PubMed Health⌕ Search

Biomedical subjects

R P Singh

Publications and source records attributed to R P Singh.

At least 19 recordsLinked to original sources
PubMed1996

Detection of stylet-borne and circulative potato viruses in aphids by duplex reverse transcription polymerase chain reaction.

A reverse transcription polymerase chain reaction (RT-PCR) assay was designed to amplify stylet-borne potato virus Yo (PVYo) in aphids using primers located in the viral capsid gene. A 480 bp long product was detected in aphids exposed to PVYo-infected potato plants. Approximately 40% of Myzus persicae and 15% of Aphis nasturtii exposed briefly to PVYo-infected plants acquired the virus. This rate of acquisition by both species of aphids was typical of our earlier observation of the virus transmission tests. No significant difference in virus detection was observed whether the aphids were tested immediately after exposure to virus sources or stored for up to 45 days in ethanol at room temperature. The addition of a second pair to primers located in the capsid gene of circulative potato leafroll virus (PLRV) allowed simultaneous amplification of two viruses (duplex RT-PCR) in single aphids. Acquisition of PVYo by the aphids already viruliferous with PLRV was significantly reduced, compared to aphids not carrying PLRV. Duplex RT-PCR for PVYo and PLRV could be applied to analyze aphids collected from the field to ascertain the relative presence of both viruses in a single test.

Animals↗
PubMed1996

Factors affecting detection of PVY in dormant tubers by reverse transcription polymerase chain reaction and nucleic acid spot hybridization.

A reverse transcription polymerase chain reaction (RT-PCR) protocol was developed using two 20-mer primers located in nuclear inclusion genes NIa and NIb of potato virus Y (PVY). A 1017 bp PCR-product was detected in dormant potato tubers, infected with PVY(O), but not in tubers from healthy plants. The PCR product was specific to PVY, as determined by Southern blot detection by hybridization with a PVY(O)-specific probe. As little as 1 pg of purified PVY(O)-RNA can be detected after RT-PCR amplification. The presence of phenolics or polysaccharides in tuber nucleic acids inhibited PVY(O) amplification, which was eliminated by diluting nucleic acid preparations prior to cDNA synthesis, modifying the nucleic acid extraction procedure by isopropanol precipitation and using phosphate-buffered saline-Tween in the cDNA mix. Potato cultivars differed in PVY(O) concentration in tubers as much as 128-fold. Tuber parts used for nucleic acid extractions were important in potato cultivars with low virus titres and did not result in reduced detection of PVY(O) by both nucleic acid spot hybridization and RT-PCR, but RT-PCR band intensity was lower at longer storage periods. The primer pair developed in this study exhibited broad specificities with field isolates from Peru, Scotland and North America.

DNA, Viral↗
PubMed1995

Peptides as standards for denaturing isoelectric focusing.

A set of commercially available peptides suitable for use as standards in denaturing isoelectric focusing (IEF) is described. The peptides N-procalcitonin fragment 1-57 (pI 3.98), Gln11-amyloid beta-protein fragment 1-28 (pI 5.76), gastric inhibitory polypeptide (pI 7.14), parathyroid hormone fragment 1-34 (pI 8.64) and human beta-endorphin (pI 9.49) can be focused to their isoelectric point in the presence of 8 M urea and 2% Nonidet P-40, and subsequently fixed and stained in polyacrylamide gels. The peptides give a linear standard curve in close agreement with a slope determined with a surface pH electrode. Under the same conditions some proteins focus to positions significantly at odds with their theoretical isoelectric point. The origins of these discrepancies and the implications for the determination of isoelectric points of unknown proteins by denaturing IEF are discussed.

Animals↗
PubMed1995

Digoxigenin-labelled cDNA probes for the detection of potato virus Y in dormant potato tubers.

An isolate (Y139) of the potato virus Y strain PVYO, common in the potato-growing regions of eastern Canada, was cloned and segments of about 1800 bases from the 3'-end of the PVY-genome were sequenced. These clones were used to prepare molecular detection probes by labelling with digoxigenin. Five methods reported to yield nucleic acid preparations from tuber tissue were compared. Two of the methods failed to yield nucleic acids from tubers suitable for use in dot-blot hybridization. The other three methods yielded satisfactory nucleic acids which could be successfully detected with the PVYO probes. A method of nucleic acid extraction from tuber tissues was selected which is simple, reproducible and does not use phenol. Using this standardized procedure, PVY was detected in freshly harvested, greenhouse-grown tubers of 8 potato cultivars; the probe was specific to PVY; and the sensitivity of detection using purified viral RNA was down to 10 pg. The sensitivity of detection in tuber extracts was down to a dilution of 1:64, while that from potato leaves was to a dilution of 1:1024. Nucleic acids prepared by the standardized procedure and denatured prior to storage at 4 degrees C, -20 degrees C or at -70 degrees C remained suitable for hybridization for over 2 weeks.

DNA Probes↗
PubMed1995

Detection of potato leafroll virus in single aphids by the reverse transcription polymerase chain reaction and its potential epidemiological application.

A reverse transcription and polymerase chain reaction (RT-PCR) system was developed using two 20-mer primers located in the potato leafroll virus (PLRV) capsid gene. A 336-bp PCR product was detected from aphids (Myzus persicae) which had been fed on PLRV-infected plants. The PCR band was specific to PLRV as determined by Southern blots and detection by a PLRV-specific probe. As little as 5 min exposure of aphids to PLRV-infected leaves resulted in the presence of PLRV-specific bands in 13% of aphids. However, the percentage of PLRV-positive aphids increased with longer exposure to infected sources and reached 90% after 3-4 days of feeding. PLRV can be detected from a single viruliferous aphid or a single viruliferous aphid combined with up to 29 non-viruliferous aphids. PLRV can be detected from freshly collected aphids, those stored at -70 degrees C, or those stored in 70% ethanol at room temperature for extended periods. This method is applicable to assess the viruliferous nature of aphids caught in yellow-pan traps during the growing season or stored for over a year.

Animals↗
PubMed1995

A study of spontaneous echo contrast in patients with rheumatic mitral stenosis and normal sinus rhythm: an Indian perspective.

OBJECTIVE: To study the incidence of spontaneous echo contrast in left atrium of Indian patients with rheumatic mitral stenosis in normal sinus rhythm and to define its relations. SUBJECTS: Transthoracic and multiplane transoesophageal echocardiographic studies were performed in 89 consecutive patients with rheumatic mitral stenosis who were in normal sinus rhythm. RESULTS: Spontaneous echo contrast in the left atrium was seen in 57.3% of patients on multiplane transoesophageal echocardiography and in only 5.6% on transthoracic echocardiography. The mean mitral valve area was 1.07 (SD 0.33) cm2 and 1.32 (0.45) cm2 (P = 0.004), mean left atrial size was 4.27 (0.67) cm and 3.91 (0.5) cm (P = 0.029), mean diastolic pressure gradient was 12.64 (5.69) mm Hg and 10 (5.5) mm Hg (P = 0.049), and absence of mitral regurgitation was seen in 45% and 23% of patients respectively (P = 0.1). Among patients with spontaneous echo contrast, 31% had either left atrial/appendage thrombus or a history of embolism, upsilon 0% in patients without spontaneous echo contrast (P < 0.0001). CONCLUSIONS: There is a high incidence of spontaneous echo contrast in the left atrium in Indian patients with rheumatic mitral stenosis in normal sinus rhythm on multiplane transoesophageal echocardiography. These patients are likely to embolise or form thrombi in the left atrium. The presence of spontaneous echo contrast is also associated with significantly smaller mitral valve area, larger left atrium, and higher mean diastolic mitral pressure gradient.

Adult↗
PubMed1995

Biting activity of the malaria vector, Anopheles culicifacies, on man and cattle in Doon valley, India.

In the present study, the information about biting time and activity of a malaria vector, Anopheles culicifacies on man and cattle in Doon valley has been mentioned. The maximum biting activity, 22.4 percent in human bait indoor and 20.5 percent in human bait outdoor was noted between 22.00-23.00 h in the night. However, the biting activity on animal bait (calf) was noted maximum in the mid night at 24.00-01.00 h.

Animals↗
PubMed1994

Influence of different factors on the adsorption of carbofuran (2, 3-dihydro-2,2-dimethyl-7-benzofuranyl-N-methyl carbamate) on soils.

The effects of exchangeable cations (H+ and Na+), autoclaving, organic matter, cationic and anionic surfactants, and temperature on the adsorption of carbofuran on two different types of soils were studied. The adsorption isotherms for all effects/treatments were in close agreement with the Freundlich equation and yielded S-shaped isotherms. The amount of carbofuran adsorbed in all cases was higher in Jhansi red loam soil than in Pilibhit sandy loam soil and was related to organic matter content, clay content, CaCO3 content, surface area, and cation-exchange capacity of the soils. The adsorption on soils from both sites follows the order H soil-->Na soil-->natural soil at 25 degrees C-->autoclaved soil-->soil from which organic matter had been removed-->cationic surfactant-->anionic surfactant-->natural soil at 50 degrees C and was in accordance with Freundlich constant K values and distribution coefficient Kd values. The adsorptive capacity of carbofuran for organic matter and clay content for both the Jhansi and the Pilibhit soils was also evaluated by calculating Kom and Kc values, and it was found that the carbofuran adsorption was better correlated with clay content than with organic matter content of soils. On the basis of adsorption isotherms, various thermodynamic parameters such as the thermodynamic equilibrium constant Ko, standard free energy (delta G zero) changes standard ethalpy (delta H zero) changes, and standard entropy (delta S zero) changes have been calculated in order to predict the nature of isotherms.

Adsorption↗
PubMed1994

Multimeric non-radioactive cRNA probes improve detection of potato spindle tuber viroid (PSTVd).

Experimental data showed that multimeric, complementary RNA (cRNA) probes, labelled with non-radioactive digoxigenin (DIG), improved sensitivity of detection of the potato spindle tuber viroid (PSTVd) RNA by 2- to 30-fold as compared with corresponding multimeric cDNA probes. The degree of PSTVd detectability improvement depended upon the type of alkaline phosphatase substrate (colorimetric vs. chemiluminescent) used. Use of hexameric DIG-labelled cRNA probes in combination with chemiluminescent (Lumi-Phos 530) substrate resulted in detection of 0.48 pg of PSTVd RNA. The size of the synthesized cRNA probes corresponded to the size of the respective PSTVd cDNA templates. Interestingly, there was no relationship between the size of the synthesized, DIG-labelled DNA probes and that of the PSTVd cDNA template. This type of anomaly was not observed with other plant viral cDNA templates. Monomeric or multimeric cDNA probes detected both a mild and a severe PSTVd strain in viroid-infected potato leaf extracts diluted 1024 to 2048 times. In comparison, cRNA probes exhibited a much greater dilution end point; PSTVd RNA was detectable in viroid-infected potato leaf tissue diluted up to 16,384 times. Comparable levels of PSTVd sensitivity of detection were obtained with viroid-infected potato tuber tissue.

Alkaline Phosphatase↗
PubMed1994

Improvement in the sensitivity of PVYN detection by increasing the cDNA probe size.

Cloned cDNA to a North American isolate of tobacco veinal necrotic strain of potato virus Y (PVYN) RNA was used for the detection of PVYN in diseased samples. Digoxigenin-labelled cDNA probes were prepared from approximately 0.56 kb, approximately 1.2 kb, approximately 2.5 kb, and approximately 3.25 kb overlapping clones representing the 3'-terminus of the PVYN genome. It was shown that the probe sizes, as determined by alkaline gel-electrophoresis, generally corresponded to the size of the template cDNA used. The sensitivity of detection of PVYN was maximum using the largest probe and the sensitivity generally decreased with a decrease in probe size. Five pg of homologous purified PVYN RNA was detected with the approximately 3.25 kb probe, and 1000 pg of PVYN RNA was required for the approximately 0.56 kb probe. The different levels of sensitivity of detection were also apparent when crude nucleic acid extracts from PVYN and PVYO infected plants were used. In comparison with tobacco bioassay, and dot immunobinding assay nucleic acid hybridization was found to be more sensitive.

DNA Probes↗
PubMed1994

Molecular cloning and sequencing of the capsid and the nuclear inclusion protein genes of a North American PVYN isolate.

The sequence of the 3'-terminal 3258 nucleotides of a tobacco veinal necrosis strain of a potato virus Y (PVYN) isolate from North America was determined. The sequence revealed an open reading frame of 2931 nucleotides, of which the start codon was not identified. The nontranslated region contains 327 nucleotides upstream of a poly(A) tract. The open reading frame encodes a large polyprotein containing 976 amino acids. The data indicate that the coat protein (CP) and the nuclear inclusion protein (NIb) are derived from the large polypeptide by proteolytic cleavage similar to many other potyviruses. The putative cleavage sites of NIa/NIb and NIb/CP correspond to Q/A and Q/G sequences, respectively, as in other PVYN isolates. The CP and NIb contain 267 and 519 amino acid residues, respectively. Higher sequence homology of CP was observed with other PVYN isolates than with the PVYO isolates.

Amino Acid Sequence↗
PubMed1994

Dermal toxicity of hexachlorocyclohexane and pirimiphos-methyl in female rats.

Technical hexachlorocyclohexane (100 mg/kg/d) and pirimiphosmethyl EC 50 (250 mg/kg/d) given individually and in combination to female rats for 7, 15 or 30 d by skin application caused poisoning, pathomorphological changes in vital organs, and significant enzymatic changes in liver and serum. The changes produced by the 2 compounds in combination did not suggest potentiation at the tested dose levels.

Administration, Cutaneous↗
PubMed1993

Interaction of technical hexachlorocyclohexane and oxydemeton methyl 25 EC to female rats after dermal application.

Technical hexachlorocyclohexane (HCH, 100 mg/kg/day) and oxydemeton methyl 25 EC (125 mg/kg/day) to female rats for 7, 15 and 30 days individually and in combination through skin application caused pathomorphological changes in vital organs and significant enzymatic changes in liver and serum. However changes produced by the two compounds in combination were not suggestive of potentiation effect at the tested dose level in female rats.

Administration, Cutaneous↗