Multichannel piezoelectric genesensor for the detection of human papilloma virus.
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Abstract:
To establish a method for rapid detection and sub-typing of human papilloma virus (HPV).We utilized the piezoelectric genosensor (PG) technique, which is a combination of the piezoelectric biosensor and gene chips for HPV identification in 22 recurrent biopsy specimens and 22 corresponding original biopsy specimens. The control samples came from normal tissue of healthy persons. A combined reaction took place on the sensor surface between the target genes and probes. The frequency of the piezoelectric sensor will decrease when such reactions occur, and the frequency decrease depends on the concentration of the target gene. Specimens were also analyzed with conventional PCR and dot blot.Of the 22 recurrent specimens, 15 contained HPV6 DNA, 2 HPV11 DNA, and 4 HPV16 DNA. Only one specimen was negative. All the 22 original specimens were positive: 17 harbored HPV6 DNA, 3 sequence homologous HPV11 DNA, and 2 HPV16 DNA. No HPV18 DNA was detected in any specimen. When compared with PCR and dot blot analysis, the results were essentially the same except for one specimen, which was shown to contain other sub-types of HPV.Our results show that the piezoelectric genosensor technique is a rapid and specific method to analyze HPV.Keywords:
Dot blot
Southern blot
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To establish a method for rapid detection and sub-typing of human papilloma virus (HPV).We utilized the piezoelectric genosensor (PG) technique, which is a combination of the piezoelectric biosensor and gene chips for HPV identification in 22 recurrent biopsy specimens and 22 corresponding original biopsy specimens. The control samples came from normal tissue of healthy persons. A combined reaction took place on the sensor surface between the target genes and probes. The frequency of the piezoelectric sensor will decrease when such reactions occur, and the frequency decrease depends on the concentration of the target gene. Specimens were also analyzed with conventional PCR and dot blot.Of the 22 recurrent specimens, 15 contained HPV6 DNA, 2 HPV11 DNA, and 4 HPV16 DNA. Only one specimen was negative. All the 22 original specimens were positive: 17 harbored HPV6 DNA, 3 sequence homologous HPV11 DNA, and 2 HPV16 DNA. No HPV18 DNA was detected in any specimen. When compared with PCR and dot blot analysis, the results were essentially the same except for one specimen, which was shown to contain other sub-types of HPV.Our results show that the piezoelectric genosensor technique is a rapid and specific method to analyze HPV.
Dot blot
Southern blot
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Objective To establish a method for rapid detection and sub typing of human papilloma virus (HPV) in biomedical samples. Methods We utilized the piezoelectric genosensor technique, a combination of the piezoelectric biosensor and gene chips for HPV identification, in 22 cases of recurrent biopsy specimens and 22 cases of corresponding original biopsy specimens. The specimens were also analyzed by conventional PCR and Dot blot methods.Results In the 22 recurrent cases, 15 had HPV6 DNA, 2 contained HPV11 DNA, and 4 harbored HPV16 DNA. Only one case was negative. In the 22 original cases, all were positive: 17 harbored HPV6 DNA, 3 contained sequence homologous HPV11 DNA, and 2 harbored HPV16 DNA. No HPV18 DNA was detected in any specimen. When compared with the PCR and Dot blot analysis, the results were essentially the same except one case, which was shown to contain other sub typing of HPV.Conclusion Our results showed that piezoelectric genosensor technique is a rapid and specific method to analyze HPV.
Dot blot
Southern blot
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A rapid and sensitive method for detecting and typing human papillomaviruses (HPVs) in cell scrapings is presented. DNA from scrapings is extracted and bound to nitrocellulose filters (Slot-Blot). By DNA-DNA hybridization with specific32P-labelled HPV-probes (types 6÷11 or 16÷18) the patient’s DNA is then analyzed for the presence of, and for the type of, HPV DNA sequences. A parallel hybridization with a human repetitive element (Alu sequence) allows quantitation of the different hybridization results. Experiments with HeLa cell DNA show that as little as 104HPV sequences can be detected and typed specifically with this test. Evaluation of this test is completed within 6 to 7 days after cell collection. This Slot-Blot method was used to analyse 1330 specimens taken at the Bernese Dysplasia Outpatient Clinic. The results reveal a very high percentage (90%) of HPV-positive cases in the patient group examined.
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DNA–DNA hybridization
Hybridization probe
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Human papillomaviruses (HPVs) are associated with benign and malignant neoplasms of the cervix. One of the criteria for their etiologic role requires an assessment of whether virtually all or only a small fraction of lesions contain viral genomes. DNA preparations from colposcopically directed punch biopsies of cervical lesions were analyzed by Southern blot hybridization and the polymerase chain reaction (PCR) for the presence of HPV DNA. The biopsy specimens represented different pathologic entities (koilocytosis, condyloma, cervical intraepithelial neoplasia, and invasive carcinoma). In Southern blot hybridization with radioactive probes for HPV 11, 16, 18, 31, and 33, HPV DNA was detected in 74% of the biopsy specimens (42 of 57 cases), with the predominant types being HPV 16 and HPV 18. In contrast, after PCR amplification with primers yielding fragments of characteristic size for HPV 11, 16, and 18, the analysis of the same 57 biopsy specimens revealed that all samples were positive for at least one HPV type. To exclude false-positive PCR results, controls without HPV DNA were interspersed at regular intervals, and results were evaluated only if these controls remained HPV negative. To exclude false-negative results due to failure of the reaction, a target sequence within the c-Ha-ras-1 gene was used as an internal control. All HPV typing results obtained by Southern blot hybridization were in agreement with HPV typing by PCR. The higher number of positive samples in the latter analysis stems from the increased sensitivity of PCR, which was which was effective in identifying as few as 10-100 HPV DNA molecules; in contrast, the sensitivity of Southern blot hybridization was 1 pg, or approximately 10(5) molecules of HPV DNA. The authors conclude that, with sufficiently sensitive diagnostic methods, HPV DNA can be detected in most, if not all, neoplastic cervical lesions.
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Large-scale screening for human parvovirus B19 (B19) DNA in serum samples was carried out by both dot blot hybridization and the polymerase chain reaction (PCR). Dot blot hybridization was undertaken with a digoxigenin-labeled DNA probe. Serum samples from four patients were pooled and tested by a dot blot hybridization assay. When a dot was positive, each of the four samples was tested separately to identify the positive sample. The PCR template was the DNA extracted from mixed serum samples from 10 patients. When B19 DNA was positive by PCR, each of the ten samples was tested separately. A total of 7,969 serum samples were tested by dot blot hybridization and 15 samples (11 patients) were positive for B19 DNA; 7,038 serum samples were tested by PCR and 71 samples (50 patients) were positive. Large-scale screening for B19 DNA by PCR suggested a broader spectrum of clinical manifestations associated with B19 infection.
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Southern blot
Digoxigenin
Hybridization probe
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Ninety-six colposcopically directed biopsies from squamous epithelial carcinoma of the uterine cervix and 22 age-matched normal control biopsy specimens were examined by both Southern blot hybridization and polymerase chain reaction (PCR) for the presence of different human papillomavirus (HPV) DNA types. Cancer of the uterine cervix, which is the most common malignant disease in Indian women, showed a high frequency (98%) of HPV as compared to those reported from other parts of the world. HPV type 16 was found to be the dominant (64%) type while the frequency of HPV type 18 was very low (3%). On individual typing of HPV, no biopsy was found to contain any other known HPV types under stringent conditions of hybridization except a single case of HPV type 11. Only one case of double infection with HPV types 16 and 18 was recorded. Under low stringency conditions of hybridization with a mixed probe of HPV types 16 and 18, 29 additional biopsies were found to be positive. Southern blot hybridization alone detected HPV DNA in 92% of the cases but none in the controls. By PCR, six (6.25%) more cases and four (18.18%) healthy women were found to be positive for HPVs. Analysis of the physical state of HPV 16 indicated integration in about 70% of carcinoma cases while 30% of them were in episomal form. The findings suggest that infection with HPV is an important etiologic factor for the development of cervical cancer, that a number of such tumours may arise without HPV infection, and that integration of the viral DNA into host genome is not always essential for malignant progression.(ABSTRACT TRUNCATED AT 250 WORDS)
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Polymerase chain reaction (PCR) amplification was used to detect cytomegalovirus (CMV) in tissue culture and in urine specimens from newborns. Synthetic oligonucleotide primer pairs were used to amplify DNA from the major immediate-early and the late antigen genes of CMV. Amplified products were detected by gel electrophoresis and by dot-blot hybridization with oligonucleotide probes. Using one or both of the primer pairs and associated probes, we found 46 different tissue culture isolates of CMV that were positive; no reaction products were detected when the same primers and probes were used to amplify other herpes family viruses or human genomic DNA. Urine samples from 44 congenitally infected infants were positive when tested with one or both primer pairs and probes. When compared with tissue culture, detection by gel electrophoresis provided a sensitivity of 93%, a specificity of 100%, and a predictive value of a positiveresult of 100%. Dot-blot analysis raised the sensitivity to 100%. We conclude that PCR amplification may be a valuable tool for diagnosing congenital CMV infection.
Cytomegalovirus
Multiple displacement amplification
Hot start PCR
Ligase chain reaction
Betaherpesvirinae
Recombinase Polymerase Amplification
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Dot blot
Multiple displacement amplification
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Isolation and Characterization of Gastric Carcinomaassociated DNA Fragments by U~ng Optimized AP-PCR
The molecular mechanism of Gastric cancer (GC) is gaining ground rapidly. Cytological study has identified the multiple changes of gene, to isolate GC associated genes, optimized arbitrarily primed polymerase chain reaction (AP-PCR) is used in present study matching GC tissues and non-cancerous gastric tissues. The present study includes three parts: the first step is acquirement of the differential bands. The DNA is isolated from matched GC tissues and non-cancerous gastric tissues. DNA fingerprinting generated by using optimized APPCR. The second step is identification of the DNA. The DNA extracted from the differential bands are amplified and identified by dot blot hybridization,
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