A one-step reaction for the rapid identification of Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16S-23S rRNA intergenic sequences

Aims:  Species-specific primers targeting the 16S–23S ribosomal DNA (rDNA) intergenic spacer region (ISR) were designed to rapidly discriminate between Lactobacillus mindensis , Lactobacillus panis , Lactobacillus paralimentarius , Lactobacillus pontis and Lactobacillus frumenti species recently isolated from French sourdough.
Methods and Results:  The 16S–23S ISRs were amplified using primers 16S/p2 and 23S/p7, which anneal to positions 1388–1406 of the 16S rRNA gene and to positions 207–189 of the 23S rRNA gene respectively, Escherichia coli numbering (GenBank accession number V00331 ). Clone libraries of the resulting amplicons were constructed using a pCR2·1 TA cloning kit and sequenced. Species-specific primers were designed based on the sequences obtained and were used to amplify the 16S–23S ISR in the Lactobacillus species considered. For all of them, two PCR amplicons, designated as small ISR (S-ISR) and large ISR (L-ISR), were obtained. The L-ISR is composed of the corresponding S-ISR, interrupted by a sequence containing tRNA^Ile and tRNA^Ala genes. Based on these sequences, species-specific primers were designed and proved to identify accurately the species considered among 30 reference Lactobacillus species tested.
Conclusions:  Designed species-specific primers enable a rapid and accurate identification of L. mindensis , L. paralimentarius , L. panis , L. pontis and L. frumenti species among other lactobacilli.
Significance and Impact of the Study:  The proposed method provides a powerful and convenient means of rapidly identifying some sourdough lactobacilli, which could be of help in large starter culture surveys.     

Development of a PCR assay for identification of the Bacillus cereus group species

Aims:  A PCR technique was developed as a reliable and rapid identification method for the Bacillus cereus group species, based on a unique conserved sequence of the motB gene (encoding flagellar motor protein) from B. cereus , Bacillus thuringiensis and Bacillus anthracis .
Methods and Results:  Primer locations were identified against eight strains of the B. cereus group spp. from nucleotide sequences available in the National Centre for Biotechnology Information database. The PCR assay was applied for the identification of 117 strains of the B. cereus group spp. and 19 strains from other microbial species, with special emphasis on foodborne pathogens.
Conclusion:  The designed cross-species primers are group specific and did not react with DNA from other Bacillus and non- Bacillus species either motile or not. The primers system enabled us to detect 10³ CFU of B. cereus cells per millilitre of sample.
Significance and Impact of the Study:  Bacillus cereus group spp. belongs to one of the most prevalent foodborne pathogens. Bacterial growth results in production of different toxins; therefore, consumption of food containing >10⁶ bacteria per gram may result in emetic and diarrhoeal syndromes. A rapid and sensitive bacterial detection method is significant for food safety.     

Detection of Phytophthora nicotianae in Soil with Real-time Quantitative PCR

Phytophthora nicotianae is one of the most important soil-borne plant pathogens. A rapid, specific and sensitive real-time polymerase chain reaction (PCR) detection method for P. nicotianae was established, which used primers targeting the internal transcribed spacer (ITS) regions of rDNA genes of Phytophthora spp. Based on the nucleotide sequences of ITS2 of 15 different species of Phytophthora , the primers and probe were designed specifically to amplify DNA from P. nicotianae. With a series of 10-fold DNA dilutions extracted from P. nicotianae pure cultures, the detection limit was 10 pg/μl in conventional PCR, whereas in SYBR Green I PCR the detection limit was 0.12 fg/μl and in TaqMan PCR 1.2 fg/μl, and real-time PCR was 10⁴–10⁵ times more sensitive than conventional PCR. The simple and rapid procedures maximized the yield and quality of recovered DNA from soil and allowed the processing of many samples in a short time. The direct DNA extractions from soil were utilized to yield DNA suitable for PCR. By combining this protocol with the real-time PCR procedure it has been possible to specifically detect P. nicotianae in soil, and the degree of sensitivity was 1.0 pg/μl. The system was applied to survey soil samples from tobacco field sites in China for the presence of P. nicotianae and the analyses of naturally infested soil showed the reliability of the real-time PCR method.     

Population dynamics of a motile and a non-motile Azospirillum lipoferum strain during rice root colonization and motility variation in the rhizosphere

Abstract: Azospirillum lipoferum 4B and non-motile A. lipoferum 4T have been simultaneously isolated from rice rhizosphere at the same frequency. A. lipoferum 4T showed stable morphological and metabolic traits which are atypical for A. lipoferum species such as lack of motility, carbohydrate metabolism and laccase activity. Inoculation experiments showed that A. lipoferum 4T, but not A. lipoferum 4B, needed rice roots to stabilize in sterile soil. Both strains were able to colonize efficiently rice roots (10⁸ cfu g⁻¹ fresh roots) but motile form 4B remained dominant. In spite of their phenotypical differences, A. lipoferum 4B and 4T co-existed without exclusion in sterile soil (planted or not) and rice rhizosphere. Inoculation of rice roots with A. lipoferum 4B showed that rice rhizosphere enhanced the frequency of appearance of stable non-motile forms (40%). This percentage was weaker in plantlet growth medium (4%). However, these non-motile bacteria kept the same biochemical traits than the motile parental strain 4B (carbohydrates metabolism, laccase activity).     

Rapid and sensitive detection of Vibrio cholerae by loop-mediated isothermal amplification targeted to the gene of outer membrane protein ompW

Aims:  The present study was aimed to develop a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Vibrio cholerae .
Methods and Results:  A set of five designed primers that recognized specifically the V. cholerae ompW gene was used. The optimized time and temperature conditions for the LAMP assay were 75 min at 65°C, respectively. The LAMP method accurately identified 16 isolates of V. cholerae but did not detect 28 non- cholerae Vibrio isolates and 37 non- Vibrio bacterial isolates. The sensitivity of LAMP for V. cholerae detection in pure cultures was 2·2 × 10³ CFU ml⁻¹ or equivalent to 8 CFU per reaction. In the case of spiked shrimp samples without enrichment, the detection limit for V. cholerae was 2·2 × 10⁴ CFU g⁻¹ or equivalent to 20 CFU per reaction, while that of PCR was 100 CFU per reaction.
Conclusion:  The developed LAMP assay targeting ompW gene was rapid, specific and sensitive for V. cholerae detection.
Significant and Impact of the study:  The developed LAMP assay appears to be precise, accurate and a valuable tool for detection of V. cholerae . This assay can replace laborious biochemical tests for the identification of V. cholerae in contaminated food sample.     

A new rapid and sensitive detection method for cereulide-producing Bacillus cereus using a cycleave real-time PCR

Aims:  To develop a rapid and sensitive detection method for cereulide-producing Bacillus cereus using a real-time PCR based on the sequence of the cereulide synthesis gene.
Methods and Results:  A total of 56 cereulide-producing B. cereus and 15 cereulide-negative strains were tested. We designed specific primers and probes for the detection of cereulide-producing B. cereus . The new cycleave real-time PCR assay gave positive detections for all of 56 cereulide-producing B. cereus strains, whereas all other strains including 10 systemic infectious disease strains were negative. No cross-reaction was observed and the internal control showed positive for all samples.
Conclusions:  The performance of the assay was highly reproducible and specific for cereulide-producing B. cereus . The positive detection was obtained within only 2 h for cereulide-producing strains. The detection limit of this assay was evaluated as 10⁴ CFU g⁻¹ food sample. The assay also confirmed that strains from systemic infectious cases were cereulide-negative.
Significance and Impact of the Study:  This assay is applicable for contaminated foods as well as specimens from infectious disease cases. We recommend this assay for routine examination of suspected B. cereus food poisonings.     

Molecular detection of nifH gene-containing Paenibacillus in the rhizosphere of sorghum (Sorghum bicolor) sown in Cerrado soil

Aims:  To develop a polymerase chain reaction (PCR)-based approach for the detection of nifH gene-containing Paenibacillus in environmental samples.
Methods and Results:  The primers, nifHPAENf and nifHPAENr, were designed and tested with DNA from: (i) strains of different nitrogen-fixing Paenibacillus species, (ii) strains of other nitrogen-fixing genera and (iii) rhizosphere of sorghum sown in Cerrado soil amended with either 12 or 120 kg ha⁻¹ of nitrogen fertilizer. All nitrogen-fixing Paenibacillus strains tested and the DNA samples from rhizosphere soil were amplified when these primers were used, generating a 280 bp fragment. When the PCR products obtained from both sorghum rhizospheres were cloned and sequenced, the majority of the clones analysed could be identified as Paenibacillus durus . Moreover, a greater diversity in the nifH sequences could be observed in the rhizosphere treated with a high amount of nitrogen fertilizer.
Conclusions:  Nitrogen fertilization slightly influenced the structure of the nifH gene-containing Paenibacillus community in sorghum rhizospheres cultivated in Cerrado soil.
Significance and Impact of the Study:  The PCR detection method developed is adequate to assess the presence of nifH gene-containing Paenibacillus in the environment and can be used in future to determine the ecological role of this group of micro-organisms for the nitrogen input to the plants.     

Rapid detection of pathogenic Vibrio parahaemolyticus by a sensitive and specific duplex PCR-hybridization probes assay using LightCycler

Aims:  To develop a real-time polymerase chain reaction (PCR) hybridization probe assay for rapid and specific detection of thermostable direct haemolysin-producing Vibrio parahaemolyticus.
Methods and Results:  Primers and hybridization probes were designed to target the toxR and tdh2 genes. Mismatches were introduced in the tdh2 primers for specific amplification of the target. The 3' ends of donor probes for both genes were labelled with fluorescein. The 5' ends of recipient probes for tdh2 and toxR were labelled with LC Red 640 and LC Red 705, respectively. The real-time assay was evaluated against conventional biochemical tests and the KAP-RPLA kit (Kanagawa phenomenon detection kit by reverse passive latex agglutination). toxR and tdh2 were detected in 100% and 91% of clinical V. parahaemolyticus isolates ( n  = 118), respectively. Specificity and sensitivity of the real-time assay for toxR and tdh2 were 100%, respectively. Dynamic range of detection for toxR was 10⁷–10¹ CFU ml⁻¹ and that for tdh2 was 10⁷–10⁴ CFU ml⁻¹.
Conclusions:  The LightCycler assay described is sensitive and highly specific for detection of pathogenic V. parahaemolyticus in a single reaction tube within 80 min.
Significance and Impact of the Study:  The assay developed allows accurate detection of pathogenic V. parahaemolyticus , which is valuable for rapid tracing of infection source during outbreaks.     

Multiplex real-time PCR for exhaustive detection of diarrhoeagenic Escherichia coli

Aims:  The source and routes of diarrhoeagenic Escherichia coli (DEC) have not been clarified because it is difficult to detect these organisms in samples with numerous coliform bacteria. We have developed multiplex real-time PCR assays for exhaustive detection of DEC.
Methods and Results:  Primers and TaqMan probes were designed to amplify and quantify one gene ( eae , stx1 , stx2 , elt , est , virB , aggR , astA, and afaB ) from each of seven pathotypes of DEC, in duplex or triplex reactions under the same PCR cycling conditions. Specificity was confirmed using 860 strains including 88 DEC strains. The fluorescence threshold cycle and DNA concentrations correlated with decision coefficients of more than 0·99. Subsequently, meat samples and enrichment broths were spiked with DEC and the assays used to detect the genes. The detection limits varied from 7·1 × 10² to 1·1 × 10⁴ CFU ml⁻¹, depending on the target genes. All meat samples spiked with a variety of DEC (more than 10 CFU 10 g⁻¹) were found to be positive by the method.
Conclusions:  The present system allows for the efficient and simultaneous determination of various DEC pathotypes.
Significance and Impact of the Study:  This system makes epidemiological investigations for DEC sensitive and quick, and is a useful tool to clarify the source and routes of DEC.     

Design and evaluation of malolactic enzyme gene targeted primers for rapid identification and detection of Oenococcus oeni in wine

Rapid identification and detection of Oenococcus oeni was achieved by species-specific PCR. Two primers flanking a 1025 bp region of the O. oeni gene encoding the malolactic enzyme were designed. The expected DNA amplificate was obtained only when purified DNA from O. oeni was used. The identity of PCR product was confirmed by nested PCR and restriction analysis. Within 8 h, 10³ cfu ml⁻¹ of oenococci were detected in fermenting grape must containing 10⁷ yeast cells, whereas the detection limit in wine was 10⁴ cfu ml⁻¹. The rapidity and reliability of the PCR procedure established suggests that the method may be profitably applied in winery laboratories for quality control.     

Persistence of Escherichia coli O157:H7 on the rhizosphere and phyllosphere of lettuce

Aims:  The major objective of this study was to determine the effects of low levels of Escherichia coli O157:H7 contamination on plant by monitoring the survival of the pathogen on the rhizosphere and leaf surfaces of lettuce during the growth process.
Methods and Results:  Real-time PCR and plate counts were used to quantify the survival of E. coli O157:H7 in the rhizosphere and leaf surfaces after planting. Real-time PCR assays were designed to amplify the stx 1, stx 2 and the eae genes of E. coli O157:H7. The detection limit for E. coli O157:H7 quantification by real-time PCR was 2·4 × 10³ CFU g⁻¹ of starting DNA in rhizosphere and phyllosphere samples and about 10² CFU g⁻¹ by plate count. The time for pathogens to reach detection limits on the leaf surface by plate counts was 7 days after planting in comparison with 21 days in the rhizosphere. However, real-time PCR continued to detect stx 1, stx 2 and the eae genes throughout the experimental period.
Conclusion:  Escherichia coli O157:H7 survived throughout the growth period as was determined by real-time PCR and by subsequent enrichment and immunomagnetic separation of edible part of plants.
Significance and impact of the Study:  The potential presence of human pathogens in vegetables grown in soils contaminated with E. coli O157:H7 is a serious problem to our national food supply as the pathogen may survive on the leaf surface as they come in contact with contaminated soil during germination.     

Quantitative strain-specific detection of Lactobacillus rhamnosus GG in human faecal samples by real-time PCR

Aims:  To develop a strain-specific rapid assay for identification and quantification of Lactobacillus rhamnosus GG in human faecal samples.
Methods and Results:  A unique random amplified polymorphic DNA (RAPD) band of the L. rhamnosus GG strain was isolated and sequenced. Strain-specific polymerase chain reaction (PCR) primers and probes were designed based on the sequence. Quantification was performed by the real-time PCR using a fluorescent resonance energy transfer (FRET) system. The specificity of the assay was tested with DNA isolated from a set of known strains and human faecal samples. The analytical sensitivity of the method for L. rhamnosus GG was about 10 CFU per assay, which corresponds to 10⁵ CFU g⁻¹ of wet faeces.
Conclusions:  Quantitative real-time PCR is a suitable method for strain-specific identification of L. rhamnosus GG in human faecal samples.
Significance and Impact of the Study:  Lactobacillus rhamnosus GG is one of the most studied probiotic strains in clinical trials but still lacks a DNA-based identification method. This study describes a real-time PCR method for strain-specific identification and quantification of L. rhamnosus GG in human faecal samples.     

Specific and sensitive detection of Ralstonia solanacearum in soil with quantitative, real-time PCR assays

Aims:  The aim of this study was to develop a sensitive and an effective method suitable for large-scale detection and quantification of Ralstonia solanacearum in soil.
Methods and Results:  Based on the specific sequence of R. solanacearum strain G1000, the primer pair R.sol1-R.sol2 and the TaqMan probe Rs-pro were designed, and specific and sensitive PCR detection methods were successfully established. The detection limit was 100 fg μl⁻¹ DNA in conventional PCR and 1·2 fg μl⁻¹ in real-time PCR. By combining real-time PCR with the modified protocols to extract DNA from soil, it was possible to achieve real-time detection of R. solanacearum in soil, and the degree of sensitivity was 100 fg μl⁻¹. To detect inhibition in soil samples, an exogenous internal positive control (IPC) was included preventing false negative results, and IPC was successfully amplified from all samples tested. The methodology developed was used to detect the presence of R. solanacearum in tobacco fields in China.
Conclusions:  The real-time PCR combined with the protocol to extract DNA from soil led to the development of a specific, sensitive and rapid detection method for R. solanacearum in soil.
Significance and Impact of the Study:  The real-time PCR improves the detection sensitivity and specificity and provides an important tool for routine detection of R. solanacearum in soil samples and for epidemiological and ecological studies.     

Automated concentration and recovery of micro-organisms from drinking water using dead-end ultrafiltration

Aims:  Concentration of pathogens diluted in large volumes of water is necessary for their detection. An automated concentration system placed online in drinking water distribution systems would facilitate detection and mitigate the risk to public health.
Methods and Results:  A prototype concentrator based on dead-end hollow fibre ultrafiltration was used to concentrate Bacillus atrophaeus spores directly from tap water. Backflush was used to recover accumulated particulates for analysis. In field tests conducted on a water utility distribution system, 3·2 × 10⁴–1·4 × 10⁶ CFU ml⁻¹ (6·1 × 10⁶–3·0 × 10⁸ CFU) were recovered from the filter when 2·9 × 10⁷–1·0 × 10⁹ CFU were spiked into the system. Per cent recovery ranged from 21% to 68% for flow volumes of 15–21 l. Tests using spore influent levels <10 CFU l⁻¹ (spike < 1000 CFU) yielded 23–40% recovery for volumes >100 l.
Conclusions:  B. atrophaeus spores at levels <10 CFU l⁻¹ were concentrated directly from tap water using an automated dead-end hollow-fibre ultrafiltration system.
Significance and Impact of the Study:  The prototype concentrator represents a critical step towards an autonomous system that could be installed in drinking water distribution lines or other critical water lines to facilitate monitoring. Recovered samples can be analysed using standard or rapid biosensor methods.     

Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum, Clostridium baratii and Clostridium butyricum

Aims:  To develop real-time PCR assays for tracking and tracing clostridia responsible for human botulism.
Methods and Results:  Real-time PCR assays based on the detection of the genes ntnh encoding the nontoxin-nonhaemagglutinin (NTNH) proteins or the most homologous regions of the botulinum neurotoxin ( bont ) genes have been developed together with four real-time PCR assays, each being specific of the genes bont/A , bont/B , bont/E , bont/F and enables a toxin type-specific identification. The specificity of the assays was demonstrated using a panel of botulinum toxin producing clostridia (29 strains), nonbotulinum toxin producing clostridia (21 strains) and various other bacterial strains. The toxin type-specific assays had a sensitivity of 100 fg–1000 fg of total DNA in the PCR tube (25–250 genome equivalents) which correspond to 10³ to 10⁴ cells ml⁻¹. After a 48 h enrichment in anaerobic conditions, these PCR assays allowed the detection of Clostridium botulinum type A in a naturally contaminated sample of 'foie gras' suspected in a C. botulinum outbreak.
Conclusion:  These PCR tests are specific and reliable for detection of heterogeneous BoNT producing clostridia responsible for human botulism.
Significance and Impact of the Study:  Adoption of these PCR assays is a step forward a reliable and rapid detection of these clostridia in food samples.     

A one step PCR-based method for the detection of economically important soft rot Erwinia species on micropropagated potato plants

A simple, rapid and sensitive PCR-based method was developed for the detection of all five subspecies of Erwinia carotovora , including subsp. carotovora and subsp. atroseptica , and all pathovars/biovars of Erwinia chrysanthemi , on plant tissue culture material. Primers SR3F and SR1cR, based on a conserved region of the 16S rRNA gene, amplified a DNA fragment of 119 bp from all 65 such strains tested. Detection limits of the method in vitro were 2·0 × 10²–3·4 × 10³ cfu ml⁻¹ (equivalent to 1–17 cfu per PCR) and, following extraction of genomic DNA from plant extract, detection limits were 2·3 × 10²–1·9 × 10⁴ cfu per microplant sample (equivalent to 5 cfu – 3·8 × 10² cfu per PCR). To improve the sensitivity of the method in planta , to obviate the need for complex and laborious DNA extractions, and to remove inhibitory substances present in the plant extract, an enrichment step was included prior to PCR. Following enrichment, the sensitivity of detection was <10 cfu per microplant sample. This method provides the first sensitive means of detecting latent infection caused by several economically important soft rot erwinias simultaneously on potato tissue culture material.     

Molecular typing and identification of Bdellovibrio-and-like organisms isolated from seawater shrimp ponds and adjacent coastal waters

Aims:  To apply and compare two PCR-based methods for typing saltwater Bdellovibrio- and-like organisms (BALOs) and to understand ecological and phylogenetic aspects of the BALOs isolated from shrimp mariculture systems.
Methods and Results:  Using double-layer agar technique, the numbers of culturable BALOs that lyse Vibrio alginolyticus were found to be 10–10³ PFU ml⁻¹ in the surface water samples. A total of 130 BALOs isolates were differentiated into five phylotypes by denaturing gradient gel electrophoresis targeting the 16S rDNA V3 region and four phylotypes by amplified rDNA restriction analysis of the Bacteriovoracaceae -specific 16S rDNA fragment respectively. Phylogenetic analysis of representative isolates showed that all of them were identified as Bacteriovorax spp., but affiliated with four different clusters in the family Bacteriovoracaceae.
Conclusions:  The two PCR-based methods both can be chosen to rapidly type the saltwater BALOs at cluster level. And the relatively large numbers of BALOs with various phylotypes recovered from the same habitats suggested that these predators might play important ecological role in shrimp mariculture environments.
Significance and Impact of the Study:  We proposed two effective methods to distinguish rapidly large numbers of BALOs isolates and our results would be helpful to understand the diversity and function of BALOs in mariculture environments.     

Longevity of the freshwater anostracan Streptocephalus mackini (Crustacean: Anostraca) in relation to food (Chlorella vulgaris) concentration

SUMMARY 1. Temporary ponds are inhabited by a variety of invertebrates, of which anostracans are an important group. We studied the lifetables of male and female anostracan Streptocephalus mackini at 3 algal concentrations (0.5 × 10⁶, 1.0 × 10⁶ and 1.5 × 10⁶ cells mL⁻¹).
2. Regardless of sex, S. mackini showed better survivorship at lower food levels. The longest average lifespan observed was 85 ± 2 days for males fed Chlorella at 0.5 × 10⁶ cells mL⁻¹.
3. Both net reproductive rate and generation time decreased with increasing food level. The highest net reproductive rate was about 120 cysts per female. The longest generation time of about 40 days, observed at 0.5 × 10⁶ cells mL⁻¹, was more than three times that at 1.5 × 10⁶ cells mL⁻¹.
4. The rate of population increase ( r ) was nearly the same (0.31 ± 0.06) at high (1.5 × 10⁶ cells mL⁻¹) and intermediate (1.0 × 10⁶ cells mL⁻¹) food levels. The r -value at low food level (0.5 × 10⁶ cells mL⁻¹ of Chlorella ) was 0.20 ± 0.01 per day.     

Persistence of Shiga toxin-producing Escherichia coli O26 in various manure-amended soil types

Aims:  To evaluate the behaviour of Shiga toxin-producing Escherichia coli (STEC) O26 strains inoculated in manure-amended soils under in vitro conditions.
Methods and Results:  Four green fluorescent protein (GFP)-labelled STEC O26 strains were inoculated in duplicate (at 10⁶ CFU g⁻¹) in three different manure-amended soil types, including two loam soils (A and B) and one clay loam soil (C), and two incubation temperatures (4 and 20°C) were tested. STEC counts and soil physical parameters were periodically monitored. STEC O26 cells were able to persist during extended periods in soil even in the presence of low moisture levels, i.e. less than 0·08 g H₂O g⁻¹ dry soil. At 4 and 20°C, STEC could be detected in soil A for 288 and 196 days, respectively, and in soils B and C for at least 365 days postinoculation at both temperatures. The ambient temperature (i.e. 20°C) was significantly associated with the highest STEC count decline in all soils tested.
Conclusions:  The temperature and soil properties appear to be contributory factors affecting the long-term survival of STEC O26 in manure-amended soils.
Significance and Impact of the Study:  This study provides useful information regarding the ecology of STEC O26 in manure-amended soils and may have implications for land and waste management.     

Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to detect ultra low population of Ralstonia solanacearum (Smith 1896) Yabuchi et al. (1996)

Aims:  To develop a reliable and sensitive protocol for detection of Ralstonia solanacearum using MDA-PCR (Multiple displacement amplification–PCR amplification).
Methods and Results:  MDA-PCR technique was performed on pure cell lysates as well as soil samples. Pure cell lysate as well as that of soil DNA was used as template in MDA reaction. MDA of template DNA was carried out in the presence of sample buffer, reaction buffer and enzyme mix (Φ 29 DNA polymerase and random hexamers). The MDA amplified DNA was used for PCR amplification using R. solanacearum -specific PCR primers. MDA-PCR could detect as low as 1 colony forming unit (CFU ml⁻¹) of bacteria within 8 h including DNA isolation.
Conclusion:  MDA followed by standard PCR facilitated the detection of pathogen from very low count samples. The method is of great importance in managing the brown rot disease of potato.
Significance and Impact of study:  The ultrasensitive detection technique developed in the present study is sensitive and speedy enough to be included into integrated wilt disease control programmes.