No abstract

We determined the complete nucleotide sequence of baboon endogenous virus (BaEV) strain M7. The total length of the retroviral DNA is 8,507 base pairs. The order of viral proteins encoded is N112-p12-p15-p30-p10-COON in the gag, NHZ-protease-reverse transcriptase-endonuclease-COON in the pol, and N112-gp70-p20E-COON in the env genes. The transmembrane env protein, p20E, contains an amino acid sequence closely related to the "immunosuppressive peptide." Uncharacteristically, the pot and env genes do not overlap and are translated in the same frame. The deduced amino acid sequence of BaEV is related to 1) reticuloendotheliosis virus strain A in both the pol and env regions, 2) murine leukemia viruses in the pol but not in the env region, 3) simian type D retroviruses in the env but not in the pot region. This suggests that env genes in retroviruses may have been independently interchanged during evolution.

Blood and other animal fluids contain a variety of substances that inhibit the polymerase chain reaction (PCR), so that isolation of DNA is generally necessary prior to PCR. We have developed a novel reagent cocktail that effectively suppresses these inhibitory substances and makes DNA isolation from blood unnecessary for PCR. When this reagent was included in the PCR mixture, DNA fragments of the beta-globin gene could be efficiently amplified directly from human blood samples treated with various anticoagulants or PCR-inhibitory substances. We confirmed the usefulness of this cocktail by examining a large number of blood samples with various PCR primer sets. In addition to fresh blood, this method enabled PCR amplification from blood samples stored at 4 degrees C, -20 degrees C or -80 degrees C for a minimum of 1 year.

No abstract

Direct amplification of DNA from clinical specimens, such as blood and faeces, by polymerase chain reaction (PCR) is most often hindered by endogenous inhibitory substances, including haemoglobin and bile acids. We tested whether Ampdirects' A (Shimadzu), a novel reagent cocktail that has been shown to suppress the inhibitors in blood, is also useful for faecal samples, and found that the vero toxin genes (VTl and VT2) of Escherichia coli 0157 could be efficiently amplified from the supernatant of boiled faeces by PCR in the presence of this cocktail without prior extraction of DNA. We compared the efficiency of amplification with and without the cocktail, using the supernatant of boiled normal faeces supplemented with E. coli 0157. PCR without the cocktail failed to amplify the vero toxin genes from the supernatant diluted < 6400-fold or containing >0•02% (final concentration) of boiled faeces. By contrast, PCR with Ampdirect A amplified the toxin genes in the mixture containing as much boiled faeces as 0•5% and as few E. coli as 4 to 8 colony-forming units (CFU). The minimum limit for E. coli 0157 detection by this method was estimated to be about 10 4 CFU!g faeces. The results obtained by this direct method agreed well with those obtained by the indirect method using DNA preextracted from patients' faeces (the detection limit being 10 3 CFU!g faeces).