Simultaneous Detection of RNA and DNA Targets Based on Multiplex Isothermal Amplification

The detection of pathogenic microorganisms present in food, feed, plant, and other samples is important for providing safe food as well as for preventing the spread of microbes. The genome of pathogens is made of DNA or RNA, therefore a multiplex diagnostics tool would ideally be able to amplify and detect both RNA and DNA targets in parallel. With this goal we have developed an isothermal nucleic acid sequence based amplification [NASBA] implemented microarray analysis (NAIMA) procedure, suitable for the simultaneous multiplex amplification of RNA and DNA targets, coupled with the detection on ArrayTubes. The method is demonstrated to be very sensitive and specific for the detection of two economically important quarantine plant pathogens of potato, the <i>potato spindle tuber viroid</i> (RNA target) and Ralstonia solanacearum (DNA target). Because of its isothermal amplification and simple detection equipment, the method is also applicable for on-site analyses. NAIMA can be used in any domain where there is the need to detect RNA and DNA targets simultaneously

Loop-Mediated Isothermal Amplification of Specific Endoglucanase Gene Sequence for Detection of the Bacterial Wilt Pathogen <i>Ralstonia solanacearum</i>

<div><p>The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP) assays that target 16S rRNA, <i>fli</i>C and <i>egl</i> genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the <i>egl</i> gene, which shows high analytical specificity for diverse strains of the betaproteobacterium <i>Ralstonia solanacearum</i>, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the <i>egl</i> LAMP assay requires minimum sample preparation (a few minutes of boiling) for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of <i>R. solanacearum</i> strains of different phylotypes.</p></div

Primers used in the <i>egl</i> LAMP assay.

<p>*Position of the primer on <i>R. solanacearum</i> strain GMI100 endoglucanase precursor (<i>egl</i>) gene (GenBank accession number DQ657595) sequence.</p

Melting temperature ranges of the LAMP products obtained on <i>R. solanacearum</i> strains using the <i>egl</i> LAMP assay.

<p>PI, PIIA, PIIB, PIII, PIV: phylotypes I, IIA, IIB, III and IV, respectively. The melting temperatures were measured on a SmartCycler apparatus (Cepheid, Sunnyvale, CA).</p

Comparison of the amplification speeds of the different LAMP assays.

<p>When tested on <i>R. solanacearum</i> strain NCPPB 3997 (10⁸ cells/mL), the <i>egl</i> LAMP assay (blue) was the fastest assay, compared with the modified <i>fli</i>C (green) and 16S rRNA (violet) assays.</p

Analytical sensitivity of the <i>egl</i> LAMP assay.

<p>Data are means (±SD), calculated from three independent runs.</p><p>“-”: negative result (absence of signal).</p>a<p>, detected once out of three replicates.</p>b<p>, detected twice out of three replicates.</p><p>Data obtained on different <i>R. solanacearum</i> phylotypes samples, from boiled bacterial suspensions.</p

The most prominent BMI-related changes in gene expression were found for interleukin 7 receptor (IL7R), insulin-like growth factor binding protein 3 (IGFBP3), defensin 3 (DEFA3) and DO beta major histocompatibility complex, class II (HLA-DOB).

<p>CD4⁺ T cells - open squares, NK cells - closed circles. Error bars represent intra-individual standard error.</p

Overview of metabolite profiles variability found in plasma samples of healthy volunteers by principle component analysis score plot.

<p>Triangle – male volunteer, circle – female volunteer, coloured according to volunteers, labels shows sampling number within one volunteer. PC - principal component.</p

The ontology enrichment analysis of metabolites that were identified as gender, age and BMI related or were identified as highly variable (HV, CV>0.5) in the analysed healthy population subset.

<p>Total number of differentially present (DP) metabolites (p<0.05) related on gender, age, BMI, BMI within male individuals, age within male volunteers (:male), BMI within female volunteers and age within female volunteers (:female) are also given. Fisher's exact test was used to calculate significance of ontology enrichment (p-value is reported; ns – not significant). ALL - all metabolites within the ontology group. Number of differentially present metabolites in each category is given in brackets within each category.</p

Average concentrations of two soluble sugars, glucose and mannose, are significantly increased in plasma samples of healthy volunteers with higher BMI values.

<p>p_Glucose = 0.0014, p_mannose = 0.0001. Error bars represent standard error within monthly measurements.</p