PCR/LDR/universal array platforms for the diagnosis of infectious disease

Abstract

Infectious diseases account for between 14 and 17 million deaths worldwide each year. Accurate and rapid diagnosis of bacterial, fungal, viral, and parasitic infections is therefore essential to reduce the morbidity and mortality associated with these diseases. Classical microbiological and serological methods have long served as the gold standard for diagnosis but are increasingly being replaced by molecular diagnostic methods that demonstrate increased sensitivity and specificity and provide an identification of the etiologic agent in a shorter period of time. PCR/LDR coupled with universal array detection provides a highly sensitive and specific platform for the detection and identification of bacterial and viral infections.

Fig. 1

Schematic representation of a PCR/LDR/universal array assay. In the example shown, the assay is used to detect the identity of a single base variant (A/C) within a gene of interest. The target gene is amplified using gene specific PCR primers. The PCR amplicon is subjected to an LDR reaction using two upstream primers, one ending in T (complementary to the A allele) with the complement to zip-code 2 attached to its 5′-end and the other ending in G (complementary to the C allele) with the complement to zip-code1 attached to its 5′-end. If the allele present in the sample is C (as shown), then only the “T” primer will ligate to the downstream fluorescently labeled primer. When the ligation products are hybridized to the universal array, a positive signal is seen at zip-code address 1

Fig. 2

Quality control and validation of arrays post spotting. (Panel a) Hybridization of ROX-labeled fiducial complement to the array. Since the fiducial oligonucleotide is included at every location, all spots should provide a positive fluorescent signal of equal intensity. Note that we double spot each address on the array. (Panel b) Hybridization of FAM-labeled zip-code complements to the addresses in every fourth column (Columns 4, 8, 12, 16, 20, and 24) of the array (addresses 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, and 96), When no other columns or addresses provide a positive signal, the print run is considered successful. Three other hybridization experiments are carried out using FAM-labeled zip-code complements to the addresses in Columns 1, 5, 9, 13, 17, and 21 combined, Columns 2, 6, 10, 14, 18, and 22 combined, and Columns 3, 7, 11, 15, 19, and 23 combined (data not shown)

Fig. 3

Representative images of universal array detection of Dengue virus serotypes I–IV following hybridization of LDR products from a PCR/LDR assay. LDR primers for each serotype bear zip-code complements to distinct addresses. For correct identification only the addresses specific to a given serotype should provide a positive signal. In this assay, a minimum of two signals are required to identify a given serotype. (see ref. (22) for details)