Saibal K. Poddar

Detection of adenovirus using PCR and molecular beacon

The polymerase chain reaction (PCR) and a molecular beacon probe were used for the detection of Adenovirus. A 307 bp DNA fragment from a conserved region of the hexon gene was amplified. The specific molecular beacon was characterized with respect to its efficiency of quenching, and signal to noise ratio by spectrofluorometric analysis of its hybridization with virus specific complementary single stranded oligonucleotide target. Amplification was carried out in the presence of the molecular beacon probe, and the amplified target was detected by measurement of fluorescence signal in the post PCR sample. Separately, a 32P-labeled linear probe (having the same sequence as that of molecular beacon probe) was liquid-phase hybridized with the product of PCR performed in the absence of the molecular beacon. The virus specific target was then detected by electrophoresis of the hybridized product in a nondenaturing polyacrylamide gel and subsequent autoradiographic analysis. The detection limit of adenovirus by PCR in the presence of the molecular beacon probe was found to be similar to that obtained by labeled linear probe hybridization following PCR.

Influenza virus types and subtypes detection by single step single tube multiplex reverse transcription-polymerase chain reaction (RT-PCR) and agarose gel electrophoresis

Influenza virus type and subtype specific primers were selected for use in reverse transcription polymerase chain reaction (RT-PCR). The selected primer sets were used in a single step RT-PCR of influenza virus RNA in multiplex format for the detection of virus type and subtypes. Three one step reaction conditions are optimized: (1) multiplex typing only, (2) multiplex subtyping of influenza A, and (3) multiplex typing and subtyping simultaneously. RNA from strains of influenza virus type A of subtypes H1N1, H3N2 and H5N1 and influenza virus type B was used for amplification and detection. The amplified DNA fragments were size analyzed for the presence of specific type and subtypes target DNA by agarose gel electrophoresis. The sensitivity of detection was about 0.01 TCID(50) for both influenza type A and B when amplification was for typing alone. In multiplex subtyping or multiplex typing and subtyping simultaneously, the sensitivities for types and subtypes specific bands were 0.01-0.1 TCID (50) in the tested volume.

Effect of inhibitors in clinical specimens on Taq and Tth DNA polymerase-based PCR amplification of influenza A virus

Fifteen randomly selected nasopharyngeal (NP) swab specimens (culture-negative for influenza A virus) were spiked with influenza A virus and the nucleic acids were extracted and subjected to PCR amplification with Thermus aquaticus (Taq) and T. thermophilus (Tth) DNA polymerases. Products of the expected size, and giving equivalent band intensities, were obtained from four specimens with both polymerases. Fox six specimens, less products were obtained with Taq DNA polymerase than with Tth DNA polymerase. Products were detected from five NPs only by PCR with Tth DNA polymerase. The transport medium and the calcium alginate swab fibre of the specimens were shown not to be the source of the inhibitors. The incorporation of 32P-dCTP into cDNA, and the yield of PCR products of cDNA made from control RNA template (purified from H2O spiked virus suspension) were decreased in the presence of inhibitory extracts, showing that both the reverse transcription (RT) and PCR steps in amplification with Taq DNA polymerase were sensitive to the inhibitors. In contrast, Tth DNA polymerase was more resistant to the inhibitors and viral nucleic acid from all the specimens examined could be amplified and detected in a single step by RT-PCR with Tth DNA polymerase.

Detection of type and subtypes of influenza virus by hybrid formation of FRET probe with amplified target DNA and melting temperature analysis

Fluorescence resonance energy transfer probes were selected for three sets of reverse transcription -polymerase chain reaction (RT-PCR), each in duplex format to detect: (1). Influenza virus type A or B; (2). Neuraminidase subtypes N1 or N2; and (3). Hemagglutinin subtypes H1 or H3 using LightCycler Instrument. A pair of probes targeted a type or subtype specific RT-PCR amplified gene segment. The presence of a target in a set of amplification reaction was detected by increased fluorescence over background emitted from the appropriate reporter fluorophore on probe-target hybrid formation and by melting temperature (T(m)) analysis of probe-target hybrid. The T(m) of a probe-target in a duplex amplification was distinctly different from the other, and thus T(m) value allowed specific detection of a target. Amplified product in each set of amplification was also examined by conventional agarose gel electrophoresis. The sensitivities of detection by fluorescence signal analysis were equal or ten times better than that detected by agarose gel electrophoresis.

Evaluation of PCR assays in presence of antibody to thermostable DNA polymerases for detection of microbial agents: Avoiding false negative results for specimen containing low-titer agent

The serial low‐titer specimens of Influenza A virus and Adeno virus type 7 were tested for the presence of virus specific genes by PCR based on Tth DNA polymerase and by that based on Taq DNA polymerase, in the absence and presence of antibody to the respective DNA polymerases. Increased product DNA synthesis and higher sensitivity of detection were observed in the presence of antibody compared to those in the absence of antibody. 10‐ to 100‐fold lower titer specimen of Influenza A virus and 10‐fold lower titer specimen of Adeno virus could be detected in the presence of antibody than those detected in the absence of antibody to the appropriate DNA polymerase, in a PCR. J. Clin. Lab. Anal. 12:238–241, 1998.

Nonspecific primer and PCR generated hybridization probes for physical ordering large restriction fragments in complex genome of S. aureus.

Pulsed field gel electrophoresis (PFGE) allows separation of large restriction fragments from bacterial genome. Restriction fragments obtained by digestion of Staphylococcus aureus DNA with rare cutting enzymes (Sma I, and Csp I) were separated by PFGE. To arrange the physical order of the fragments generated by digestion with one enzyme, probes were prepared by nonspecific priming and polymerase chain reaction (PCR), using individual fragments of the other enzymatic digest as a template. Probes were then used for Southern hybridization to the PFGE separated fragment distribution of the two infrequent cleaving enzymes (Sma I and Csp I). Using probes generated from four Sma I fragments and five Csp I fragments as individual templates, a partial physical order of Csp I fragments of the genome of S. aureus ISP8 has been determined in relation to a previously published Sma I map of S. aureus genome.