Reliability of nested polymerase chain reaction in the diagnosis of bacterial endophthalmitis.
OBJECTIVE
To test the utility of DNA Taq polymerase (Taq) treated with 8-methoxypsoralen with ultraviolet irradiation and Taq treated with restriction endonuclease in a nested polymerase chain reaction test for the diagnosis of bacterial endophthalmitis.


METHODS
Prospective, comparative study. Vitreous biopsy fluid from 32 cases of clinically diagnosed bacterial endophthalmitis and 10 noninfective controls were cultured for aerobic/anaerobic bacteria and tested by nested polymerase chain reaction using two sets of universal eubacterial primers in triplicate with untreated Taq, Taq treated with 8-methoxypsoralen with ultraviolet irradiation, and Taq treated with Sau 3A1.


RESULTS
Using untreated Taq, false-positive results were obtained in nested polymerase chain reaction with all 10 control samples, which were not seen with the other two methods of nested polymerase chain reaction. However, the sensitivity of nested polymerase chain reaction using Sau 3A1 was the same sensitivity as the conventional culture (34.4%), whereas the sensitivity of the nested polymerase chain reaction using 8-methoxypsoralen was 46.9% higher than in the conventional culture.


CONCLUSIONS
To eliminate the problem of false positives in bacterial nested polymerase chain reaction, we recommend the routine utilization of Taq treated with 8- methoxypsoralen and ultraviolet irradiation.
Sharma
Savitri
S
Jhaveri Microbiology Centre, Hyderabad Eye Research Foundation, L.V. Prasad Eye Institute, L.V. Prasad Marg, Banjara Hills, Hyderabad, India. savitri@lvpeye.stph.net
Das
Debashish
D
Anand
Raj
R
Das
Taraprasad
T
Kannabiran
Chitra
C
D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis; D015252:Deoxyribonucleases, Type II Site-Specific / Q000494:pharmacology; D009877:Endophthalmitis / Q000175:diagnosis* / Q000382:microbiology*; D015818:Eye Infections, Bacterial / Q000175:diagnosis* / Q000382:microbiology; D005189:False Positive Reactions; D006801:Humans; D008730:Methoxsalen / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011237:Predictive Value of Tests; D011446:Prospective Studies; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000187:drug effects / Q000528:radiation effects; D014466:Ultraviolet Rays; D014822:Vitreous Body / Q000382:microbiology*
D004269:DNA, Bacterial; D019914:Taq Polymerase; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases; D008730:Methoxsalen