Detection of Huanglongbing (Citrus Greening) Disease by Nucleic Acid Spot Hybridization

2009 ◽  
Vol 64 (9-10) ◽  
pp. 711-716 ◽  
Author(s):  
Kuraba Gopal ◽  
Sundeep Sudarsan ◽  
Venati Gopi ◽  
Latchireddy Naram Naidu ◽  
Maniyaram Ramaiah ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Sweet Orange ◽  
Pcr Amplification ◽  
Positive Control ◽  
Infected Tissue ◽  
Citrus Greening Disease ◽  
Pcr Product ◽  
Spot Hybridization ◽  
Total Dna ◽  
Nucleic Acid Spot Hybridization

Polymerase chain reaction (PCR) amplification with primers specific to the rDNA region successfully amplified the 1160-bp DNA fragment from a Huanglongbing (HLB)-infected sweet orange sample with mottling symptoms leaves, but not from healthy sweet orange plants. The PCR product of 1160-bp was used as probe labeled with biotin for detection of the HLB pathogen in the nucleic acid spot hybridization (NASH) test. It was found that the HLB pathogen could be detected up to 1:100 dilution in HLB-infected tissue. Total DNA extracted from HLB-infected tissue was diluted 2-fold as 900 ng in TE buffer and spotted on a nitrocellulose membrane. Strong signals were observed up to 225 ng of DNA dilution, whereas a moderate signal was recorded at 112 ng. No hybridization signal was observed in the healthy samples, while strong signals were observed in the positive control

Improvements in the RT-PCR detection of enteric viruses in environmental samples

1998 ◽  
Vol 38 (12) ◽  
pp. 83-86 ◽  
Author(s):  
K. J. Schwab ◽  
F. H. Neill ◽  
M. K. Estes ◽  
R. L. Atmar
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcriptase ◽  
Environmental Samples ◽  
Internal Standard ◽  
Pcr Amplification ◽  
Enteric Viruses ◽  
New Method ◽  
Rt Pcr ◽  
Pcr Product ◽  
Enzyme Method

Current methods for the detection of nucleic acid from enteric viruses in environmental samples usually involve extensive concentration and purification of target viruses followed by RT-PCR amplification using two enzymes, reverse transcriptase and Taq polymerase. We have developed a modified method that improves RT-PCR assays by: (i) the use of an RT-PCR internal standard control RNA to identify potential false negative results caused by inhibition of RT-PCR enzymes; (ii) the use of rTth (Perkin-Elmer, Foster City, CA), a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase in a single-tube, single-buffer, elevated-temperature reaction; and (iii) the use of thermolabile uracil N-glycosylase (HK-UNG) (Epicentre Technologies, Madison, WI) to prevent PCR product carryover contamination. The new method was compared to the traditional two-enzyme, RT-PCR method for detection of Norwalk virus (NV) and hepatitis A virus (HAV) in buffer, stool, clam and oyster samples. The new method was at least as sensitive in NV and HAV detection compared to the traditional two-enzyme method. The internal standard control successfully detected inhibitors to RT-PCR amplification. NV and HAV PCR products generated with dUTP replacing dTTP during amplification were seeded into subsequent samples to test the prevention of PCR product carryover contamination by HK-UNG. The new method successfully eliminated PCR product carryover contamination in contrast to the traditional two-enzyme method. These improvements to viral nucleic acid detection have the potential to improve sensitivity, specificity and confidence in RT-PCR results.

Typing of herpes simplex virus isolates with monoclonal antibodies and by nucleic acid spot hybridization

1985 ◽  
Vol 12 (1-2) ◽  
pp. 169-177 ◽  
Author(s):  
Thedi Ziegler ◽  
Veijo Hukkanen ◽  
Pertti Arstila ◽  
Petri Auvinen ◽  
Annika Jalava ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nucleic Acid ◽  
Spot Hybridization ◽  
Simplex Virus ◽  
Nucleic Acid Spot Hybridization ◽  
Virus Isolates

scholarly journals Single-Cell Analysis of the t(14;18)(q32;q21) Chromosomal Translocation in Hodgkin's Disease Demonstrates the Absence of This Translocation in Neoplastic Hodgkin and Reed-Sternberg Cells

Blood ◽  
1998 ◽  
Vol 91 (8) ◽  
pp. 2866-2874 ◽  
Author(s):  
Sylvia Gravel ◽  
Georges Delsol ◽  
Talal Al Saati
Keyword(s):  
Lymph Nodes ◽  
Single Cell ◽  
Hodgkin's Disease ◽  
Single Cell Analysis ◽  
Hodgkin’S Disease ◽  
Single Cells ◽  
Chromosomal Translocation ◽  
Pcr Amplification ◽  
Positive Control ◽  
Total Dna

Using the polymerase chain reaction (PCR) technique and total DNA extracts of Hodgkin's disease (HD)-involved lymph nodes, the t(14;18)(q32;q21) translocation was detected in 37 of 115 (32.2%) cases studied. No correlation was found between the presence of this translocation and bcl-2 protein expression in Hodgkin and Reed-Sternberg (HRS) cells detected by immunohistochemistry in 58 of 96 (60.4%) cases. To identify the cells carrying the t(14;18) translocation, single-cell DNA from HRS cells isolated by micromanipulation from frozen tissue sections of lymph nodes was investigated by PCR amplification. Eleven cases showing a positive band of the same size in at least two of five PCR experiments performed on the same total DNA extract were selected for single-cell PCR. We postulated that this repeated successful amplification could be indicative of the presence of the t(14;18) translocation in the neoplastic HRS cells. Single cells from frozen tumor sections of the t(14;18)-positive OCI LY8 cell line grafted into nude mice served as a positive control. The bcl-2/JH rearrangement, involved in this translocation, could be amplified from single-cell DNA of the latter tumor, whereas, in all of the HD cases, HRS cells were found to be negative. We conclude that the t(14;18) translocation is not localized in HRS cells, but in nonmalignant B bystander lymphocytes, admixed with these neoplastic cells.

Detection of adenoviruses in stool specimens by nucleic acid spot hybridization

1985 ◽  
Vol 16 (3) ◽  
pp. 213-218 ◽  
Author(s):  
Per Stålhandske ◽  
Timo Hyypiä ◽  
Annika Allard ◽  
Pekka Halonen ◽  
Ulf Pettersson
Keyword(s):  
Nucleic Acid ◽  
Spot Hybridization ◽  
Stool Specimens ◽  
Nucleic Acid Spot Hybridization
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