BACKGROUND Buruli ulcer (BU) caused by Mycobacterium ulcerans (M. ulcerans) has emerged as an important public health problem in several rural communities in sub-Saharan Africa. Early diagnosis and prompt treatment are important in preventing disfiguring complications associated with late stages of the disease progression. Presently there is no simple and rapid test that is appropriate for early diagnosis and use in the low-resource settings where M. ulcerans is most prevalent. METHODOLOGY We compared conventional and pocket warmer loop mediated isothermal amplification (LAMP) methods (using a heat block and a pocket warmer respectively as heat source for amplification reaction) for the detection of M. ulcerans in clinical specimens. The effect of purified and crude DNA preparations on the detection rate of the LAMP assays were also investigated and compared with that of IS2404 PCR, a reference assay for the detection of M. ulcerans. Thirty clinical specimens from suspected BU cases were examined by LAMP and IS2404 PCR. PRINCIPAL FINDINGS The lower detection limit of both LAMP methods at 60°C was 300 copies of IS2404 and 30 copies of IS2404 for the conventional LAMP at 65°C. When purified DNA extracts were used, both the conventional LAMP and IS2404 PCR concordantly detected 21 positive cases, while the pocket warmer LAMP detected 19 cases. Nine of 30 samples were positive by both the LAMP assays as well as IS2404 PCR when crude extracts of clinical specimens were used. CONCLUSION/SIGNIFICANCE The LAMP method can be used as a simple and rapid test for the detection of M. ulcerans in clinical specimens. However, obtaining purified DNA, as well as generating isothermal conditions, remains a major challenge for the use of the LAMP method under field conditions. With further improvement in DNA extraction and amplification conditions, the pwLAMP could be used as a point of care diagnostic test for BU.

We developed and evaluated the specificity and sensitivity of a loop-mediated isothermal amplification (LAMP) method for rapid detection of the food-borne Escherichia coli O157 strains. Six primers, including outer primers, inner primers and loop primers, were specially designed for recognizing eight distinct sequences on three targets, which were rfbE, stx1 and stx2. The detection limits were found to be 100, 100 and 10 fg DNA/tube for rfbE, stx1 and stx2, respectively. Application of LAMP assays were performed on 417 food-borne E. coli strains, the sensitivity of LAMP assays for the rfbE, stx1 and stx2 was 100, 95.3 and 96.3%, and the negative predictive value was 100, 96.7 and 97.1%, respectively; with a 100% specificity and positive predictive value for all three targets.

Class 1 and 2 integrons were detected in 45.8%(54/118) and 19.5%(23/118) of our tested Pseudomonas aeruginosa isolates, respectively. Three strains were positive for both the integrons. This is the first report of class 2 integrons in P. aeruginosa and also of isolates carrying class 1 and 2 integrons simultaneously.

Six methicillin-resistant Staphylococcus aureus MRSA strains from two nosocomial infection cases described in a previous study [15], of which two occurred in March and the other four in May, 2005, were found to possess one copy of class 1 integron with aadA2 gene cassette located on chromosomes by Southern hybridization. Polymerase chain reaction (PCR) detection of mecA and pvl, SCCmec typing, multilocus sequence typing (MLST), spaA typing and coa typing were also performed. The results revealed 6 MRSA fell into the ST239-MRSA-III group (clonal complex 8), with the spaA type GKAOMQ and coa type HIJKL, whereas the pvl locus was not detected. DNA fingerprinting analysis by random amplified polymorphic DNA-PCR using three different assays were also performed, and all strains exhibited identical patterns, indicating that they were clonally related and might be mainly due to a specific clone in the hospital. This was the first time, to our knowledge, that class 1 integron-bearing MRSA (I-MRSA), simultaneously carrying two mobile genetic elements was confirmed: class 1 integron and SCCmec.

The aim of the present study was to determine the source of nosocomial outbreak due to Burkholderia cepacia by molecular techniques. A total of 11 B. cepacia strains were isolated; nine from blood and one from sputum of patients without cystic fibrosis, and one from reverse osmosis water at a local hospital in Guangzhou, China. Analyses of 11 strains by the Sau-PCR assay and pulsed-field gel electrophoresis revealed that nine strains obtained from the blood of outpatients in a hemodialysis unit and one strain from reverse osmosis water had identical DNA profiles, indicating that the reverse osmosis water supply could be a source of infection.

A total of 53 methicillin-resistant coagulase-negative staphylococci strains isolated in a hospital in Guangzhou, China, were analyzed to detect class 1 integrons and SCCmec typing. Thirty strains had the class 1 integrase (intI1) gene and 26 strains possessed the 3' conserved region of qacEDelta1-sul1. Four different types of gene cassette arrays were found and a highly prevalent array of dfrA12-orfF-aadA2 gene cassettes was observed. Thirty class 1 integron-positive coagulase-negative staphylococci strains were subjected to Southern hybridization analysis; the result showed that class 1 integrons were located on chromosome, not plasmid. According to the results of SCCmec typing for 30 integron-bearing MRCNS strains, five, 15 and five strains belonged to type I, II and III SCCmec, respectively, and five strains were untypeable. For 23 non-integron-bearing methicillin-resistant coagulase-negative staphylococci strains, four, nine and seven strains belonged to type I, II and III SCCmec, respectively, and three strains were untypeable. None of the strains belonged to type IV or V. Twenty-three coagulase-negative staphylococci isolates of three Staphylococcal species that contained the dfrA12-orfF-aadA2 gene cassette array were phylogenetically unrelated to each other by randomly amplified polymorphic DNA, indicating that the gene cassettes might be disseminated in the clinical strains by a horizontal gene transfer.

During 2003 to 2004, class 1 integron was detected in 8 out of 10 tested enterococci isolates, with 2 of them positive for class 2 integron. This is the first report of class 2 integron in Enterococcus faecalis and class 1 integron in Enterococcus faecium.

A total of 46 gram-positive bacteria isolated from clinical specimens collected in China were subjected to PCR analysis with the intI1-specific primers, and the intI1-positive strains were further analyzed for their resistance gene cassette. All isolates possessed the class 1 integron in their genomes and the array of gene cassettes was dhfrXII-orfF-aadA2, which is very similar to other organisms except in one isolate carrying an additional copy of the class 1 integron containing the aadA2 gene cassette. Altogether, the results indicate that the class 1 integron is widespread in gram-positive clinical strains isolated in Guangzhou, China.

Twenty-three strains of Salmonella spp. isolated from healthy humans in Guangdong, China, were examined for their susceptibility to ten common antibiotics and the presence of antibiotic resistance integrons. All the strains were resistant to at least one antibiotic, and 4 strains were positive for the intI1 gene. Polymerase chain reaction using in-F and in-B primers showed the existence of amplicons of 1,009 bp in two, 1,664 bp in one, and 1,009 bp and 1,664 bp in one of the intI1 -positive isolates, respectively. Sequence analysis revealed that the 1,009-bp amplicon harbored gene cassette aadA2, conferring resistance to spectinomycin, and the 1,664-bp amplicon harbored genes aadA5 and dfr17, conferring resistance to spectinomycin, streptomycin and trimethoprim. Meanwhile the experiments of plasmid conjugation and Southern hybridization with intI1 as the DNA probe indicated that all the integrons found in these strains were chromosomal. Because the strains carrying class 1 integrons were isolated from healthy humans, it suggests the need for all-round surveillance of the antibiotic resistance of pathogens.

Laribacter hongkongensis is a novel emerging pathogen associated with human gastroenteritis. We aimed to investigate the prevalence, antimicrobial resistance and genotypic relationship of 199 L. hongkongensis isolates from 690 intestinal samples of fish and frogs. These samples were collected from retail markets in the city of Guangzhou in southern China from October 2008 to September 2009. L. hongkongensis was detected in from 80 (16.3%) out of 490 freshwater fish, and this number included 76 (32.3%) out of 235 grass carp and 4 (14.8%) out of 27 bighead carp. A higher isolation rate of 59.5%(119 out of 200) was observed in edible frogs. The isolation rate was highest in the spring in comparison with other seasons. Notably, 63.8% of the isolates were resistant to at least one class of antimicrobial agents. Analysis by pulsed-field gel electrophoresis (PFGE) revealed that the isolates could be grouped into three clusters. Isolates from fish intestines were grouped into two clusters: cluster I and II. Isolates of frog-origin and several fish-origin isolates were grouped into cluster III. Two patient-derived strains could be classed into cluster III. Extensive genetic heterogeneity among the isolates was observed. The results indicate that L. hongkongensis isolates exhibits host tropism, extensive resistance to widely used antimicrobials and diverse biological evolution in an aquatic environment. The frog is more likely than the freshwater fish to be the potential source for human infection with L. hongkongensis.

A loop-mediated isothermal amplification (LAMP) method for rapid detection of various Staphylococcus strains and associated antibiotic resistance determinant had been developed and evaluated in this study. Six primers, including outer primers, inner primers and loop primers, were specially designed for recognizing eight distinct sequences on three targets: 16SrRNA, femA and mecA.. Forty-one reference strains, including various species of gram-negative and -positive isolates, were included in this study to evaluate and optimize LAMP assays. The optimal reaction condition was found to be 65 °C for 45 min, with detection limits at 100 fg DNA/tube and 10 CFU/reaction for 16S rRNA, 100 fg DNA/tube and 10 CFU/reaction for femA, 1 pg DNA/tube and 100 CFU/reaction for mecA, respectively. Application of LAMP assays were performed on 118 various types of Staphylococcus isolates, the detection rate of LAMP assays for the 16SrRNA, femA and mecA was 100%(118/118), 98.5%(64/65) and 94.3%(66/70), and the negative predictive value (NPV) was 100%, 98.1% and 92.3% respectively; with a 100% positive predictive value (PPV) for all three targets. In conclusion, LAMP assays were demonstrated to be useful and powerful tools for rapid detection of various Staphylococcus strains, and undoubtedly, the rapidness, technical simplicity, and cost-effectiveness of LAMP assays will demonstrate broad application for bacteriological detection of food-borne Methicillin-resistant Staphylococcus (MRS) isolates.

Nucleotide-based analytical approaches are indispensable and effective, targeting for the transgenic ingredients in biotechnical products in terms of safety assessment. In this study, a loop-mediated isothermal amplification method was developed for the specific detection of exogenous nucleic acids of hLTF/hLALBA-induced transgenic cattle. The detection limit of the LAMP method was proved to be as low as 10 copies of target molecules in optimized systems, and to be 10-100 times more sensitive than the conventional PCR. Furthermore, fluorescent dye SYBR Green I was used to visualize the color changes of LAMP products by naked eyes in daylight, which resulted in distinct colors between positive and negative reactions. For the detection of transgenes, all the transgenic samples collected from hLTF and hLALBA-induced cattle were amplified by LAMP in 1 h, followed by direct visual SYBR Green I dying or gel electrophoresis. Results showed that transgenic and non-transgenic samples exhibited distinct properties in colors or electrophoresis profiles. Thus, all the results indicated that the LAMP assay was a simple and convenient method for the test of transgenic animals.

A loop-mediated isothermal amplification (LAMP) assay was developed for detection of bovine arvovirus (BPV) DNA. Four primers were designed to recognize six distinct regions on the target DNA based on a highly conserved sequence in the VP2 region of the BPV genome. The optimized LAMP reaction conditions were 8mM Mg(2+), 1.2mM betaine, and an incubation at 63°C for 45min. After amplification the products were detected either by observing a ladder pattern following gel electrophoresis, observation of turbidity, or a color change with the addition of SYBR Green I to the reaction tube. The detection limit of the LAMP assay was 9 copies of BPV-DNA and was 100 times more sensitive than conventional PCR. A ladder pattern of bands after gel electrophoresis was observed for only BPV isolates and showed that the BPV LAMP assay was highly specific without any cross-reactivity with other related viruses. The LAMP assay was evaluated further using 59 field samples and the results were comparable to conventional PCR. The LAMP assay is a simple, rapid and economic detection method; it can provide a useful technique suitable for detection of BPV infection in both field conditions and laboratory settings.

We developed and evaluated the specificity and sensitivity of a loop-mediated isothermal amplification (LAMP) method for rapid detection of the multidrug-resistance gene cfr. A pair of outer primers and a pair of inner primers and one loop primer were specially designed for recognizing seven distinct sequences on the target cfr gene. The amplification reaction was performed within only 35min under isothermal conditions at 63°C in a regular water bath with visual measurement. The LAMP assay showed higher sensitivity than the conventional PCR, with a detection limit of 1pg per tube of chicken Staphylococcus sciuri genomic DNA. The detection rate of cfr gene for 50 Staphylococcus clinical strains by LAMP assays was 16% and appeared 100% consistence with the result by PCR method. The LAMP method reported here demonstrated a potential and valuable means for detection of the multidrug-resistance gene cfr: easy, rapid, visual, specific, accurate and sensitive, especially useful for on-the-spot investigation.

A rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) method was developed and evaluated for the detection of Marek's disease virus (MDV) by amplification of conserved MDV meq gene sequences. LAMP is an innovative technique that allows the rapid detection of targeted nucleic acid sequences under isothermal conditions without the need for complex instrumentation. In this study, meq gene sequences were amplified successfully from different MDV strains by LAMP within 60min and no cross-reactivity was observed in a panel of related viruses that were associated with diseases of chickens. The detection limit of LAMP was 3.2copies/million cells compared with 320copies/million cells required for conventional PCR. Positive detection rates were assessed using either LAMP or PCR by examination of feather follicles that were collected from chickens infected experimentally with either strain J-1 (n=20) or strain Md5 (n=17), In addition to these samples, three isolates that were suspected to have been infected in the clinic were also tested. Results showed that the positive detection rate for LAMP was 95%(38/40), compared with 87.5%(35/40) and 90%(38/40) for strains J-1 and Md5 by PCR, respectively. These results indicated that the LAMP assay was more sensitive, rapid and specific than conventional PCR for the detection of MDV. This easy-to-perform technique will be useful for the detection of MDV and will aid in the establishment of disease control protocols.

In recent years, the large yellow croaker (Pseudosciaena crocea), an important marine fish farmed in the coastal areas of Zhejiang province, east China, has become severely endangered as a result of the bacterial pathogen Pseudomonas putida. This paper reports the development of a visual loop-mediated isothermal amplification (LAMP) assay for rapid detection of the pathogen. Four primers, F3, B3, FIP and BIP, were designed on the basis of DNA sequence of the rpoN gene of P. putida. After optimization of the reaction conditions, the detection limit of LAMP assay was 4.8cfu per reaction, 10-fold higher than that of conventional PCR. The assay showed high specificity to discriminate all P. putida isolates from nine other Gram-negative bacteria. The assay also successfully detected the pathogen DNA in the tissues of infected fish. For visual LAMP without cross-contamination, SYBR Green I was embedded in a microcrystalline wax capsule and preset in the reaction tubes; after the reaction the wax was melted at 85°C to release the dye and allow intercalation with the amplicons. The simple, highly sensitive, highly specific and cost-effective characteristics of visual LAMP may encourage its application in the rapid diagnosis of this pathogen.

A loop-mediated isothermal amplification (LAMP) method was developed for the rapid and sensitive detection of the emerging resistance gene New Delhi Metallo-β-lactamase-1 (NDM-1), with its specificity and sensitivity having been evaluated. Six primers, including a pair of outer primers, a pair of inner primers, and a pair of loop primers, were specially designed for recognizing eight distinct sequences on the target NDM-1 gene. The amplification reaction was performed within only 40 min under isothermal conditions at 65°C in a regular water bath. The LAMP assay showed good specificity and higher sensitivity than the conventional polymerase chain reaction (PCR), with a detection limit of 1 pg genomic DNA per tube of one NDM-1-positive reference strain. The detection result for the 345 clinical samples showed 100% consistence with the result by the PCR method, and three contaminated samples could be detected correctly by LAMP assays, while they could not be detected by PCR. The LAMP method reported here demonstrated a potential and valuable means for detection of the NDM-1 gene: easy, rapid, visual, specific, accurate, and sensitive, especially useful for on-the-spot investigation.

Loop-mediated isothermal amplification (LAMP) allows a rapid amplification of nucleic acids under isothermal conditions. It can be combined with a rhodamine-based dual chemosensor for much more efficient, field-friendly detection of Listeria monocytogenes. In this report, LAMP was performed at 63 °C for 10 min, followed by a rapid reaction of DNA amplification and the byproduct, pyrophosphate ion, with a rhodamine-based dual chemosensor and Cu(2+) is visualized as a disappearance of red color. The detection limit of L. monocytogenes by the LAMP-chemosensor was 8 to 10 cells per reaction tube, and the total assay time including 10 min for rapid DNA extraction was approximately 30 min. Data on naturally contaminated raw milk samples indicated that the LAMP method was highly specific and sensitive, giving 100% concordance with the ISO 10560 reference method. The results showed that the LAMP-chemosensor method has the advantages of better sensitivity and speed and less dependence on equipment than the standard Polymerase Chain Reaction for specifically detecting low levels of L. monocytogenes DNA, and this can be useful in the field as a routine diagnostic tool. PRACTICAL APPLICATION: The LAMP-chemosensor method reported here provided a powerful tool for detection of L. monocytogenes in raw milk samples due to its specificity, sensitivity, and rapidity.

Legionella pneumophila is accounted for more than 80% of Legionella infection. However it is difficult to discriminate between the L. pneumophila and non-L. pneumophila species rapidly. In order to detect the Legionella spp. and distinguish L. pneumophila from Legionella spp., a real-time loop-mediated isothermal amplification (LAMP) platform that targets a specific sequence of the 16S rRNA gene was developed. LS-LAMP amplifies the fragment of the 16S rRNA gene to detect all species of Legionella genus. A specific sequence appears at the 16S rRNA gene of L. pneumophila, while non-L. pneumophila strains have a variable sequence in this site, which can be recognized by the primer of LP-LAMP. In the present study, 61 reference strains were used for the method verification. We found that the specificity was 100% for both LS-LAMP and LP-LAMP, and the sensitivity of LAMP assay for L. pneumophila detection was between 52 and 5.2 copies per reaction. In the environmental water samples detection, a total of 107 water samples were identified by the method. The culture and serological test were used as reference methods. The specificity of LS-LAMP and LP-LAMP for the samples detection were 91.59%(98/107) and 93.33%(56/60), respectively. The sensitivity of LS-LAMP and LP-LAMP were 100%(51/51) and 100%(18/18). The results suggest that real-time LAMP, as a new assay, provides a specific and sensitive method for rapid detection and differentiation of Legionella spp. and L. pneumophila and should be utilized to test environmental water samples for increased rates of detection.

Streptococcus iniae is a major pathogen that causes sever economic losses in tilapia aquaculture. A set of four specific primers was designed by targeting lctO gene. With Bst DNA polymerase, the target DNA can be clearly amplified for 60 min at 64 °C in a simple water bath. The sensitivity of the LAMP assay for the detection of S. iniae is about 12.4 cells per reaction in both of pure cultures and added fish tissues cultures. LAMP products could be judged with agar gel or naked eye after addition of SYBR Green I. There were no cross-reactions with other bacterial strains indicating high specificity of the LAMP. The LAMP method was also applied to detect S. iniae-infected tilapia tissues effectively. The LAMP assay reported here indicates the potential usefulness of the technique as a valuable simple, rapid alternative procedure for the detection of S. iniae during streptococcicosis monitoring of cultured fish.

Anaplasma ovis is an intraerythrocytic rickettsial pathogen of small ruminants. Loop-mediated isothermal amplification (LAMP) is a nucleic acid detection method in which the target DNA can be efficiently amplified with high specificity and sensitivity under isothermal conditions. In this study, a LAMP method was developed for the specific detection of A. ovis, using LAMP primers designed on the basis of the major surface protein 4 gene. LAMP was performed at 65 °C for 30 min. Its specificity was confirmed by successful amplification of several A. ovis isolates and through EcoRI restriction analysis of LAMP products. No cross-reactivity with the A. marginale Lushi isolate, Mycoplasma mycoides subsp. capri, Chlamydophila psittaci, Theileria ovis, T. luwenshuni, T. uilenbergi, or the Babesia sp. Xinjiang isolate was observed. Detection using the LAMP method was compared with that using conventional PCR in 227 field samples; LAMP demonstrated a sensitivity of 95.45%. In summary, LAMP is a specific, sensitive, and rapid test for the diagnosis of A. ovis infection, with the potential to be standardized as a detection method for A. ovis in areas of endemicity.