Onchocerca ochengi: morphological identification of the L3 in wild Simulium damnosum s.l., verified by DNA probes

Parasitology ◽  
1998 ◽  
Vol 116 (4) ◽  
pp. 337-348 ◽  
Author(s):  
G. WAHL ◽  
J. M. SCHIBEL
Keyword(s):  
Body Length ◽  
Developmental Stages ◽  
Infective Larva ◽  
Blot Hybridization ◽  
Dna Probe ◽  
Morphological Identification ◽  
Dot Blot ◽  
Infective Larvae ◽  
Simulium Damnosum ◽  
Onchocerca Ochengi

In order to assess the prevalence of the cattle filaria Onchocerca ochengi in onchocerciasis vectors (Simulium damnosum s.l.) in North Cameroon, we searched for a means to morphologically identify its developing larvae, which closely resemble those of O. volvulus. To this end microfilariae of the 2 Onchocerca species were isolated from slaughter cattle in Ngaoundéré and injected into neonate Simulium species. Whereas the early developmental stages (sausage stage, L2 and pre-infective larva) were indistinguishable, the infective larvae (L3) of O. ochengi were longer (median: 740 μm), more slender (diameter = 19·3 μm = 2·6% of body length) and had a relatively shorter tail (4·9% of body length) than those of O. volvulus (680 μm, 20·5 μm, 3·0% and 5·8% respectively). The tail of O. ochengi L3 was thick and rounded, whereas it was slightly tapering in O. volvulus L3. O. ochengi L3 produced by feeding flies on infected cattle in a different area in North Cameroon (Sora Mboum) showed the same features as intrathoracically produced O. ochengi L3 from Ngaoundéré, but were even longer (785 μm). On the basis of the differences in length, relative diameter, length of the tail and shape of the tail, a simple key for the separation of O. volvulus and O. ochengi L3 was elaborated, and 248 L3 found in wild S. damnosum s.l. were separated into ‘O. ochengi’ (160 L3) and ‘O. volvulus’ (88 L3) following this key. Sequential dot blot hybridization of each of the 248 larvae with a DNA probe which reacts with O. ochengi and O. volvulus but not with other Onchocerca species (pOo5/1) and with an O. volvulus-specific DNA probe (pOv12) revealed that the morphological identification had been correct in 86–91% of the cases. Only a small proportion (6–9%) of the dot blots did not react with either probe. Since this proportion was equal in experiments using experimentally produced L3 and in experiments using wild L3, the non-hybridization was certainly due to a loss of L3 during washing of the filters and not due to the presence of other unknown L3 species resembling O. volvulus and O. ochengi. Our study shows that in Cameroon it is possible to identify O. volvulus and O. ochengi infective larvae during routine fly dissections by morphology alone.

Characterization of a fusion cDNA (RARA/myl) transcribed from the t(15;17) translocation breakpoint in acute promyelocytic leukemia

1992 ◽  
Vol 12 (2) ◽  
pp. 800-810
Author(s):  
K S Chang ◽  
S A Stass ◽  
D T Chu ◽  
L L Deaven ◽  
J M Trujillo ◽  
...  
Keyword(s):  
Cdna Library ◽  
Acute Promyelocytic Leukemia ◽  
Blot Analysis ◽  
Blot Hybridization ◽  
Promyelocytic Leukemia ◽  
Dna Probe ◽  
Translocation Breakpoint ◽  
Chromosome 15 ◽  
Dot Blot ◽  
Mrna Species

A nonrandom chromosomal translocation breakpoint, t(15;17)(q22;q21), is found in almost all patients with acute promyelocytic leukemia (APL). Most of these breakpoints occur within the second intron of the retinoic acid receptor-alpha (RARA) gene. We screened a cDNA library of APL and have identified and sequenced a cDNA transcribed from the t(15;17) translocation breakpoint. The 5' end of cDNA p1715 consists of 503 bp of the RARA exon II sequence. A 1.76-kb cDNA without homology to any known gene available in GenBank was found truncated downstream. This cDNA sequence was assigned to chromosome 15 by dot blot hybridization of the flow cytometry-sorted chromosomes. We designate this fusion cDNA RARA/myl, which is different from myl/RARA reported by de The et al. (H. de The, C. Chomienne, M. Lanotte, L. Degos, and A. Dejean, Nature (London) 347:558-561, 1990). This result demonstrates that the two different types of hybrid mRNA can be transcribed from this breakpoint. We screened a non-APL cDNA library and identified a 2.8-kb myl cDNA. This cDNA is able to encode a polypeptide with a molecular weight of 78,450. Alternative splicing of the myl gene which resulted in myl proteins with different C terminals was found. Southern blot analysis of the genomic DNA isolated from 17 APL patients by using the myl DNA probe demonstrated that the myl gene in 12 samples was rearranged. Northern (RNA) blot analysis of RARA gene expression in two APL RNA samples showed abnormal mRNA species of 4.2 and 3.2 kb in one patient and of 4.8 and 3.8 kb in another patient; these were in addition to the normal mRNA species of 3.7 and 2.7-kb. The myl DNA probe detected a 2.6-kb abnormal mRNA in addition to the normal mRNA species of 3.2, 4.2, and 5.5 kb. Using the polymerase chain reaction, we demonstrated that both RARA/myl and myl/RARA were coexpressed in samples from three different APL patients. From this study, we conclude that the t(15;17) translocation breakpoint results in the transcription of two different fusion transcripts which are expected to be translated into fusion proteins.

Primers and a Specific DNA Probe for Detecting Lactic Acid Bacteria Producing 3-Hydroxypropionaldehyde from Glycerol in Spoiled Ciders

2001 ◽  
Vol 64 (6) ◽  
pp. 833-837 ◽  
Author(s):  
OLIVIER CLAISSE ◽  
ALINE LONVAUD-FUNEL
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Polymerase Chain Reaction Amplification ◽  
Klebsiella Oxytoca ◽  
Blot Hybridization ◽  
Dna Probe ◽  
Dot Blot ◽  
Glycerol Dehydratase ◽  
Polymerase Chain ◽  
Dna Primers

Of the 40 strains isolated from several spoiled ciders where glycerol was degraded, 36 were identified as Lactobacillus collinoides, three were Lactobacillus hilgardii, and one was Lactobacillus mali. However, only 30 L. collinoides and two L. hilgardii could degrade glycerol. The glycerol dehydratase activity was shown. The main product of the transformation was 1,3 propanediol. Two DNA primers GD1 and GD2 were chosen in the region encoding one of the subunits of glycerol dehydratase of Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella Typhimurium, and Clostridium pasteurianum. A 279-bp amplicon in polymerase chain reaction amplification was obtained with the genomic L. collinoides IOEB 9527 DNA as template. The amino acid sequence deduced from the amplicon DNA sequence showed a very high similarity and identity with the gene of gram-negative and C. pasteurianum species. After labeling, the amplicon was used as DNA probe in dot-blot hybridization with the genomic DNA of all the tested strains. Only strains that could degrade glycerol hybridized. Moreover, polymerase chain reactions using GD1 and GD2 revealed only glycerol dehydratase genes of positive L. collinoides and L. hilgardii strains. The primers and the amplicon proved to be suitable and reliable tools to detect the lactic acid bacteria involved in the deterioration of cider.

DNA probe for Lactobacillus delbrueckii subsp. lactis

1999 ◽  
Vol 66 (2) ◽  
pp. 303-311 ◽  
Author(s):  
GIORGIO GIRAFFA ◽  
DIEGO MORA
Keyword(s):  
Dairy Products ◽  
Blot Hybridization ◽  
Dna Probes ◽  
Dna Probe ◽  
Starter Cultures ◽  
Lactobacillus Delbrueckii ◽  
Rrna Genes ◽  
Universal Primers ◽  
Dot Blot ◽  
Dna Fragment

Lactobacilli are of great commercial value because they are widely used as starters in food fermentation. The species Lactobacillus delbrueckii, which comprises the three subspecies bulgaricus, lactis and delbrueckii, is important in dairy products and vegetables. The subspecies bulgaricus is present in yogurt, and subspecies lactis is recovered from whey, starter cultures and cheeses (Stiles & Holzapfel, 1997). It is unusual to recover Lb. delbrueckii subsp. delbrueckii (Lb. delbrueckii) from dairy products, from which Lb. delbrueckii subsp. lactis (Lb. lactis) and subsp. bulgaricus (Lb. bulgaricus) are typically isolated. However, given the similarity of these two latter subspecies, a clear identification of isolates on the basis of phenotype criteria alone is often problematic (Dellaglio, 1989; Millière et al. 1996).The use of DNA probes and methods based on polymerase chain reaction (PCR) have greatly facilitated diagnostic identification of bacteria. For the lactobacilli, DNA probes have been described for Lb. helveticus, Lb. acidophilus, Lb. fermentum, Lb. plantarum and most of the other Lactobacillus species (Pot et al. 1994; Tailliez et al. 1994; Quere et al. 1997). For Lb. delbrueckii, an EcoRI DNA fragment of the plasmid pY85 was used as a probe for this species, although it was not able to discriminate its three subspecies (Delley et al. 1990).In our laboratory, a specific amplification of a DNA fragment of ∼1·7 kbp using universal primers for the amplification of ribosomal RNA (rRNA) genes was found only for dairy isolates of Lb. lactis and not for Lb. bulgaricus, Lb. delbrueckii, Lb. helveticus and Lb. acidophilus (G. Giraffa, P. de Vecchi and L. Rossetti, unpublished results). This prompted us to test the possible use of this fragment as a specific DNA probe for Lb. lactis. To this end, Southern and dot blot hybridization experiments were carried out with total DNA of several strains belonging to different lactic acid bacteria (LAB) species.

Detection of varicella-zoster virus by dot-blot hybridization using a molecularly cloned viral DNA probe

1984 ◽  
Vol 13 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Mindell Seidlin ◽  
Howard E. Takiff ◽  
Holly A. Smith ◽  
John Hay ◽  
Stephen E. Straus
Keyword(s):  
Varicella Zoster Virus ◽  
Blot Hybridization ◽  
Dna Probe ◽  
Dot Blot ◽  
Viral Dna ◽  
Varicella Zoster ◽  
Dot Blot Hybridization

scholarly journals DETECTION OF SAG1 AND BAG1 Toxoplasma gondii DNA PROBES LABELLED WITH DIGOXIGENIN-11-dUTP

2013 ◽  
Vol 7 (1) ◽  
Author(s):  
Ida Ayu Pasti Apsari ◽  
Wayan Tunas Artama ◽  
Sumartono S ◽  
I Made Damriyasa
Keyword(s):  
Toxoplasma Gondii ◽  
Blot Hybridization ◽  
Dna Probes ◽  
Dna Probe ◽  
Hybridization Method ◽  
Dot Blot ◽  
Radioactive Label ◽  
Minimum Concentration ◽  
Dot Blot Hybridization ◽  
Labeling Method

The objective of this research was to detect a minimum concentration of the probes that could be used for dot blot hybridization analysis. The method required labeled DNA probes. In this study a non-radioactive label of Digoxigenin-11-dUTP was used for labeling the Sag1 and the Bag1 of Toxoplasma gondii DNA probe. Labeling method for the probes was done according to the random primed labeling technique. The result showed that 0.67 pg/µl Sag1 probe and 0.58 pg/µl Bag1 probe could be detected by anti-Dig-antibody. It could be concluded that 0.67 pg/µl Sag1 probe and 0.58 pg/µl Bag1 probe could be used to diagnose toxoplasmosis by dot blot hybridization method.

scholarly journals Penggunaan Pelacak DNA untuk Deteksi Papaya ringspot virus dengan Metode Hibridisasi Asam Nukleat

2018 ◽  
Vol 14 (3) ◽  
pp. 89
Author(s):  
Irsan Nuhantoro ◽  
Sri Hendrastuti Hidayat ◽  
Kikin Hamzah Mutaqin
Keyword(s):  
Nucleic Acid ◽  
Detection Method ◽  
Blot Hybridization ◽  
High Specificity ◽  
Dna Probe ◽  
Ringspot Virus ◽  
Nucleic Acid Hybridization ◽  
Disease Spread ◽  
Papaya Ringspot Virus ◽  
Dot Blot

Use of DNA Probe for Detection of Papaya ringspot virus Using Nucleic Acid Hybridization MethodPapaya ringspot caused by Papaya ringspot virus (PRSV) is one of the most destructive diseases of papaya. The disease had not been found in Indonesia, until disease outbreak in Nangroe Aceh Darussalam was reported in 2012. Since then, the disease spread rapidly in most papaya growing areas in Sumatera, Java and Bali. Papaya ringspot virus (PRSV) is generally detected using serological or polymerase chain reaction methods. Improvement in detection method is necessary to facilitate a more reliable tool for controlling the spread of PRSV. The aim of the research was to construct DNA probe for development of detection method based on nucleic acid hybridization. Molecular characterization based on HCPro gene sequence indicated high homology (97.88 to 99.05%) among PRSV isolates from Boyolali (Central Java), Medan (North Sumatera), Sleman (Yogyakarta) and Tabanan (Bali). Two DNA clones of HCPro gene were selected for probe construction and the probes were then labeled using PCR DIG-dioxigenin. Optimization of nucleic acid dot blot hybridization method to achieve strongest positive reaction was developed, i.e. using stringency washes at 1×SSC, 0.1% SDS, incubation at 60 oC for 15’. The DNA probe for PRSV has a high specificity and sensitifity; it could detect PRSV at the lowest concentration of nucleic acid (0.062 µg µL-1).

Detection of enteric adenoviruses by dot-blot hybridization using a molecularly cloned viral DNA probe

1985 ◽  
Vol 16 (2) ◽  
pp. 107-118 ◽  
Author(s):  
Howard Takiff ◽  
Mindell Seidlin ◽  
Philip Krause ◽  
James Rooney ◽  
Stephen E. Straus ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Dna Probe ◽  
Dot Blot ◽  
Viral Dna ◽  
Dot Blot Hybridization ◽  
Enteric Adenoviruses

scholarly journals Characterization of a fusion cDNA (RARA/myl) transcribed from the t(15;17) translocation breakpoint in acute promyelocytic leukemia.

1992 ◽  
Vol 12 (2) ◽  
pp. 800-810 ◽  
Author(s):  
K S Chang ◽  
S A Stass ◽  
D T Chu ◽  
L L Deaven ◽  
J M Trujillo ◽  
...  
Keyword(s):  
Cdna Library ◽  
Acute Promyelocytic Leukemia ◽  
Blot Analysis ◽  
Blot Hybridization ◽  
Promyelocytic Leukemia ◽  
Dna Probe ◽  
Translocation Breakpoint ◽  
Chromosome 15 ◽  
Dot Blot ◽  
Mrna Species

A nonrandom chromosomal translocation breakpoint, t(15;17)(q22;q21), is found in almost all patients with acute promyelocytic leukemia (APL). Most of these breakpoints occur within the second intron of the retinoic acid receptor-alpha (RARA) gene. We screened a cDNA library of APL and have identified and sequenced a cDNA transcribed from the t(15;17) translocation breakpoint. The 5' end of cDNA p1715 consists of 503 bp of the RARA exon II sequence. A 1.76-kb cDNA without homology to any known gene available in GenBank was found truncated downstream. This cDNA sequence was assigned to chromosome 15 by dot blot hybridization of the flow cytometry-sorted chromosomes. We designate this fusion cDNA RARA/myl, which is different from myl/RARA reported by de The et al. (H. de The, C. Chomienne, M. Lanotte, L. Degos, and A. Dejean, Nature (London) 347:558-561, 1990). This result demonstrates that the two different types of hybrid mRNA can be transcribed from this breakpoint. We screened a non-APL cDNA library and identified a 2.8-kb myl cDNA. This cDNA is able to encode a polypeptide with a molecular weight of 78,450. Alternative splicing of the myl gene which resulted in myl proteins with different C terminals was found. Southern blot analysis of the genomic DNA isolated from 17 APL patients by using the myl DNA probe demonstrated that the myl gene in 12 samples was rearranged. Northern (RNA) blot analysis of RARA gene expression in two APL RNA samples showed abnormal mRNA species of 4.2 and 3.2 kb in one patient and of 4.8 and 3.8 kb in another patient; these were in addition to the normal mRNA species of 3.7 and 2.7-kb. The myl DNA probe detected a 2.6-kb abnormal mRNA in addition to the normal mRNA species of 3.2, 4.2, and 5.5 kb. Using the polymerase chain reaction, we demonstrated that both RARA/myl and myl/RARA were coexpressed in samples from three different APL patients. From this study, we conclude that the t(15;17) translocation breakpoint results in the transcription of two different fusion transcripts which are expected to be translated into fusion proteins.

Detection of Hepatitis a Virus and other Enteroviruses in Wastewater and Surface Water Samples by Gene Probe Assay

1991 ◽  
Vol 24 (2) ◽  
pp. 267-272 ◽  
Author(s):  
S. Dubrou ◽  
H. Kopecka ◽  
J. M. Lopez Pila ◽  
J. Maréchal ◽  
J. Prévot
Keyword(s):  
Surface Water ◽  
Cell Cultures ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Limit Of Detection ◽  
Blot Hybridization ◽  
Noncoding Region ◽  
Gene Probe ◽  
Dot Blot ◽  
Lower Efficiency

Enteroviruses were specifically detected by dot blot hybridization when using poliovirus type 1 (PV1) derived subgenomic radiolabeled cRNA probes (riboprobes) in environmental water specimens and in the cell cultures in which the viruses were amplificated. The riboprobe corresponding to the 5' noncoding sequence detected the majority of enteroviruses. Hepatitis A virus (HAV) was specifically detected by an HAV cRNA probe corresponding to the 5' noncoding region of its genome. By this test, the limit of detection of coxsackievirus B5 and echovirus 7 seeded in mineral water was 103 to 104 PFU/spot. In cell cultures, positive signals were observed in the lysates of cells infected by one PFU. Higher positive signals were obtained with a short PV1 probe (nt 221-670) corresponding to the 5' noncoding region, which is a well preserved sequence among the enteroviruses, than with PV1 genomic probe. Hybridization allowed a good detection of enteroviral RNAs in wastewater specimens, but with a lower efficiency in surface water. In this case, amplification of viruses in the cell cultures gave significant hybridization results.

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