Experimental Diagnosis of HFRS with Photobiotin-Labelled cDNA Probe
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Dot blot
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Digoxigenin
Caliciviridae
Hybridization probe
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A nonradioactive dot blot hybridization assay for human parvovirus B19 DNA was set up by using a biotin-labeled DNA probe and streptavidin-alkaline phosphatase conjugate. The assay was used to examine 4,895 specimens referred for B19 virus diagnosis during 1987. Of 48 specimens that gave positive reactions for B19 DNA, 41 were confirmed virus positive by electron microscopy (n = 36), radioimmunoassay (n = 26), or counterimmunoelectrophoresis (n = 20). In 7 samples which were not confirmed and in 11 samples giving weak reactions for B19 DNA, there was serological or epidemiological evidence of recent B19 infection. A further 70 specimens gave weak, apparently false-positive reactions. By electron microscopy, 13 of 16 were contaminated by bacteria, and plasmid DNA was demonstrated in one specimen. Of 55 specimens tested, 52 reacted with streptavidin-alkaline phosphatase conjugate alone. These were probable sources of nonspecificity in an otherwise practical and economic screening method for B19 virus.
Dot blot
Hybridization probe
Streptavidin
Counterimmunoelectrophoresis
Southern blot
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N protein is the major forming element of nucleocapsid of CDV and is also a conservative immunogenicity protein.A digoxin labeled probe prepared by enzyme digesting recombinant plasmid of CDV N protein and gel reclaiming targeting DNA was used to detect blenna narium of 40 suspected cases of CD and to detect spleen,liver,heart,lung,kidney and blood of 20 dead cases of CD by dot blotting hybridization.Results showed that among 40 suspected cases of CD,33 cases were positive and positive rate was 82.5%.This rate was higher than that of colloid gold test paper detection.Virus relevance ratio of spleen was the highest among different tissues of detection.Above results showed that dot blotting hybridization had very important application value in early diagnosis and epidemiological investigation of CDV.
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A rapid dot blot hybridization assay for the detection of B19 parvovirus DNA in human sera was developed. Small portions of four serum samples were mixed, filtered onto a nylon membrane, and hybridized with a digoxigenin-labeled DNA probe; for each membrane, 380 serum samples could be tested. When a dot was positive by the hybridization assay, the four serum samples dotted together were separately tested to identify the sample positive for B19 DNA. A total of 10,150 serum samples submitted for viral serological and laboratory investigation with no specific requests for B19 testing were analyzed. Nine serum samples were positive for B19 DNA by dot blot hybridization assay, and the results were confirmed by electron microscopy. This method has proven to be reliable, economical in terms of time and costs, and useful for large-scale screening of clinical specimens, both for diagnostic work and for a source of antigen.
Digoxigenin
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Southern blot
DNA–DNA hybridization
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A hybridization assay for the detection of cytomegalovirus (CMV) in urine specimens was established. Two different DNA fragments were used as hybridization probes: the HindIII L fragment (11.7 kilobases) and the EcoRI J fragment (10.6 kilobases) of the human CMV strain AD169. These probes were used in an isolated and highly purified form and therefore did not cross hybridize with vector sequences. As shown by hybridization with DNA from CMV-infected and uninfected cells, the assay was highly CMV specific and sensitive (detection limit, 750 to 500 fg of CMV DNA). A total of 122 urine specimens were examined by DNA hybridization, virus isolation, and the detection of CMV-induced early nuclear protein. The results coincided in 91% of the samples. The application of DNA hybridization to urine samples, however, is not without problems, and some of the pitfalls and drawbacks are discussed.
Southern blot
Hybridization probe
Dot blot
DNA–DNA hybridization
Cytomegalovirus
Betaherpesvirinae
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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
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Abstract Nucleic acid hybridization was used for the detection of adenovirus DNA in stool specimens, and the results were compared with those obtained by a radioimmunoassay (RIA) for adenovirus hexon antigen. DNA from 40 specimens, 18 of which were positive by RIA, were spotted onto nitrocellulose filters and analyzed by hybridization using radioactively labeled adenovirus‐2 DNA or a cloned DNA fragment from enteric adenovirus‐41 as probes. With the adenovirus‐2 DNA probe, 15 of the 18 RIA‐positive specimens were also positive in the hybridization assay, and one of the RIA negative specimens was also scored as positive. The cloned adenovirus‐41 fragment gave a positive signal with five specimens, all of which were also detected with the adenovirus‐2 DNA probe. The results show that hybridization is an alternative method for detection of adenovirus in stool specimens. The sensitivity of the assay is comparable to that of the RIA.
Hybridization probe
DNA–DNA hybridization
Adenovirus genome
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Coxsackievirus
Cytopathic effect
Hybridization probe
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Detection of human cytomegalovirus (CMV) DNA from peripheral blood and urine specimens taken from renal transplant and bone marrow transplant recipients was examined by using nested polymerase chain reaction (PCR). Results by the PCR were compared with those of healthy subjects. Oligonucleotide primers were used to amplify the immediate-early (IE) and the late antigen (V) genes of CMV. Amplified DNA products were identified by 2% agarose gel electrophoresis and by Southern hybridization with alkaline phosphatase-labeled probes. Twenty eight blood specimens from 118 healthy subjects were positive for the V gene fragment of CMV, whereas the IE gene fragment was more associated with a negative result (116 of 118 specimens). When amplified with mixed primer pairs, no blood and urine specimens simultaneously produced the PCR products of two separate CMV genes. In the case of transplant patients, however, 50 of 139 blood specimens and 34 of 117 urine specimens were positive with the V primers, and 23 blood and 17 urine specimens were positive with the IE primers. Using mixed primer pairs, amplification of two CMV genes was detected in 14 blood and 10 urine specimens. In three cases of bone marrow transplant recipients, amplification of two CMV genes was detectable prior to anti-CMV IgM development. From these results, we suggest that detection of CMV-DNA by nested PCR with mixed primer pairs may be a valuable tool for diagnosing CMV infections.
Primer (cosmetics)
Agarose gel electrophoresis
Betaherpesvirinae
Cytomegalovirus
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Virus isolation
Medical microbiology
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