Biomedical subjects
Publications and source records attributed to R Sooknanan.
A nucleic acid sequence-based amplification system primarily targeting mRNA from the Listeria monocytogenes hlyA gene was developed. This system enabled the detection of low numbers (< 10 CFU/g) of L. monocytogenes cells inoculated into a variety of dairy and egg products after 48 h of enrichment in modified listeria enrichment broth.
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The Nucleic Acid Sequence-Based Amplification (NASBA) process involves alternate steps of DNA synthesis from an RNA template and RNA synthesis from a DNA template, using avian myeloblastosis virus (AMV) reverse transcriptase and T7 RNA polymerase, respectively. The overall fidelity of the amplification process was determined by sequence analysis of cloned DNA products of NASBA reactions. An error frequency of less than 0.3% was observed in cloned DNA products from two different segments of the HIV-1 gag gene. Partial substitution of GTP with ITP in the NASBA reaction did not significantly change the fidelity of the process. An error rate of 2 x 10(-4) was calculated for the combined effects of both polymerases.
Quantification of HIV-1 viral RNA in plasma was achieved by competitive co-amplification of a dilution series of in vitro generated RNA using the nucleic acid sequence based amplification (NASBA) technology. This 1.5 kilobase in vitro RNA, comprising the gag and part of the pol region, differs only by sequence-randomization of a 20 nt fragment from the wild-type RNA, ensuring equal efficiency of amplification. In model systems the accuracy of this method is within one log. Application of the Q-NASBA to plasma samples of a patient with a primary HIV-1 infection shows good concordance of the HIV-1 RNA profile with the p24 antigen profile. However, the HIV-1 RNA determination is more sensitive than the p24 antigen determination. Peak values of HIV-1 RNA are around 10(8) RNA molecules per ml plasma at the moment of seroconversion. Quantitative nucleic acid detection methods, like Q-NASBA, allow the monitoring of HIV-1 RNA during the course of infection which might have predictive value for disease development.
The reverse transcriptase-polymerase chain reaction (RT-PCR) for BCR-ABL mRNA is increasingly used to diagnose and monitor patients with Ph+ chronic myeloid leukemia (CML). We investigated an alternative approach to detect BCR-ABL mRNA in CML in order to overcome some of the potential drawbacks of RT-PCR. Nucleic acid sequence based amplification (NASBA) is a homogeneous, isothermal, in vitro process that provides the direct amplification of RNA. Peripheral blood from seven patients with Ph+ CML and Ph+ EM-2 cells were investigated by NASBA and RT-PCR. A nested set of four primers flanking the BCR-ABL junction was used in two serial NASBA reactions performed for 2 hours. The two methods were fully concordant for detection of transcripts with bcr3-abl2 and bcr2-abl2 junctions. Ethidium bromide fluorescence with NASBA indicated in repeated experiments that similar quantities of total RNA from patient material contained different amounts of BCR-ABL mRNA. The data suggest that direct amplification of RNA is suitable for identifying and monitoring patients with Ph+ CML and may provide a means to quantify BCR-ABL mRNA levels.
The aim of this study was to evaluate the effect of interferon-alpha therapy on serum and liver HBV DNA in 20 patients with chronic hepatitis B and to correlate the presence or absence of HBV DNA with the clinical response. There were 11 responders and all lost HBV DNA from the serum. Ten of the 11 were followed for 36 months following IFN treatment and remained well with absence of HBeAg and HBV DNA from the serum and with normal ALT. Five also lost HBsAg. HBV DNA became undetectable in the liver of nine of 10 of these patients in whom liver tissue was available for study. HBV DNA persisted in the liver of seven of nine nonresponders and was not detected in two in spite of the presence of HBV DNA and HBeAg in the serum of these two patients. We conclude that IFN may induce long remissions in patients with chronic hepatitis B with loss of HBV DNA from the serum and that occasionally HBV DNA may persist in the liver of such patients.
Isothermal nucleic acid amplification of target RNA or DNA sequences is accomplished by the simultaneous enzymatic activity of AMV reverse transcriptase, T7 RNA polymerase and RNase H. Amplification factors of the nucleic acid sequence based amplification (NASBA) method range from 2 x 10(6) to 5 x 10(7) after 2.5 h incubation at 41 degrees C. During NASBA there is a major accumulation of specific single stranded RNA. RNA:DNA hybrid and double stranded DNA are also synthesized, although to a minor extent. The system is optimized for the detection of HIV-1 sequences in in vitro infected cells, blood and plasma. Detection levels are 10 molecules of HIV-1 in a model system with in vitro generated HIV-1 RNA as input and 5 infected cells on a background of 5 x 10(4) non-infected cells. Blood and plasma can also be used as the source of nucleic acid for detection of HIV-1 sequences using a specifically developed sample preparation method. Using NASBA it is possible to amplify specifically RNA or DNA from a pool of total nucleic acid, which permits the investigation of the expression of specific genes involved in pathogenesis of infectious agents. The combination of NASBA with a rapid and user-friendly nucleic acid extraction method makes the whole procedure suitable for large scale diagnosis of infectious agents (e.g. HIV-1).
HBV DNA was measured in the sera of 69 patients with hepatitis B virus infections. Sixteen patients had acute hepatitis B, 24 had chronic active hepatitis (CAH), 6 had chronic persistent hepatitis (CPH), 5 had cirrhosis without CAH and 18 were asymptomatic HBsAg carriers. In patients with acute hepatitis B who recovered, HBV DNA was present in the serum transiently early in the illness. HBV DNA persisted in the serum in the two patients who developed chronic hepatitis. Sera of 23 of 24 patients with CAH were persistently positive for HBV DNA. There was no relationship between the quantity of HBV DNA in the serum and the histological intensity of activity. Thirteen of the 24 patients with CAH had histological evidence of cirrhosis in addition to CAH and HBV DNA was detected in the sera of all 13. The sera of 2 of 6 patients with CPH were positive for HBV DNA. In one it was positive only where there was clinical evidence of reactivation of HBV infection. The other patient subsequently developed CAH. Sera of 5 patients with established HBsAg positive cirrhosis but without evidence of CAH were negative for HBV DNA. Two of these patients had hepatocellular carcinoma. Sera of 18 asymptomatic anti-HBe positive carriers with normal ALT were negative for HBV DNA. HBeAg and HBV DNA were not always found in the serum together. In acute hepatitis 5 patients with HBV DNA in the serum were HBeAg positive, but in 6 patients the sera were HBeAg positive inthe absenceof HBV DNA.
Sera of 17 HBeAg positive and 104 anti-HBe positive asymptomatic HBsAg carriers from two cohorts were tested for HBV DNA. HBV DNA was found in 13 of 17 HBeAg positive carriers (76.5%) and in only 7 of 104 of anti-HBe positive carriers (6.7%). Eleven of the 17 HBeAg positive carriers were retested for HBV DNA over a period of 7 to 36 months after the initial test. HBV DNA disappeared from the serum in 2 patients in spite of persistence of the HBe antigen. Of the 104 anti-HBe carriers, 89 were retested for HBV DNA over a period of 6 to 52 months after the initial test. HBV DNA disappeared from the serum in 5 of the 7 who were previously positive for HBV DNA, and persisted in 2. These findings indicate that there is an inconstant relationship between the time of seroconversion of HBeAg to anti-HBe and the disappearance of HBV DNA. In one HBeAg positive patient, HBV DNA, which was absent in the serum on first testing, was present on retesting. This suggests that the presence of HBV DNA in the serum of some patients may be intermittent. The presence of HBV DNA in the serum of some anti-HBe positive carriers accounts for the finding that they may be infective. All but one of the HBV DNA positive anti-HBe carriers were born outside North America, most in Asia. HBV DNA were found more frequently in the serum of anti-HBe positive carriers who had biochemical and histological evidence of liver disease than in carriers without such evidence.
Hepatitis B viral (HBV) DNA was detected in a hepatoma cell line which produces hepatitis B surface antigen (HBsAg) and in patients with acute hepatitis B. The serum of one patient with acute hepatitis B was found to be infectious when injected i.v. into a chimpanzee up to a dilution of 10(-8). Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) were detectable in the same serum sample by radioimmunoassay up to a dilution of 10(-5) and of 10(-3), respectively. Using DNA: DNA hybridization on nitrocellulose membranes, HBV DNA sequences were detectable up to 10(-8) dilution corresponding to the infectivity level. Based on this finding, it appears that DNA: DNA hybridization is the most sensitive method for detecting hepatitis B virus (HBV) infection. In situations with low virus levels it may be the only indicator of the presence of infectious hepatitis B virus. The use of a tritium-labelled probe makes the method economical and adaptable to hospital laboratories.