[The detection and analysis of leptospiral DNA in patients' serum of early leptospirosis by polymerase chain reaction and DNA hybridization with digoxigenin-AMPPD].
We have detected and analysed the leptospiral DNA in serum of patients with early Leptospirosis from the epidemic area of China by PCR and DNA hybridization with Digoxigenin (Dig)-3-(2'-Spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)- phenyl-1,2-dioxetane (AMPPD) to develop a sensitive, specific and reliable technique for the early diagnosis of leptospirosis, and full satisfactory results have obtained. Fourteen serum specimens from patients with leptospirosis proven by blood culture and serological test were prepared according to Boom's methods for PCR test, and oligonucleotide primers, named G1 G2, were obtained from a genomic library of leptospira interrogans. PCR amplification with serum specimens was performed. Each cycle of amplification consisted of denaturation at 94 degrees C for 1 min, annealing at 55 degrees C for 1 min and elongation at 72 degrees C for 2 min. Each sample was subjected to 32 cycles. The amplified DNA were separated by electrophoresis with 2% agarose gel and hybridized with the homologous DNA probe labelling with Dig-AMPPD by means of Southern blotting. All of 14 samples revealed the presence of leptospira and the strong signals were visualized with homologous DNA probe hybridization by Southern blotting. Negative and positive controls appeared correctly. The DNA fragment generated from PCR amplification homologically hybridized with the DNA of 16 strains which are from Yasudas' genomic species and represent the different genomic groups of leptospires. The single recognized band (about 400 bps) from 6 out of the 16 strains has come out which are representative of the principal strains in Sichuan, China.(ABSTRACT TRUNCATED AT 250 WORDS)
Bao
L
L
WHO/FAO Collabrative and Research Centre of Leptospirosis, Royal Tropical Institute, The Netherlands.
Dai
B
B
Gravekamp
C
C
Hartskeerl
R
R
Terpstra
W
W
D000218:Adamantane / Q000031:analogs & derivatives*; D015139:Blotting, Southern; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis*; D004076:Digoxigenin; D006801:Humans; D007919:Leptospira / Q000302:isolation & purification*; D007922:Leptospirosis / Q000175:diagnosis*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction
D015342:DNA Probes; D004269:DNA, Bacterial; C061523:3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane; D004076:Digoxigenin; D000218:Adamantane