Rapid and sensitive detection of Acidovorax citrulli in cucurbit seeds by visual loop‐mediated isothermal amplification assay

A rapid, sensitive and visual loop‐mediated isothermal amplification (LAMP) method for detecting Acidovorax citrulli in cucurbit seed was developed in this study. The LAMP primers were designed to recognize the non‐ribosomal peptide synthetase (NRPS) gene (locus tag: Aave_4658) from A. citrulli. The LAMP assay was conducted at 64°C in 1 hr with calcein as an indicator. The sensitivity and specificity of the LAMP assay were further compared with those of a conventional polymerase chain reaction (PCR). The LAMP assay is highly specific to A. citrulli, and no cross‐reaction was observed with other bacterial pathogen. The sensitivity of the LAMP assay was 100‐fold higher than that of conventional PCR with a detection limit of 1 pg of genomic DNA. Using the LAMP assay, 7 of 12 cantaloupe seedlots collected from Xinjiang province were determined to be positive for A. citrulli. In contrast, only 2 of 12 seedlots showed positive for the pathogen with conventional PCR. Moreover, A. citrulli was detected in 100% of artificially infested seedlots with 0.01% infestation or greater. Our results demonstrated that the LAMP assay was simple, visual and sensitive for detecting A. citrulli, especially in seed health testing. Hence, this method has great potential application in routine detecting seed‐borne pathogens and reducing the risk of epidemics.     

Rapid and Sensitive Detection of Listeria ivanovii by Loop-Mediated Isothermal Amplification of the smcL Gene

A loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of the L. ivanovii strains had been developed and evaluated in this study. Oligonucleotide primers specific for L. ivanovii species were designed corresponding to smcL gene sequences. The primers set comprise six primers targeting eight regions on the species-specific gene smcL. The LAMP assay could be completed within 1 h at 64°C in a water bath. Amplification products were directly observed by the Loopamp Fluorescent Detection Reagent (FD) or detected by agarose gel electrophoresis. Moreover, the LAMP reactions were also detected by real-time measurement of turbidity. The exclusivity of 77 non-L. ivanovii and the inclusivity of 17 L. ivanovii were both 100% in the assay. Sensitivity of the LAMP assay was 250 fg DNA and 16 CFU per reaction for detection of L. ivanovii in pure cultures and simulated human stool. The LAMP assay was 10 and 100-fold more sensitive than quantitative PCR (qPCR) and conventional PCR assays,respectively. When applied to human stool samples spiked with low level (8 CFU/0.5 g) of L. ivanovii strains, the new LAMP assay described here achieved positive detection after 6 hours enrichment. In conclusion, the new LAMP assay in this study can be used as a valuable, rapid and sensitive detection tool for the detection of L. ivanovii in field, medical and veterinary laboratories.     

Development and application of a simple loop-mediated isothermal amplification method on rapid detection of Listeria monocytogenes strains

A loop-mediated isothermal amplification (LAMP) method for rapid detection of the food-borne L. monocytogenes strains had been developed and evaluated in this study. The optimal reaction condition was 65°C for 45 min, with the detection limit as 1 pg DNA/tube and 100 CFU/reaction. Application of the established LAMP assay was performed on 182 food-borne L. monocytogenes strains using a rapid procedure and easy result confirmation, with the sensitivity of LAMP versus PCR assays as 96.7% (176/182) and 91.2% (166/182), respectively; with 100% specificity, positive predictive value (PPV) and negative predictive value (NPV) for both assays.     

Development of a loop-mediated isothermal amplification assay for detection of <Emphasis Type="Italic">Cronobacter</Emphasis> spp. (<Emphasis Type="Italic">Enterobacter sakazakii</Emphasis>)

Contamination of Cronobacter spp. (Enterobacter sakazakii) in infant formulas and other food products is a severe problem. Here a loop-mediated isothermal amplification (LAMP) assay was developed for rapidly detecting Cronobacter spp. in powdered infant formula. Sequences of 16S/23S rDNA internal intergenic spacer of Cronobacter spp. were used as the target template to design LAMP primers. The detection outcome can be evaluated by the white precipitate or the fluorescence intensity under ultraviolet irradiation, both visible to naked eyes. The sensitivity and specificity of the LAMP assay was further analyzed in comparison with that of regular PCR and real time quantitative PCR. The results showed that all of Cronobacter spp. strains display positive reaction to the detections while all of the non-Cronobacter spp. strains were negative, and that the LAMP assay exhibits a high sensitivity of 9.1 fg/μL (The sensitivity of regular PCR and real time quantitative PCR is 91 and 9.1 pg/μL, respectively.). The amplified reaction could be accomplished in about 1 h, with the results visible to naked eyes. Hence, the LAMP assay developed by this study can provide a rapid and simple approach for the detection of Cronobacter spp. in infant formula.     

Real-time loop-mediated isothermal amplification assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder

Loop-mediated isothermal amplification (LAMP) assay is a powerful and innovative gene amplification technique that specifically amplifies the target gene under isothermal conditions with a high degree of sensitivity, rapidity and specificity. The major advantage of the LAMP assay is monitoring of amplified products without the requirement of any sophisticated equipment. In the present study a real time LAMP assay was employed for rapid and real time detection of Bacillus anthracis spores spiked in 0.1 g of soil and talcum powder ranging from 2 to 10⁷ spores. DNA was isolated from spiked soil and talcum powder using PBS containing 1% Triton X-100, and heat treatment. Isolated DNA was used as template for LAMP and PCR. LAMP amplification was obtained in 60 min under isothermal condition at 63°C by employing a set of six primers targeting the pag gene of B. anthracis. The detection limit of LAMP assay in soil and talcum powder was found to be as low as 5 spores, compared to 10³ spores and 10⁴ spores by PCR in talcum powder and soil, respectively. The findings suggest that LAMP is a more rapid and sensitive assay than PCR for detecting anthrax spores, additionally the methodology to prepare DNA from spiked samples is simple, rapid and cost effective.     

Specific and Sensitive Detection of Phytophthora nicotianae by Nested PCR and Loop‐mediated Isothermal Amplification Assays

Phytophthora nicotianae is an important soilborne plant pathogen. It causes black shank in tobacco and other commercially important crop diseases. Early and accurate detection of P. nicotianae is essential for controlling these diseases. In this study, primers based on the Ras‐related protein gene (Ypt1) of P. nicotianae were tested for their specific detection of the pathogen using nested PCR and LAMP assays. For specificity testing, DNA extracts from 47 P. nicotianae isolates, 45 isolates of 16 different oomycetes and 25 isolates of other fungal species were used; no cross‐reaction with other pathogens was observed. The sensitivity assay showed that the nested PCR and LAMP assays had detection limits of 100 fg and 10 fg genomic DNA per 25‐μl reaction, respectively. Furthermore, the nested PCR and LAMP assays were used for the detection of DNA from naturally P. nicotianae‐infected tobacco tissues and soil. Our results suggest that the LAMP assay has the greatest potential for the specific detection of P. nicotianae in regions that are at risk of contracting tobacco black shank disease and that the Ypt1 gene is a novel and effective target of P. nicotianae LAMP visual detection.     

Development of a new loop-mediated isothermal amplification assay for <Emphasis Type="Italic">prt</Emphasis> (<Emphasis Type="Italic">rfbS</Emphasis>) gene to improve the identification of <Emphasis Type="Italic">Salmonella</Emphasis> serogroup D

Loop-mediated isothermal amplification (LAMP) is a promising nucleic acid assay for rapid and cost-effective detection of pathogen-specific sequences within a sample. Development of an appropriate taxonomic group-specific LAMP assay highly relies on the design of proper primers to cover all major members of the taxon. Regarding this fact, we designed and evaluated a new LAMP primer set specific to prt (rfbS) gene for rapid identification of Salmonella serogroup D serotypes. Unlike the previously reported LAMP assay for serogroup D which detects solely the non-typhoidal serotypes; the new LAMP primers set detects both typhoidal and non-typhoidal serotypes of this serogroup with a detection limit of 10 CFU/rection. Furthermore, the technique was successfully applied to artificially contaminated meat samples with an inoculation level of 1–5 CFU/250 ml of Salmonella Enteritidis, following a 5-h pre-enrichment step in tryptic soy broth. Overall, the new LAMP assay and its optimized setup would be useful for fast diagnosis of food poisoning incidents caused by these bacteria.     

Development of a Real‐time Fluorescence Loop‐mediated Isothermal Amplification Assay for Detection of Burkholderia gladioli pv. alliicola

Burkholderia gladioli pv. alliicola is a causal agent of rot on a wide range of hosts including onion and tulip. It is one of quarantine phytopathogenic bacteria in China. To reduce the economic losses associated with this pathogen, simple and rapid detection methods are needed. In this study, an efficient loop‐mediated isothermal amplification (LAMP) assay with a real‐time fluorometer was developed. The analysis of 16S‐23S rRNA intergenic transcribed spacer (ITS) sequences showed considerable variability between different Burkholderia species and B. gradioli pathovars. A set of LAMP primers was designed based on the ITS region. The sensitivity and specificity of the developed assay were evaluated at the optimal temperature of 65°C. The primers were specific for B. gladioli pv. alliicola and did not react to strains of others species and other pathovars in the species B. gladioli. The sensitivity of the real‐time LAMP assay was 1 fg DNA which was 100 times higher than that of conventional PCR. The method was verified by testing natural samples and inoculated onion seeds, and it showed effectiveness. The real‐time LAMP assay established in this study is an effective method for detection of B. gladioli pv. alliicola.     

Rapid Diagnosis of Soya Bean Root Rot Caused by Fusarium culmorum Using a Loop‐Mediated Isothermal Amplification Assay

We report a rapid diagnosis of soya bean (Glycine max L.) root rot caused by Fusarium culmorum, using a loop‐mediated isothermal amplification (LAMP) assay. We used the CYP51C gene sequence to design LAMP assay primers specific for F. culmorum. The LAMP assay amplified the target gene efficiently in 60 min at 63°C. The sensitivity of the assay was 100 pg/μl of genomic DNA. Among the tested soya bean pathogens, a positive colour (sky blue) was only observed in the presence of F. culmorum with the addition of hydroxynaphthol blue (HNB) dye prior to amplification, whereas other species isolates showed no colour change. Suspected diseased soya bean samples collected in the field from Jiangsu, Shandong and Anhui provinces and Beijing were diagnosed successfully using the LAMP assay reported here. This study provides a new and readily available method for rapid diagnosis of soya bean root rot caused by F. culmorum.     

Rapid Real-Time PCR Assay for Culture and Tissue Identification of <Emphasis Type="Italic">Geomyces destructans</Emphasis>: the Etiologic Agent of Bat Geomycosis (White Nose Syndrome)

Geomyces destructans is the etiologic agent of bat geomycosis, commonly referred to as white nose syndrome (WNS). This infection has caused severe morbidity and mortality in little brown bats (Myotis lucifugus) and has also spread to other bat species with significant decline in the populations. Currently, G. destructans infection is identified by culture, ITS–PCR, and histopathology. We hypothesized that a real-time PCR assay would considerably improve detection of G. destructans in bats. The 100 bp sequence of the Alpha-L-Rhamnosidase gene was validated as a target for real-time PCR. The assay sensitivity was determined from serial dilution of DNA extracted from G. destructans conidia (5 × 10⁻¹–5 × 10⁷), and the specificity was tested using DNA from 30 closely and distantly related fungi and 5 common bacterial pathogens. The real-time PCR assay was highly sensitive with detection limit of two G. destructans conidia per reaction at 40 PCR cycles. The assay was also highly specific as none of the other fungal or bacterial DNA cross-reacted in the real-time PCR assay. One hundred and forty-seven bat tissue samples, suspected of infection with G. destructans, were used to compare the real-time PCR assay to other methods employed for the detection of G. destructans. Real-time PCR was highly sensitive with 80 of 147 (55%) samples testing positive for G. destructans DNA. In comparison, histopathology examination revealed 64/147 (44%) positive samples. The internal transcribed spacer (ITS)–PCR yielded positive amplicon for G. destructans from 37 tissue samples (25%). The least sensitive assay was the fungal culture with only 17 tissue samples (12%) yielding G. destructans in culture. The data suggested that the real-time PCR assay is highly promising for rapid, sensitive, and specific identification of G. destructans. Further trials and inter-laboratory comparisons of this novel assay are recommended to improve the diagnosis of bat geomycosis.     

Rapid detection of Listeria monocytogenes in dairy samples utilizing a PCR-based fluorogenic 5′ nuclease assay

The presence of Listeria monocytogenes as a dairy food contaminant is a lethal threat to dairy industrialists; therefore, products tainted with L. monocytogenes must be quickly detected and removed from production. This fluorogenic PCR-based assay was developed to rapidly detect L. monocytogenes contamination in dairy samples before a final product is distributed. The detection method employed uses a PCR primer pair and a fluorogenic TaqMan probe which bind to a region of a virulence determinant gene specific to L. monocytogenes. As the DNA target is amplified, the 5′ nuclease activity of Taq DNA polymerase hydrolyzes the internal fluorogenic probe creating a change in fluorescence that can be monitored and automatically analyzed with a fluorometer. Sensitivity studies indicated a lower detection limit of under 10 CFU for pure culture extracts and spiked dairy enrichments. A study was performed on 266 dairy product samples obtained from Central California dairy production plants. Eighty-three of these samples were artificially spiked with both high and low concentrations of L. monocytogenes before an overnight enrichment in TSB/LiCl/colostin sulfate/moxalactam media. DNA from enriched samples was obtained using a rapid Chelex extraction specifically designed for dairy sample enrichments and automated analysis. The extraction was followed by the fluorogenic PCR assay and measurement of fluorescence increase. The assay was completed within 24 h, with an observed 95.2% sensitivity, 96.7% specificity, 92.9% positive predictive value, 97.8% negative predictive value, and 96.2% accuracy. According to specificity studies, five other bacterial species cross-reacted with the fluorogenic 5′ nuclease PCR. However, only one of these strains (Listeria grayi) was able to grow in the enrichment medium employed, and was not isolated from any of the 266 dairy product enrichments evaluated in this study. Therefore, this method provides a rapid, sensitive, and automatable analysis alternative to standard culture techniques for the detection of Listeria monocytogenes in dairy samples. Received 4 February 1998/ Accepted in revised form 1 October 1998     

Sensitive and rapid identification of Vibrio vulnificus by loop-mediated isothermal amplification

Vibrio vulnificus is a serious bacterial pathogen for humans and aquatic animals. We developed a rapid, sensitive and specific identification method for V. vulnificus using loop-mediated isothermal amplification (LAMP) technique. A set of primers, composed of two outer primers and two inner primers, was designed based on the cytolysin gene sequence of V. vulnificus. The LAMP reaction was processed in a heat block at 65 °C for 60 min. The amplification products were detected by visual inspection using SYBR Green I, as well as by electrophoresis on agarose gels. Our results showed that the LAMP reaction was highly specific to V. vulnificus. This method was 10-fold more sensitive than conventional PCR. In conclusion, the LAMP assay was extremely rapid, simple, cost-effective, sensitive and specific for the rapid identification of V. vulnificus.     

Loop‐mediated isothermal amplification method for rapid detection of Vibrio alginolyticus,the causative agent of vibriosis in mariculture fish

Aims: The purpose of this study was to develop a loop‐mediated isothermal amplification (LAMP) method for the rapid, sensitive and simple detection of Vibrio alginolyticus in mariculture fish. Methods and Results: LAMP primers were designed by targeting the gyrB gene. With Bst DNA polymerase, the target DNA can be clearly amplified for 60 min at 64°C in a simple water bath. The detection sensitivity of the LAMP assay for the detection of V. alginolyticus is about 3·7 × 10² CFU ml^?1 (3·7 CFU per reaction). LAMP products could be judged with agar gel or naked eye after the addition of SYBR Green I. There were no cross‐reactions with other bacterial strains indicating a high specificity of the LAMP. The LAMP method was applied to detect V. alginolyticus‐infected fish tissues effectively. Conclusions: The LAMP established in this study is a simple, sensitive, specific, inexpensive and rapid protocol for the detection of V. alginolyticus. Significance and Impact of the Study: This LAMP method provides an important diagnostic tool for the detection of V. alginolyticus infection both in the laboratory and field.     

Rapid detection of Trichinella spiralis larvae in muscles by loop-mediated isothermal amplification

Trichinella spiralis is a tissue-dwelling nematode parasite. A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the sensitive and rapid detection of T. spiralis larvae in muscle samples. Sixteen sets of primers were designed to recognise distinct sequences of a conserved gene, a 1.6 kb repetitive element of the Trichinella genome. One set of primers was selected as the most appropriate for rapid detection. The specificity and sensitivity of the primers in LAMP reactions for T. spiralis larvae and muscle samples of mice infected with T. spiralis were determined. Another 10 heterologous parasites were selected for specificity assays. The results showed that target DNA was amplified and visualised by monitoring turbidity and adding calcein detection methods within 70 min at an isothermal temperature of 63 °C. The sensitivity of LAMP with the detection limit of 362 fg/μl was >10 times higher than that for PCR. The designed primers had a good specificity. No cross-reactivity was found with the DNA of any other parasites. The assay was able to detect T. spiralis in all mouse muscle samples infected with 10 T. spiralis larvae on day 20 p.i. We believe this is the first report regarding the application of the LAMP assay for detection of T. spiralis larvae in muscle samples from experimentally infected mice. This method demonstrates a potentially valuable means for the direct detection of T. spiralis larvae in meat inspection.     

Rapid detection of Phytophthora nicotianae by simple DNA extraction and real‐time loop‐mediated isothermal amplification assay

Phytophthora nicotianae is a phytopathogenic oomycete with a wide host range and worldwide distribution. Rapid detection and diagnosis at the early stages of disease development are important for the effective control of P. nicotianae. In this study, we designed a simple and rapid loop‐mediated isothermal amplification (LAMP)‐based detection method for P. nicotianae. We tested three DNA extraction methods and selected the Kaneka Easy DNA Extraction Kit version 2, which is rapid and robust for LAMP‐based detection. The designed primers were tested using mycelial DNA from 35 species (81 isolates) of Phytophthora, 12 species (12 isolates) of Pythium, one isolate of Phytopythium and one isolate each from seven other soil‐borne pathogens. All of the 42 P. nicotianae isolates were detected by these primers, and no other isolates gave positive results. Three isolates were tested for the sensitivity of the reaction, and the lowest amounts of template DNA that could be detected were 10 fg for two isolates and 1 fg for the third. The target was detected within 25 min in all tested samples, including DNA extracted from both inoculated and naturally infected plants. In contrast, PCR assays with P. nicotianae‐specific primers failed or showed weakened detection in several samples. Thus, we found that the rapid DNA extraction and LAMP assay methods developed in this study can be used to detect P. nicotianae with high sensitivity, specificity and stability.     

Development of A Loop-Mediated Isothermal Amplification (LAMP) for the Detection of F5 Fimbriae Gene in Enterotoxigenic Escherichia coli (ETEC)

The objective of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the detection of F5 fimbriae gene in Enterotoxigenic Escherichia coli. A set of four primers were designed based on the conservative sequence of coding F5 fimbriae. Temperature and time condition, specificity test, and sensitivity test were performed with the DNA of Escherichia coli (F5+). The results showed that the optimal reaction condition for LAMP was achieved at 61 °C for 45 min in a water bath. Ladder-like products were produced with those F5-positive samples by LAMP, while no product was generated with other negative samples. The assay of LAMP had a detection limit equivalent to 72 cfu/tube, which was more sensitive than PCR (7.2 × 10² cfu/tube). The agreement rate between LAMP and PCR was 100 % in detecting simulation samples. Thus, the LAMP assay may be a new method for rapid detection of F5 fimbriae gene of ETEC.     

Rapid Detection of Banana Streak Virus by Loop‐mediated Isothermal Amplification Assay in South China

Banana streak virus (BSV) is a significant constraint to banana production and genetic improvement. It is necessary to develop and use BSV detection strategies that are both reliable and sensitive for the management of the virus. A loop‐mediated isothermal amplification (LAMP) assay was developed and evaluated for the detection of BSV. Four primers matching a total of six sequences of the conserved ORF III polyprotein genes were synthesized for developing a specific and sensitive LAMP for DNA extracts from field‐infected banana plants. LAMP assay could detect as low as 1 pg/μl template DNA. Test results of all field samples collected from different regions of South China showed that LAMP is more sensitive than PCR. This relatively simple and sensitive technique showed excellent potential with field‐collected samples and for routine screening of tissue culture materials in South China.     

A real‐time PCR assay for detection and quantification of Lactococcus garvieae

Aims: To develop a rapid, sensitive, specific tool for the detection and quantification of Lactococcus garvieae in food and environmental samples. Methods and Results: A real‐time quantitative PCR (qPCR) assay with primers for CAU12F and CAU12R based on the 16S rRNA gene of L. garvieae was successfully established. The limit of detection for L. garvieae genomic DNA was 1 ng DNA in conventional PCR and 32 fg with a mean C_T value of 36·75 in qPCR. Quantification of L. garvieae vegetative cells was linear (R² = 0·99) over a 7‐log‐unit dynamic range down to ten L. garvieae cells. Conclusions: This method is highly specific, sensitive and reproducible for the detection of L. garvieae compared to gel‐based conventional PCR assays, thus providing precise quantification of L. garvieae in food and natural environments. Significance and Impact of the Study: This work provides efficient diagnostic and monitoring tools for the rapid identification of L. garvieae, an emerging pathogen in aquaculture and an occasional human pathogen from other members of the genus Lactobacillus.