates from diverse geographic origins. Vir typing has been found to
be highly discriminatory and very useful in epidemiological studies
(9).
The size of the PCR product is in the range of 4 to 7 kb. To
consistently obtain PCR products corresponding to the mga
regulon, template DNA was routinely prepared in gel blocks
(8). On average this procedure added a delay of at least 2 days to the time until typing results could be obtained. For a quick
epidemiological or hospital surveillance, this delay is not acceptable.
In our hands, a DNA template from boiled colony materials provided
variable results. Furthermore, the PCR using such templates often gave a background smear upon gel electrophoresis. We report here the use of
alkali lysis, which has been successfully used for PCR before
(2), to obtain good templates for long PCR.
Template DNA was obtained either by the procedure described by Gardiner
et al. (8) or by alkali lysis as described below. A single
colony from a plate containing Todd-Hewitt broth plus 0.2% yeast
extract in agar, a horse blood agar (HBA) plate, or a plate containing
HBA and colistin sulfate plus nalidixic acid (a primary plate used for
streptococcus/staphylococcus isolation) was picked up and suspended in
100 µl of 50 mM sodium hydroxide. The suspension was incubated at
95°C for 1 min, cooled to 4°C, and then neutralized with 16 µl of
1 M Tris-HCl (pH 8.0). After centrifugation for 2 min at high speed in
a microcentrifuge, the template was ready for the amplification
reaction. The PCR conditions were essentially as described before
(8). Briefly, the 50-µl reaction mixture contained 5 µl
of template DNA, 0.4 µM primers (VUF and SBR), 5 µl of the template
preparation, 200 µM deoxynucleotide triphosphates, and 0.2 µl of an
8:1 mixture of Taq and Pfu thermostable polymerases, in PC2 buffer (1). The reaction included 1 cycle at 95°C for 1 min and 30 cycles at 95°C for 15 s, 60°C
for 2 min, and 68°C for 6 min. The reaction product was digested at
37°C for 1 h by simply adding 2 U of HaeIII. RFLP was
determined by size fractionation by electrophoresis.
Fifty-two isolates corresponding to different Vir types (VTs) were
analyzed by the old and modified methods. In all cases, the two methods
gave identical results. A further 30 isolates were successfully typed
by the modified method only, yielding RFLP patterns identical to those
for members of the same VT typed by the old method. Thus, the
modification gave reproducible results. Comparison of the two methods
for a subset of 18 VTs is shown in Fig.
1. Colony material from all three media
could be used directly for Vir typing using this simplified protocol
(results not shown). Figure 1 presents results for colonies grown on
HBA plates. The presence of contaminants, such as hemoglobin (from the
HBA plate), other medium components, and other microbiological contaminants, on the primary plate did not interfere with the PCR.
Minor Staphylococcus contamination, often found in
impetiginous specimens, did not interfere. Prior to digestion with
HaeIII, the PCR product of GAS (3.5 to 7.5 kb) can be easily
distinguished from the products of other hemolytic streptococci, such
as those of groups G and C, which yield a much smaller product (1.5 to 1.8 kb) (16). Thus, this procedure is highly specific,
robust, and applicable to all GASs.
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Simplification of a Locus-Specific DNA Typing Method (Vir
Typing) for Streptococcus pyogenes
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FIG. 1.
Ethidium bromide-stained agarose gels containing
HaeIII-digested PCR products corresponding to the
mga regulon of GAS isolates. Lanes M contain lambda
HindIII markers. Band sizes, in kilobases, are indicated
in the left margin. The remaining lanes contain paired
preparations of the same local isolate representing a particular VT.
The VT is indicated by the number above the bar. The patterns in lanes
a are from template DNA obtained by the gel block method
(8), and those in lanes b are from template DNA obtained by
the alkali lysis method.
Alkali lysis is commonly used in plasmid DNA purification, wherein the large chromosomal DNA is removed with cell debris. The consistently successful amplification reported here suggests the presence in the supernatant of nicked chromosomal DNA possibly due to autolysis of some cells in a colony or due to large amounts of DNases characteristically present in streptococci.
With this simplification, Vir typing results could be obtained within 8 h. The template preparation could be stored frozen for at least 2 months without deterioration, as judged by the quality of the Vir typing profiles. With this proposed simplification, Vir typing is the fastest typing method for S. pyogenes. Moreover, there is a considerable cost savings in the preparation of the template. The universality of Vir typing, the limited number of reagents and their long shelf life, and the ease of interpretation of the RFLP patterns makes this a method of choice in routine epidemiological surveillance. This is particularly so in places such as Thailand, Malaysia, northern Australia, and India, where traditional M typing is not applicable or informative across the spectrum of endemic isolates.
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FOOTNOTES |
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* Corresponding author. Mailing address: Menzies School of Health Research, P.O. Box 41096, Causarina NT 0811, Australia. Phone: 61-8-89228599. Fax: 61-8-89275187. E-mail: jon{at}menzies.su.edu.au.
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Journal of Clinical Microbiology, May 1998, p. 1428-1429, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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