Evaluation of a Fluorescence-Based PCR Method for Identification of Serogroup A Meningococci

Abstract

Standard and fluorescence-based PCR assays were developed for the identification of serogroup A meningococci by detection of the mynA gene. This assay was evaluated using bacterial cultures but provides the sensitivity required for the detection of the mynA gene from bodily fluids during meningococcal disease.

Neisseria meningitidis is the major cause of meningitis and septicemia. Organisms of this species can be divided into antigenically distinct groups known as serogroups, of which at least 13 are currently known. N. meningitidis serogroups B and C currently predominate throughout the United Kingdom, Central Europe, and the United States (1, 4, 7), whereas serogroup A is rare in Western countries. However, serogroup A remains endemic in many parts of the developing world, particularly in the “meningitis belt” spanning central Africa, and also in areas of China and eastern Europe. Epidemics can occur in such areas and are devastating, with annual incidence rates often exceeding 100/100,000 (6). We therefore developed standard PCR and dual-labeled endpoint fluorescence PCR (DEF-PCR) assays, with the capacity for high throughput, for the identification of serogroup A meningococci.

The capsules of members of N. meningitidis serogroup A differ from those of members of serogroups B, C, Y, and W135, the other disease-associated serogroups. The capsular polysaccharide of serogroup A isolates is composed of repeating units of (α1→6)-linked-N-acetyl-d-mannosamine-1-phosphate (8). In contrast, the capsular polysaccharides of isolates of serogroups B, C, Y, and W135 are all composed of or contain sialic acid (3). The capsular polysaccharide of N. meningitidis serogroup A is encoded by an operon of four genes, mynA, -B, -C, and -D (formerly known as open reading frames 1 to 4) (8). Confusingly, these are also known as sacA to -D. However, this operon appears to be unique to members of N. meningitidis serogroup A.

The mynA gene product is responsible for the first biosynthetic step in the production of the serogroup A capsule and is probably therefore the most conserved gene in the operon. MynA primers were designed using the GeneFisher website(http://bibiserv.techfak.uni-bielefeld.de/genefisher/), and se-quences were compared against those listed in GenBank (www.ncbi.nlm.nih.gov/BLAST) to ensure a high level of specificity towards N. meningitidis serogroup A (Table 1). The DEF-PCR probe was labeled with TET reporter dye covalently linked to the 5′ ends and TAMRA quencher dye covalently linked to the 3′ ends. A collection of neisseriae and other related or clinically important bacteria was obtained from culture collections held at the National Reference Laboratories in Scotland, England, and Wales and from culture collections in Sweden and Norway, as well as from the Microbiology Department at Wishaw General Hospital, Wishaw, United Kingdom (Table 2 and Table 3). The collection of serogroup A meningococci represented a global spread of these isolates.

TABLE 1.

Primer and probe sequences for PCR and DEF-PCR assays

Primer or probe	Sequence
PCR primers
Forward (mynA-F)	5′ GTC TTA ACC GTC TTT GGC ACT 3′
Reverse (mynA-R)	5′ TGA GGC ATT ACC TAT CCC GTA 3′
Sequencing primers
Forward (mynA-FS)	5′ AGC ATT ACT GCA CAG CAT CG 3′
Reverse (mynA-RS)	5′ CAG TAG GTG CAA AAT GCC ACT 3′
DEF-PCR primers and probe
Forward primer (mynA-FF)	5′ AAC CCA ACC AGA GCC TAC AAG 3′
Reverse primer (mynA-RF)	5′ CTG CTT CAA TGT GGC CAA CAG 3′
Dye-labeled probe (mynA-P)	5′ TTT GCA GCT AGC CTT GCT GCA 3′

TABLE 2.

Bacterial strains used for determining the specificity of the mynA PCR assay

Organism (s)	No. (%) of isolates:
	Tested	Positive	Negative
N. meningitidis serogroup A	43	43 (100)
N. meningitidis serogroup B	8		8 (100)
N. meningitidis serogroup C	8		8 (100)
N. meningitidis serogroup Y	4		4 (100)
N. meningitidis serogroup W135	4		4 (100)
N. meningitidis serogroup Z1	2		2 (100)
N. meningitidis serogroup X	4		4 (100)
N. lactamica	8		8 (100)
N. mucosa	1		1 (100)
Moraxella spp.	8		8 (100)
Neisseria gonorrhoeae	8		8 (100)
Escherichia coli	5		5 (100)
Staphylococcus aureus	5		5 (100)
H. influenzae	2		2 (100)
Streptococcus pneumoniae	8		8 (100)
Group B streptococci	2		2 (100)
Total	120	43 (100)	77 (100)

TABLE 3.

Serogroup A meningococci used in the mynA PCR assay

Isolate	Lab no.	Year	Source
1	Z1466	1977	Australia
2	Z1506	1976	Brazil
3	Z4186	1990	Brazil
4	Z1269	1963	Burkina Faso
5	Z4099	1963	China
6	Z5043	1979	China
7	Z5035	1979	China
8	Z3906	1966	China
9	Z1534	1941	England
10	Z3771	1987	England
11	97-250767	1997	England
12	96-254609	1996	England
13	01-240815	2001	England
14	97-252274	1997	England
15	96-254609	1996	England
16	00-242137	2000	England
17	97-252274	1997	England
18	02-240706	2002	England
19	01-240815	2001	England
20	02-240706	2002	England
21	97-250767	1997	England
22	02-240707	2002	England
23	02-240707	2002	England
24	Z6244	1983	Gambia
25	Z5037	1985	Germany
26	Z3515	1990	Mali
27	Z5005	1967	Morocco
28	Z1275	1963	Niger
29	79-03901	1979	Scotland
30	79-09267	1979	Scotland
31	79-11005	1979	Scotland
32	81-01374	1981	Scotland
33	81-03307	1981	Scotland
34	81-38919	1981	Scotland
35	81-07534	1981	Scotland
36	82-01178	1982	Scotland
37	82-10284	1982	Scotland
38	83-16222	1983	Scotland
39	83-09976	1983	Scotland
40	83-44892	1983	Scotland
41	175/92	1992	Sweden
42	97/91	1991	Sweden
43	OR17387	1987	Sweden

Cultures of each strain were grown aerobically overnight on Columbia blood agar with horse blood or chocolate blood agar (Oxoid, Basingstoke, United Kingdom) at 37°C or at 37°C in 5% CO₂ for Neisseria spp. and Haemophilus influenzae. Crude DNA was extracted by suspending 10 single colonies in 0.5 ml of 18-MΩ sterile distilled water and heating to 100°C for 10 to 20 min, followed by centrifugation at 23,000 × g for 2 min. Supernatants were transferred into 1.8-ml non-cross-contamination tubes (Web Scientific, Crewe, United Kingdom) on a Roboseq E apparatus (MWG Biotech, Milton Keynes, United Kingdom). Standard PCR was set up as previously described (2, 5), except that each reaction was performed in a final volume of 25 μl consisting of 20 μl of 1.1 Reddymix PCR Master Mix (Abgene, Surrey, United Kingdom), 1 μl of each primer (1 pM final concentration) (MWG Biotech Ltd.), and 3 μl of crude DNA. PCR amplification was performed using a cycle of 95°C for 2 min and 45 cycles of 95°C for 1 min, 53°C for 1.5 min, and 72°C for 30 s, followed by a final extension at 72°C for 2 min.

For the DEF-PCR assays, the procedure was similar to that for standard PCR: each reaction was again performed in a final volume of 25 μl, but each reaction mixture consisted of 20 μl of 1.1 Reddymix PCR Master Mix (ABgene), 1 μl of each primer (1 pM final concentration), 1 μl of probe (0.5 pM final concentration), and 2 μl of crude DNA. The thermocycling conditions were 95°C for 2 min and 45 cycles of 95°C for 15 s, 53°C for 30 s, and 72°C for 2 min, followed by a final extension at 72°C for 3 min. To determine the assay cutoff value for negative and positive samples, five negative controls and one positive control were used. Negative controls consisted of PCR mix without target DNA but including primers, and the final volume was adjusted to 25 μl with sterile distilled water. The positive control consisted of PCR mix-2 μl of serogroup A meningococcal crude DNA extracted as described above.

The sensitivity of each PCR method was determined by serial dilution of N. meningitidis serogroup A cells in 18-MΩ sterile distilled water. Half of the total volume of the serial dilution was plated onto horse blood agar (Oxoid) and incubated overnight at 37°C. After incubation of the agar plates, the colonies were counted and the resulting data were evaluated in relation to the number of live bacteria in the original serial dilutions. Standard PCR and DEF-PCR assays were performed on the serial dilutions to determine the sensitivity of the mynA assay, and these were analyzed by gel electrophoresis on a 3% agarose gel, stained with ethidium bromide, and visualized using a UV transilluminator and a Fluoro 320 fluorescence reader (MWG Biotech Ltd.). Wavelengths of 485 and 20 nm and wavelengths of 530 and 25 nm were used for excitation and emission, respectively, to detect the fluorescence caused by TET. A total of 100 end point readings were taken from each well, and the averages were calculated using KC4 software (MWG Biotech Ltd.). The KC4 software was programmed to calculate a cutoff value of 1.4 standard deviations above the mean fluorescence for the five negative controls.

A total of 120 bacterial isolates, comprising 43 serogroup A meningococcal isolates, 30 meningococcal isolates of other serogroups, and 47 isolates of various nonmeningococcal species, were used (Table 2). Both the standard PCR and DEF-PCR assays were found to be 100% specific for the detection of N. meningitidis serogroup A (Table 2). The sensitivity of the mynA DEF-PCR assay was determined to be three viable cells (relating to three genome copies) on the basis of serial dilutions of serogroup A meningococci, but the sensitivity of the standard mynA PCR was determined to be only approximately 100 genome copies. The theoretical difference in sensitivity between the assays is therefore that the DEF-PCR assay is 30 times more sensitive than the standard PCR assay. Using oligonucleotide primers (Table 1), DNA sequencing of the mynA gene on either side of the region of the DEF-PCR probe was also performed on 33 of the 43 serogroup A meningococci (Table 3) to confirm that the nucleotides were conserved. Sequencing showed that the region where the probe annealed was 100% conserved among the strains analyzed (Fig. 1).

FIG. 1.

Nucleotide sequencing of the DEF-PCR probe region within mynA.

Because both assays were automated, they can also provide a high throughput of samples, although they can both be performed manually in a low-throughput setting. As previously described, DEF-PCR assays also eliminate the subjective visual reading of gels and exhibit greater sensitivity by providing a fluorescence value (5). DEF-PCR assays can also be performed with a manually operated fluorescence plate reader.

Standard PCR and DEF-PCR assays have therefore been developed for the identification of serogroup A meningococci. Due to their sensitivity, the assays can be applied to the detection of serogroup A meningococci in bodily fluids, although the DEF-PCR assay would be the more sensitive of the two. Both assays can be performed manually or automated and can therefore be applied in laboratories requiring either low or high throughput.