PCR screen detects multiple or circular copy of RD2. A. Primer combinations used for detection of seven potential arrangements of RD2. Thick black arrows represent RD2 element; thin gray line represents the chromosome. Red, purple, blue, and green arrows represent locations of primer #1, #2, #3, and #4, respectively. Potential arrangement variants are: arrangement as determined by genome sequencing , at least two head-to-tail copies of RD2, tail-to-tail, single copy arranged in reverse orientation than the integrative copy determined by genome sequencing, head-to-tail arrangement of copies in reverse orientation than the integrative copy determined by genome sequencing, head-to-head, and circular form. B. PCR screen detects product amplified with primer pairs #1+#2, #3+#4, and #2+#3, corresponding to arrangement variant (head-to-tail) or (circular form), and #1+#4 detecting chromosomal integration site lacking RD2. C. Primers #2+#3 detect arrangement variant 2 or 7 in multiple RD2 positive strains . Serotype M1 strain MGAS5005 (lacks RD2) was used as a negative control of amplification.