Assays

The assays

What the assays cannot resolve

These are properties of the assays, not of the implementation:

• Bruce-ladder: B. microti has the same profile as B. suis bv 2 and is not listed separately. B. suis bv 2 and bv 1/3/4 differ by a single bp (450 vs 451), which no gel and no size tolerance separates, so both are reported as suis_bv2/suis_bv1_3_4. B. pinnipedialis/B. ceti sometimes shows one extra band of roughly 1100 to 1700 bp. The vaccine strains RB51 and S19 are each resolved by a single band, but their parent 2308 is reported as abortus: Bruce-ladder never confuses RB51 with 2308, but it cannot separate 2308 from any other abortus bv 1/2/4 field strain. AMOS does separate them, by the 387 bp band, but that band reports an IS711 arrangement rather than the strain.
• AMOS: the RB51 primer lies inside IS711 itself and pairs with the universal IS711 primer across the junction of two tandem elements, so the band reports that arrangement rather than the strain. Vaccine strain RB51 and its parent 2308 are indistinguishable here and are reported as RB51_or_2308. B. ovis carries a tandem IS711 pair of its own and gives the same product; the abortus band is what keeps the two apart. S19 is called on the absence of the 178 bp eri band, which its deletion removes. Use --rb51 to separate RB51 from 2308.
• RB51 junction PCR: it answers RB51 or not RB51 and nothing else. A strain with no readable wboA locus, B. ovis included, is called N/A rather than not_RB51. Seven B. suis bv 2 genomes in the validation panel reach their not_RB51 call on a one-mismatch primer 3 site, so running this assay at --mismatches 0 turns them into N/A.

The RB51 junction assay

--rb51 is the three primer assay of Vemulapalli et al. 1999, which answers one question: is this RB51 or not. It exists because AMOS cannot tell RB51 from its parent 2308 at all, and because Bruce-ladder answers only indirectly, by reading the 1682 bp wboA band shifted to 2526 bp — a long band that a draft assembly can lose, and losing it drops the sample to N/A rather than to RB51.

The three primers are entered as two pairs sharing the forward primer:

Locus	Primers	Product	What it means
wboA	3 + 1, either side of the insertion point	456 bp intact, 1300 bp in RB51, absent when the locus is gone	wboA is there, and whether something sits inside it
wboA_IS711	3 + 2	919 bp	IS711 is inside wboA. RB51 and nothing else.

Primer 2 is the trick of the assay: it is chimeric, its 5' 22 bases wboA and its 3' 12 bases IS711, so it anneals only where the two are fused. Each half occurs on its own in the Brucella genomes that carry the locus; the fused sequence exists in RB51 and nowhere else.

Call	Read as
RB51	The 919 bp junction band is present
not_RB51	No junction band, and wboA is intact at 456 bp
N/A	Neither band: there is no wboA locus to read, so the assay cannot speak

The junction band alone makes the call, so an RB51 draft that has lost the long 1300 bp band still calls RB51, and that band is reported in extra_bands rather than required. The 456 bp band is the one doing the other half of the work: it is what separates not_RB51 from N/A, making a negative mean "wboA is intact and uninterrupted" instead of "nothing amplified".

Assay sources

Bruce-ladder

• García-Yoldi D, Marín CM, de Miguel MJ, Muñoz PM, Vizmanos JL, López-Goñi I. Multiplex PCR assay for the identification and differentiation of all Brucella species and the vaccine strains Brucella abortus S19 and RB51 and Brucella melitensis Rev1. Clin Chem. 2006;52(4):779-81. PMID 16595839
• López-Goñi I, García-Yoldi D, Marín CM, de Miguel MJ, Muñoz PM, Blasco JM, et al. Evaluation of a multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains. J Clin Microbiol. 2008;46(10):3484-7. PMID 18716225

Suis-ladder

• López-Goñi I, García-Yoldi D, Marín CM, de Miguel MJ, Barquero-Calvo E, Guzmán-Verri C, Albert D, Garin-Bastuji B. New Bruce-ladder multiplex PCR assay for the biovar typing of Brucella suis and the discrimination of Brucella suis and Brucella canis. Vet Microbiol. 2011;154(1-2):152-5. PMID 21782356

AMOS

• Bricker BJ, Halling SM. Differentiation of Brucella abortus bv. 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR. J Clin Microbiol. 1994;32(11):2660-6. PMID 7852552
• Bricker BJ, Halling SM. Enhancement of the Brucella AMOS PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51. J Clin Microbiol. 1995;33(6):1640-2. PMID 7650203

RB51 junction PCR

All three primers are taken from Fig. 1 of the paper below. Beware of secondary sources: the sequences circulated for this assay elsewhere match no Brucella genome, and the primers printed in the paper's own methods section are the cloning primers used to characterise the insertion, not the diagnostic ones. See Validation.

• Vemulapalli R, McQuiston JR, Schurig GG, Sriranganathan N, Halling SM, Boyle SM. Identification of an IS711 element interrupting the wboA gene of Brucella abortus vaccine strain RB51 and a PCR assay to distinguish strain RB51 from other Brucella species and strains. Clin Diagn Lab Immunol. 1999;6(5):760-4. doi:10.1128/CDLI.6.5.760-764.1999. PMID 10473532 · PMC95769 — free full text, Fig. 1 has the primers