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Sequential Quadriplex Real-Time PCR for Identifying 20 Common emm Types of Group A Streptococcus

We developed a sequential quadriplex real-time PCR-based method for rapid identification of 20 emm types commonly found in invasive group A Streptococcus (iGAS) strains recovered through the Centers for Disease Control and Prevention’s Active Bacterial Core surveillance. Each emm real-time PCR assay showed high specificity and accurately identified the respective target emm type, including emm subtypes in the United States.


MATERIALS AND METHODS
Bacterial isolates. All bacterial isolates were cultured on Trypticase soy agar (TSA) with 5% sheep blood plates at 37°C with 5% CO 2 . Genomic DNA was extracted from isolates using the Qiagen DNA minikit (Qiagen, Valencia, CA) as previously described (12), with a prelysis step. Briefly, a loopful of bacteria grown overnight from a blood agar plate was suspended in 50 l of lysis solution (lysozyme, 0.08 mg/ml, and mutanolysin, 150 U/ml) and incubated at 37°C for 1 h, followed by steps described in the kit manufacturer's user manual.
Optimal concentrations for primers and probes were determined by using SYBR green and TaqMan real-time PCR methods, respectively. Optimal concentrations of each oligonucleotide set were optimized with various concentrations (100, 200, 300, 400, and 500 nmol/liter) of each oligonucleotide against 10-fold dilutions of DNA from targeted emm type isolates in a Stratagene Mx3005P real-time PCR instrument (Agilent, Santa Clara, CA). The concentrations were optimized to obtain the highest DNA dilution yielding a cycle threshold (C T ) value of Յ35. Lower limits of detection for each oligonucleotide set were determined in both singleplex and multiplex formats using 10-fold serial dilutions of the targeted emm type control DNA. In total, 20 individual assays targeting 20 emm types were grouped into five quadriplex reactions (Table 2) based on their distribution in the United States (10). The reaction mixture (25 l) contained 12.5 l of 2ϫ PerfeCTa multiplex qPCR supermix, QuantaBio (VWR, Radnor, PA), optimal concentrations of oligonucleotides (Table 1), and PCR grade water and DNA (5 l). Amplification was carried out with the following cycling conditions: 1 cycle of 95°C for 10 min and 40 cycles of 94°C for 15 s and 60°C for 1 min.
We also designed a synthetic DNA plasmid (Text S1) (Thermo Fisher Scientific; GeneArt), as described elsewhere (14)(15)(16), that was used as a positive control for all real-time PCRs. The synthetic DNA was designed to contain primer and probe binding regions for all 20 emm types, S. pyogenes-specific target spy as an internal positive control, and the Erwinia xeno assay for laboratory contamination control, all concatenated in a single plasmid (14).

RESULTS
The sequential real-time PCR scheme consisted of five 4-plex reactions to identify 20 emm types, inclusive of all known subtypes within each type (see ftp://ftp.cdc.gov/pub/infectious_diseases/biotech/tsemm/ for a current listing of emm subtypes). Optimal concentrations of both primers and probes (Table 1) were determined for each assay. Monoplex real-time PCR of DNA from 106 GAS isolates from the 20 emm types showed that the assays specifically amplified all subtypes assigned to the 20 emm types, with the caveat that the emm49 reaction coidentified the closely related deletion derivative of emm49, emm151 (Table 1). No amplification was observed for the additional 66 emm types or for any of the 4 GAS emm nontypeable isolates or the 44 non-S. pyogenes species used to challenge the specificity of the assays. When 4 assays were multiplexed together in 5 individual reactions, we also observed no cross-reaction with any of the other emm types/subtypes (Table S1) or the non-S. pyogenes species. The multiplex reactions did not produce any false-positive or falsenegative results. Among 209 clinical sterile-site GAS isolates (from ABCs) used in the validation, the real-time PCR emm typing assays correctly identified respective emm types and had 100% concordance with conventional PCR and sequencing and/or WGS results. While we only tested the subtypes for the 21 emm types (including emm151) that were available within ABCs, we infer based on in silico alignment of all available subtypes in the emm database for the primer and probe designs that the PCR assays would detect all known subtypes. The sensitivity of the real-time PCR assays was determined with 10-fold serial dilutions of DNA extracted from all 21 positive-control isolates used in the validation. All newly validated assays had high sensitivity of detection (Ͻ10 genome copies/assay) in both monoplex and multiplex formats.

DISCUSSION
The CDC's Streptococcus Lab serves as a reference laboratory for streptococci and assists clinical and state public health laboratories by providing support and characterization of GAS, particularly from LTCFs and SNFs, where outbreaks among residents commonly occur (2,3,17,18). Traditionally, PCR combined with sequencing (5) along with other genotyping methods, such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), have been extensively used to study the clonal relatedness of GAS isolates for epidemiological investigations (3,17). Recently, WGS has been used for higher discriminatory genetic analysis using single nucleotide polymorphisms (SNPs) to increase resolution in GAS outbreak investigations (2,18). While all these methods provide valuable information, they either are costly or take several days to complete, and many use equipment and reagents not readily available in most laboratories. The real-time-PCR-based emm typing method developed in this study has an advantage over other methods in that it is more rapid, with results generated in a few hours rather than days. Our method identifies four emm types in a single reaction and utilizes a sequential multiplex-approach which does not require post-PCR steps for confirmation, saving time and cost. Furthermore, it employs real-time PCR, which is a technology available in many clinical, public health, and reference laboratories. This approach can provide information on the relatedness of GAS isolates circulating in a particular setting and allows for a more rapid response. We have recently found this assay very useful for typing of GAS disease cluster isolates, and we find this approach quite useful prior to proceeding with higher-resolution genomic sequencing of strains sharing the same emm types (our unpublished data). Also, this PCR-based approach could further detect GAS from culture-negative or low-DNA-copy-number clinical specimens employing the real-time PCR for the GAS-specific target gene spy (19), in which many specimens would be projected to yield emm type information from included emm type-specific targets.
In summary, a sequential quadriplex real-time PCR scheme was developed that was highly sensitive and specific for the identification of the most frequently occurring 20 emm types covering ϳ93% of iGAS isolates in the United States collected through CDC's ABCs program in 2015 (10). Also, the current real-time PCR scheme overlaps 60% of emm types (18 of 30 emm types) included in a GAS vaccine (4, 20) that has good phase I clinical trial data and targets common M serotypes in the United States, Canada, and Europe. Most clinical and public health laboratories a Each target assay identifies known subtypes tested within the emm type. b emm49/151 is considered here as one type. emm151 is a rarely encountered 11-codon deletion derivative of emm49 that occurs within the same MLST type as emm49 (ST433). routinely isolate and identify GAS but do not perform DNA-based emm typing, as the method is labor-intensive. This method will provide typing capability for these laboratories for outbreak investigation support where emm typing may be useful to determine genetic relatedness of isolates to help guide response efforts. A limitation of the method is that the real-time PCR assays do not target all known emm types and subtypes, and all subtypes within each of the 20 emm types targeted were not tested. However, while this method was developed using the most commonly occurring emm types in the United States, the assay also encompasses a large proportion of types globally (21)(22)(23) and can be adapted for use in any country where these emm types are common.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.04 MB.

ACKNOWLEDGMENT
The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.