Article Figures & Data
Figures
- Fig. 1.
Sensitivity and specificity of RT-PCR using the Tth assay. Synthetic positive and negative strands were generated by in vitro runoff transcription with T7 RNA polymerase from a vector (pGEM-3Z) containing the 5′ untranslated sequence of HCV and serially diluted in water. The number of target template copies was calculated from optical density readings. A positive-sense primer was present during cDNA synthesis, after which the enzyme was inactivated by chelation with Mn2+ and then negative-sense primer was added. Samples were amplified as described in the text. Twenty microliters (20%) of the reaction mixture was fractionated on agarose, transferred to a nylon membrane by Southern blotting, and subsequently hybridized to a 32P-labeled probe. When 1 or 6 μg of total cellular RNA extracted from normal human liver tissue was added, the sensitivity of the reactions was lowered by no more than 1 log, while the specificity of the assay was not affected.
- Fig. 2.
Detection of negative-strand HCV RNA in various tissue samples from patient 2 by the Tth-based strand-specific RT-PCR assay and by the non-strand-specific MMLV RT-based assay. The amount of RNA loaded into each reaction mixture was 1 μg; in the case of serum (S), it corresponded to 20 μl. The examined tissues included liver (Lv), spleen (Sp), bone marrow (BM), lymph node (LN), pancreas (Pn), thyroid (Th), adrenal gland (AG), kidney (Kd), lung (Lg), muscle (Ms), Skin (Sk), and spinal cord (SC). Positive or sensitivity controls (lane P) consisted of end-point dilutions of the correct synthetic strand (10 genomic eq for MMLV RT assay and 100 genomic eq for the Tth assay), and negative controls (lane N) consisted of RNA extracted from livers from uninfected subjects. While the majority of samples were positive by the MMLV RT-based RT-PCR, only bone marrow and spleen samples were positive by the strand-specific Tth-based assay.
Tables
- Table 1.
Detection of positive and negative strands of HGV RNA in serum samples and PBMCs in six HIV-infected patients
Patient CD4 cells/mm3 HGV RNA titera in: Serum (genomic eq/ml) PBMCs (genomic eq/2 × 106 to 3 × 106 cells) Positive strand Negative strand Positive strand Negative strand 1 132 5 × 107 Negb 103 Neg 2 270 5 × 105 Neg 102 Neg 3 198 5 × 104 Neg 102 Neg 4 309 5 × 104 Neg 102 Neg 5 545 5 × 103 Neg 101 Neg 6 33 5 × 103 Neg 101 Neg a The presence and titers of the positive strand were determined by the MMLV RT-based assay, while the presence of the negative strand was determined by the Tth-based assay.
b Neg, negative.
- Table 2.
Detection of positive (+) and negative (−) strands of HGV RNA in serum samples and various tissues from four patients with AIDS
Patient HGV RNA titera in: Serum (genomic eq/ml) Tissue (genomic eq of RNA/μg) Liver Spleen Bone marrow Lymph node Pancreas Thyroid Adrenal gland Kidney Lung Muscle Skin Spinal cord + − + − + − + − + − + − + − + − + − + − + − + − + − 1 5 × 104 N P N ND ND 103 102 103 N 102 N 101 N 101 N 101 N 102 N 101 N ND ND 101 N 2 5 × 104 N 102 N 103 102 105 103 101 N 101 N P N P N 101 N 101 N N N 101 N N N 3 5 × 105 N 103 102 103 102 103 P 101 N P N P N 101 N 101 N 101 N N N 101 N N N 4 5 × 104 N 102 N ND ND 102 N 103 N 102 N 101 N 102 N P N 102 N 101 N ND ND N N a The presence and titers of the positive strand were determined by the MMLV RT-based assay, while the presence and titers of the negative strand were determined by the Tth-based assay. N, negative; P, positive in 6 μg of total RNA; ND, not done.
- Table 3.
Detection of positive and negative strands of HCV RNA in serum and liver tissue samples from four patients with AIDS
Patient HCV RNA titera in: Serum (genomic eq/ml) Liver tissue (genomic eq of RNA/μg) Postive strand Negative strand Positive strand Negative strand 1 5 × 105 Negb 107 106 2 5 × 103 Neg 105 104 3 5 × 104 Neg 104 102 4 5 × 103 Neg 103 102 a The presence and titers of the positive strand were determined by the MMLV RT-based assay, while the presence and titers of the negative strand were determined by the Tth-based assay.
b Neg, negative.
