USE OF LAMBDA-PHAGE DNA AS A HYBRID INTERNAL CONTROL IN A PCR-ENZYME IMMUNOASSAY TO DETECT CHLAMYDIA-PNEUMONIAE

An inherent problem in the diagnostic PCR assay is the presence of ill -defined inhibitors of amplification which may cause false-negative re sults. Addition of an amplifiable fragment of foreign DNA in the PCR t o serve as a hybrid internal control (HIC) would allow for a simple wa y to identify specimens containing inhibitors, Two oligonucleotide hyb rid primers were synthesized to contain nucleic acid sequences of the Chlamydia pneumoniae 16S rRNA primers in a position flanking two prime rs that target the sequences of a 650-bp lambda phage DNA segment. By using the hybrid primers, hybrid DNA comprising a large sequence of la mbda phage DNA flanked by short pieces of chlamydia DNA was subsequent ly generated by PCR, cloned into a plasmid vector, and purified. Plasm ids containing the hybrid DNA were diluted and used as a HIC by adding them to each C, pneumoniae PCR test. Consequently, C. pneumoniae prim ers were able to amplify both chlamydia DNA and the HIC DNA, The produ ction of a 689-bp HIC DNA band on an acrylamide gel indicated that the specimen contained no inhibitors and that internal conditions were co mpatible with PCR, Subsequently, a biotinylated RNA probe for the HIC was transcribed from a nested sequence of the HIC and was used for its hybridization, Detection of the HIC DNA-RNA hybrid was achieved by en zyme immunoassay (EIA), This PCR-EIA system with a HIC was initially t ested with 12 previously PCR-positive and 14 previously PCR-negative s pecimens. Of the 12 PCR-positive specimens, 11 were reconfirmed as pos itive; 1 had a negative HIC value, indicating inhibition. Of the 14 pr eviously PCR-negative specimens, 13 were confirmed as true negative; 1 had a negative HIC value, indicating inhibition. The assay was then u sed with 237 nasopharyngeal specimens from patients with pneumonia, Tw enty-one of 237 (8.9%) were positive for C, pneumoniae, and 42 (17.7%) were found to inhibit the PCR, Specimens showing inhibitory activity were diluted 1:10 and were retested, Ten specimens were still inhibito ry to the PCR and required further DNA purification. No additional pos itive samples were detected and 3 nasopharyngeal specimens remained in hibitory to PCR, Coamplification of a HIC DNA can help confirm true-ne gative PCR results by ruling oat the presence of inhibitors of DNA amp lification.