A real-time PCR method to quantify spores carrying the Bacillus thuringiensis var. israelensis cry4Aa and cry4Ba genes in soil

Guidi, Valeria; Respinis, Sophie De; Benagli, Cinzia; Lüthy, Peter; Tonolla, Mauro

doi:10.1111/j.1365-2672.2010.04741.x

Cited by 21 publications

(27 citation statements)

References 37 publications

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“…The B. thuringiensis cry1b plasmid gene was used as a signature sequence for the detection of DNA from this organism's spores, which were spiked into samples to serve as controls for both DNA extraction and PCR amplification. Bacillus thuringiensis spores are highly refractory, and successful DNA extraction from these spores will ensure DNA extraction from other biological structures (10,13).…”

Section: Methodsmentioning

confidence: 99%

Detection of Coxiella burnetii in Complex Matrices by Using Multiplex Quantitative PCR during a Major Q Fever Outbreak in The Netherlands

Bruin

Groot

Heer

et al. 2011

Appl Environ Microbiol

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Q fever, caused by Coxiella burnetii, is a zoonosis with a worldwide distribution. A large rural area in the southeast of the Netherlands was heavily affected by Q fever between 2007 and 2009. This initiated the development of a robust and internally controlled multiplex quantitative PCR (qPCR) assay for the detection of C. burnetii DNA in veterinary and environmental matrices on suspected Q fever-affected farms. The qPCR detects three C. burnetii targets (icd, com1, and IS1111) and one Bacillus thuringiensis internal control target (cry1b). Bacillus thuringiensis spores were added to samples to control both DNA extraction and PCR amplification. The performance of the qPCR assay was investigated and showed a high efficiency; a limit of detection of 13.0, 10.6, and 10.4 copies per reaction for the targets icd, com1, and IS1111, respectively; and no crossreactivity with the nontarget organisms tested. Screening for C. burnetii DNA on 29 suspected Q fever-affected farms during the Q fever epidemic in 2008 showed that swabs from dust-accumulating surfaces contained higher levels of C. burnetii DNA than vaginal swabs from goats or sheep. PCR inhibition by coextracted substances was observed in some environmental samples, and 10-or 100-fold dilutions of samples were sufficient to obtain interpretable signals for both the C. burnetii targets and the internal control. The inclusion of an internal control target and three C. burnetii targets in one multiplex qPCR assay showed that complex veterinary and environmental matrices can be screened reliably for the presence of C. burnetii DNA during an outbreak.

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Section: Methodsmentioning

confidence: 99%

Detection of Coxiella burnetii in Complex Matrices by Using Multiplex Quantitative PCR during a Major Q Fever Outbreak in The Netherlands

Bruin

Groot

Heer

et al. 2011

Appl Environ Microbiol

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“…Unfortunately, the sensitivity of this approach is often too low to detect slight modifications of toxicity, as B. thuringiensis usually does not exhibit a high level of residual activity (36). Second, spores, which usually persist longer than the larvicidal activity, can be detected in field samples by culturing them on petri dishes (18,46) or by PCR and quantitative PCR, allowing the detection of potential recycling events (12,17). Finally, the fate of Cry toxins, which are mainly responsible for the toxicity of B. thuringiensis, has often been studied by using enzyme-linked immunosorbent assays (ELISAs) (1,29).…”

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confidence: 99%

Fate of Bacillus thuringiensis subsp. israelensis in the Field: Evidence for Spore Recycling and Differential Persistence of Toxins in Leaf Litter

Tetreau

Alessi

Veyrenc

et al. 2012

Appl Environ Microbiol

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Bacillus thuringiensis subsp. israelensis is a bioinsecticide increasingly used worldwide for mosquito control. Despite its apparent low level of persistence in the field due to the rapid loss of its insecticidal activity, an increasing number of studies suggested that the recycling of B. thuringiensis subsp. israelensis can occur under specific, unknown conditions. Decaying leaf litters sampled in mosquito breeding sites in the French Rhône-Alpes region several months after a treatment were shown to exhibit a high level of larval toxicity and contained large amounts of spores. In the present article, we show that the high concentration of toxins found in these litters is consistent with spore recycling in the field, which gave rise to the production of new crystal toxins. Furthermore, in these toxic leaf litter samples, Cry4Aa and Cry4Ba toxins became the major toxins instead of Cyt1Aa in the commercial mixture. In a microcosm experiment performed in the laboratory, we also demonstrated that the toxins, when added in their crystal form to nontoxic leaf litter, exhibited patterns of differential persistence consistent with the proportions of toxins observed in the field-collected toxic leaf litter samples (Cry4 > Cry11 > Cyt). These results give strong evidence that B. thuringiensis subsp. israelensis recycled in specific breeding sites containing leaf litters, and one would be justified in asking whether mosquitoes can become resistant when exposed to field-persistent B. thuringiensis subsp. israelensis for several generations.

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“…The nucleotide sequences of the primers for Bacillus sp. were as follows: direct -5`-TCACCAAGGCRACGATGCG-3`, reverse -5`-CGTATTCACCGCGGCATG-3`, for (Guidi et al, 2010, Wattiau et al, 2001). The microorganisms using organic nitrogen were grown by plate method on meatand-peptone agar (MPA).…”

Section: Methodsmentioning

confidence: 99%

Ecologically Based Disease Management Techniques in Barley Cultivation in the Central Black Soil Region

Lukin¹,

Yeprintsev²,

Федорин³

et al. 2016

AGR

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At present, there are microbiological Bacillus-based products used to preserve valuable micro biota and improving the level of biological soil productivity as well as sustaining the local environment. The research focuses on discovering how some particular biological products and autochthonous microorganisms influence the yield capacity of barley grown in the Black-Earth region of Russia. The research objectives included a search for autochthonous strains of microorganisms that improve resistance to diseases, estimation of how biological products contribute to the quality of barley seeds, biological products effect on the spread and disease resistance and estimation of how biological products enhance the yield capacity of barley. The paper describes the results of identification of an autochthonous Bacillus strains. PCR diagnostic methods were used to confirm the strain specific origin of two sample cells extracted from soil (S1 and S2). The study involved the analysis of micro biota of leached chernozem, which revealed the autochthonous strain of Bacillus S1 having a germicidal effect. The S1 strain revealed Bacillus subtilis and Bacillus сеreus, while S2 revealed only Bacillus subtilis, as detected by the method of molecular diagnostics based on using species-specific primers. Biological treatment of the seeds improved their sowing qualities, namely, germination readiness and germination capacity. In addition, it was found out that such treatment improves the resistance to disease affection and spread. Bacillus S1, in particular, reduces the disease affection by 16,5 % and the disease spread 3,5 as much. Finally, the experiment demonstrated that biological treatment can contribute to sustaining healthy environment for the plants and thus increase their yield capacity.

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A real-time PCR method to quantify spores carrying the Bacillus thuringiensis var. israelensis cry4Aa and cry4Ba genes in soil

Cited by 21 publications

References 37 publications

Detection of Coxiella burnetii in Complex Matrices by Using Multiplex Quantitative PCR during a Major Q Fever Outbreak in The Netherlands

Detection of Coxiella burnetii in Complex Matrices by Using Multiplex Quantitative PCR during a Major Q Fever Outbreak in The Netherlands

Fate of Bacillus thuringiensis subsp. israelensis in the Field: Evidence for Spore Recycling and Differential Persistence of Toxins in Leaf Litter

Ecologically Based Disease Management Techniques in Barley Cultivation in the Central Black Soil Region

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