Determination Suppressive Effect of Satellite 369 on Accumulation of Cucumber Mosaic Virus by Real-Time Reverse Transcript-Polymerase Chain Reaction

2008 ◽  
Author(s):  
Jun-li Feng ◽  
Ji-shuang Chen
Keyword(s):  
Polymerase Chain Reaction ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Real Time ◽  
Suppressive Effect ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Reverse Transcript

scholarly journals Real-Time Polymerase Chain Reaction Detection of Cauliflower mosaic virus to Complement the 35S Screening Assay for Genetically Modified Organisms

2005 ◽  
Vol 88 (3) ◽  
pp. 814-822 ◽  
Author(s):  
Katarina Cankar ◽  
Maja Ravnikar ◽  
Jana Žel ◽  
Kristina Gruden ◽  
Nataša Toplak
Keyword(s):  
Polymerase Chain Reaction ◽  
Mosaic Virus ◽  
Real Time ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Pcr Amplification ◽  
Cauliflower Mosaic Virus ◽  
Chain Reaction ◽  
Screening Assay ◽  
Polymerase Chain

Abstract Labeling of genetically modified organisms (GMOs) is now in place in many countries, including the European Union, in order to guarantee the consumer's choice between GM and non-GM products. Screening of samples is performed by polymerase chain reaction (PCR) amplification of regulatory sequences frequently introduced into genetically modified plants. Primers for the 35S promoter from Cauliflower mosaic virus (CaMV) are those most frequently used. In virus-infected plants or in samples contaminated with plant material carrying the virus, false-positive results can consequently occur. A system for real-time PCR using a TaqMan minor groove binder probe was designed that allows recognition of virus coat protein in the sample, thus allowing differentiation between transgenic and virus-infected samples. We measured the efficiency of PCR amplification, limits of detection and quantification, range of linearity, and repeatability of the assay in order to assess the applicability of the assay for routine analysis. The specificity of the detection system was tested on various virus isolates and plant species. All 8 CaMV isolates were successfully amplified using the designed system. No cross-reactivity was detected with DNA from 3 isolates of the closely related Carnation etched ring virus. Primers do not amplify plant DNA from available genetically modified maize and soybean lines or from different species of Brassicaceae or Solanaceae that are natural hosts for CaMV. We evaluated the assay for different food matrixes by spiking CaMV DNA into DNA from food samples and have successfully amplified CaMV from all samples. The assay was tested on rapeseed samples from routine GMO testing that were positive in the 35S screening assay, and the presence of the virus was confirmed.

A polymerase chain reaction assay for cucumber mosaic virus in lupin seeds

1993 ◽  
Vol 44 (1) ◽  
pp. 41 ◽  
Author(s):  
S Wylie ◽  
CR Wilson ◽  
RAC Jones ◽  
MGK Jones
Keyword(s):  
Polymerase Chain Reaction ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Large Scale ◽  
Seed Infection ◽  
Chain Reaction ◽  
Routine Testing ◽  
Source Of Infection ◽  
Lupin Seed ◽  
Polymerase Chain

Seed is the main source of infection of narrow-leafed lupin (Lupinus angustifolius) crops by cucumber mosaic virus (CMV). The ELISA procedure is currently used for large-scale, routine testing of lupin seed samples, but a more sensitive, reliable and labour-saving assay is needed which detects levels of seed infection as low as 0.1%. A Polymerase Chain Reaction (PCR) using ground dry seed samples was developed for this purpose. Primers based on concensus sequences of eight published CMV coat protein cDNAs (RNA3) of CMV subgroups 1 and 2 were used. The assay involved (1) a reverse transcription step for cDNA synthesis and (2) amplification of a specific fragment (482-501 bp depending on the strain) by PCR. Two methods of extracting virus from infected lupin material were used: (i) a rapid procedure which was effective for samples with higher levels of infection, e.g. infected leaves and 20.5% infected seed; (ii) a phenol-chloroform procedure, which led to greater sensitivity, enabling reliable detection of 0.1% seed infection. It detected CMV in 16 commercial seed samples (0.1-8% seed infection) belonging to seven cultivars from 12 different localities. Both methods were suitable for routine testing of the flour derived from grinding dry seed. On dissection of infected seeds, CMV was detected in the cotyledons and embryo and usually in or on the testa. The PCR assay detected virus from both CMV subgroups, but only subgroup 2 was found in lupin seed samples. The two CMV subgroups can be distinguished by digestion of amplified DNA with the restriction enzyme EcoRI; only CMV strains of subgroup 2 are digested to yield two fragments of size 330 and 170 bp.

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