Detection Limit Of Loop-mediated Isothermal Amplification Research Articles

Rapid and visual detection of Shiga-toxigenic Escherichia coli (STEC) in carabeef meat harnessing loop-mediated isothermal amplification (LAMP).

Shiga toxigenic E. coli are important foodborne zoonotic pathogens. The present study was envisaged to standardize loop-mediated isothermal amplification assays targeting stx1 and stx2 genes for rapid and visual detection of STEC and compare its sensitivity with PCR. The study also assessed the effect of short enrichment on the detection limit of LAMP and PCR. The developed LAMP assays were found to be highly specific. Analytical sensitivity of LAMP was 94fg/µLand 25.8fg/µL for stx-1 and stx-2 while LOD of 5 CFU/g of carabeef was measured after 6-12h enrichment. The study highlights the importance of short (6-12h) enrichment for improving the sensitivity of LAMP. The entire detection protocol could be performed within 9h yielding results on the same day. The developed LAMP assays proved to be a handy and cost-effective alternative for screening STEC contamination in meat.

Rapid Diagnostic of Streptococcus suis in Necropsy Samples of Pigs by thrA-Based Loop-Mediated Isothermal Amplification Assay.

Streptococcus (S.) suis presents a serious threat to the pig industry as well as food safety and public health. Although several LAMP assays have been developed for the identification of S. suis, no universal assay is so far available for the field-suitable examination of clinical pig specimens. Based on the thrA housekeeping gene, a new loop-mediated isothermal amplification (LAMP) assay was developed and validated for the detection of S. suis in the brain and joints of pigs. For this LAMP assay, two different methods for the extraction of DNA from brain and joint swabs were compared. Using the LPTV boiling method, the detection limit of LAMP was 1.08 CFU/reaction, while the detection limit was 53.8 CFU/reaction using a commercial DNA extraction kit. The detection limits of thrA-LAMP in combination with the LPTV boiling method were 104-105 CFU/swab in the presence of brain tissue and 103-104 CFU/swab in the presence of joint tissue. The diagnostic quality criteria of LAMP were determined by the examination of 49 brain swabs and 34 joint swabs obtained during routine diagnostic necropsies. Applying the LPTV boiling method to brain swabs, the sensitivity, specificity, and positive and negative predictive values of thrA-LAMP were 88.0, 95.8, 95.7, and 88.5% using cultural investigation as a reference method, and 76.7, 100, 100, and 73.1% using real-time PCR as a reference method. Based on these results, the thrA-LAMP assay combined with the LPTV boiling method is suitable for rapid detection of S. suis from brain swabs.

Development and clinical application of a rapid and visual loop-mediated isothermal amplification test for tetM gene in Clostridioides difficile strains cultured from feces

ObjectivesIn this study, we aimed to develop a rapid and visual loop-mediated isothermal amplification (LAMP) assay targeting the tetM gene in Clostridioides difficile strains cultured from feces. MethodsPrimers were designed to recognize the tetM gene in C. difficile by LAMP, using turbidity and visual detection. The sensitivity and specificity of LAMP primers were determined. In addition, we conducted both LAMP and polymerase chain reaction (PCR) for the tcdA, tcdB, cdtA, cdtB, ermB, and tetM genes in 300 toxigenic C. difficile strains cultured from feces. ResultsThe target DNA was amplified and visualized within 60 minutes at a temperature of 62°C. A total of 26 bacterial strains were found negative for tetM, which manifested high specificity of the primers. The detection limit of LAMP was 36.1 pg/µl, which was 100-fold more sensitive than PCR. The positive rate of tetM in toxigenic C. difficile strains cultured from feces was 93.3% by both LAMP and PCR. The proportion of toxin types in those C. difficile strains was 95.7% for A+B+CDT−, 4% for A−B+CDT−, and 0.3% for A+B+CDT+, respectively. ConclusionThis is the first study examining the tetM gene by LAMP in C. difficile strains cultured from feces. Its high specificity, sensitivity, and visual detection make the new assay a powerful diagnostic tool for rapid testing.

Development of loop mediated isothermal amplification for rapid species detection of Armillaria ostoyae using assimilating probe

We introduced here the first loop mediated isothermal amplification (LAMP) assay for the identification of honey fungus, Armillaria ostoyae, a basidiomycote playing an important role in spruce declines in the Palaearctic region. In total, 101 isolates, representing three Armillaria species, were used to develop a new LAMP assay to determine species specific identification. We have here described LAMP primers enhanced with fluorescent dye that are able to amplify A. ostoyae DNA and detect fungi in a fast single step reaction. The detection limit of LAMP was 1 pg of genomic DNA per reaction. We optimized a new LAMP assay for the rapid detection of A. ostoyae using the translation elongation factor 1-α (tef1) marker and fluorescence labelled oligonucleotide assimilating probe. The LAMP assay does not require any specialized equipment, hence it can be used in the field for the rapid detection of A. ostoyae even using the portable and mobile device. The specificity of the assay was confirmed by the use of A. ostoyae strains and Armillaria cepistipes and Armillaria gallica strains, respectively. In conclusion, the assay could be a rapid, specific, sensitive and low-cost tool for identification of A. ostoyae as well as the first step for expansion of this method in practical applications.

Detection of the Benthic Dinoflagellates, Ostreopsis cf. ovata and Amphidinium massartii (Dinophyceae), Using Loop-Mediated Isothermal Amplification

For the in situ and sensitive detection of benthic dinoflagellates, we have established an integrated loop-mediated isothermal amplification (LAMP) assay based on Ostreopsis cf. ovata and Amphidinium massartii. To detect the two species, a set of species-specific primers was constructed between the ITS gene and D1–D6 LSU gene, and the reaction temperature, time, and buffer composition were optimized to establish this method. In addition, the specificity of the LAMP primers was verified both in strains established in the laboratory and in field samples collected from the Jeju coastal waters, Korea. With the LAMP assay, the analysing time was within 45 to 60 min, which may be shorter than that with the conventional PCR. The detection sensitivity of the LAMP assay for O. cf. ovata or A. massartii was comparable to other molecular assays (PCR and quantitative PCR (qPCR)) and microscopy examination. The detection limit of LAMP was 0.1 cell of O. cf. ovata and 1 cell of A. massartii. The optimized LAMP assay was successfully applied to detect O. cf. ovata and A. massartii in field samples. Thus, this study provides an effective method for detecting target benthic dinoflagellate species, and could be further implemented to monitor phytoplankton in field surveys as an altenative.

Isothermal amplification assay for visual on-site detection of Staphylococcus aureus in Chevon

ABSTRACT The ability to rapidly detect pathogens in food is important from public health and food safety reasons. Traditional culture-based detection methods tend to be laborious, time consuming, technically demanding and may be limited due to their low sensitivity. Rapid detection methods for foodborne pathogens should be specific and sensitive to detect pathogens in low numbers. Sensitivity is important because a single pathogen present in food has the potential risk to cause infection. PCR and Real-time PCR are rapid, sensitive, and specific but require specific instruments and laboratory set-up. In the present study, a simple loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of Staphylococcus aureus. The LAMP assay was found to be ten times more sensitive than traditional end-point PCR with analytical sensitivity of 0.56 pg/μl and 5.6 pg/μl of DNA, respectively. In spiked chevon samples inoculated with S. aureus, the detection limit of LAMP and PCR assay was 3.3 × 105 CFU/g and 3.3 × 106 CFU/g, respectively, without enrichment. After enriching the samples for 6 h, the detection limit improved to 3.3 × 102 CFU/g and 3.3 × 104 CFU/g, respectively, indicating LAMP to be 100-fold more sensitive than PCR. The detection limit further improved to 3.3 CFU/g of meat after enrichment of 12 h. The developed LAMP was also found to be suitable when evaluated on field samples. The present study reports a simple LAMP assay for rapid visual detection of S. aureus which has potential for use in resource limited settings.

Lab in a tube: Isolation, extraction, and isothermal amplification detection of exosomal long noncoding RNA of gastric cancer

Tumor-derived exosomes that inherit molecular information on parental cells hold great promise for cancer diagnostics. Currently, two main technical challenges, time-consuming and labor-intensive isolation of exosome and nucleic acid extraction with limited recovery that have restricted the detection of ultralow abundance exosomal nucleic acids. Here, we proposed a simple, efficient and “lab in a tube” system for the detection of exosomal nucleic acids, which fully integrated exosomes enrichment using immunomagnetic beads (IMB) (10 min), fast exosomes lysis based on NP-40 lysate (5 min) and sensitive loop-mediated isothermal amplification (LAMP) in a tube. This method was demonstrated by detecting two exosomal long noncoding RNA biomarkers of gastric cancer (HOTTIP and lncRNA-GC1) with a dynamic detection ranging from 300 ng/μL to 10 ng/μL, and the detection limit of LAMP was 10 ng/μL. Additionally, this platform exhibited good performance in the analysis of exosomal HOTTIP RNA directly in human serum samples, which has the potential for detection of low-abundance exosomal nucleic acid biomarkers from cancers.

Field Verification of an African Swine Fever Virus Loop-Mediated Isothermal Amplification (LAMP) Assay During an Outbreak in Timor-Leste.

Recent outbreaks of African swine fever virus (ASFV) have seen the movement of this virus into multiple new regions with devastating impact. Many of these outbreaks are occurring in remote, or resource-limited areas, that do not have access to molecular laboratories. Loop-mediated isothermal amplification (LAMP) is a rapid point of care test that can overcome a range of inhibitors. We outline further development of a real-time ASFV LAMP, including field verification during an outbreak in Timor-Leste. To increase field applicability, the extraction step was removed and an internal amplification control (IAC) was implemented. Assay performance was assessed in six different sample matrices and verified for a range of clinical samples. A LAMP detection limit of 400 copies/rxn was determined based on synthetic positive control spikes. A colourmetric LAMP assay was also assessed on serum samples. Comparison of the LAMP assay to a quantitative polymerase chain reaction (qPCR) was performed on clinical ASFV samples, using both serum and oral/rectal swabs, with a substantial level of agreement observed. The further verification of the ASFV LAMP assay, removal of extraction step, implementation of an IAC and the assessment of a range of sample matrix, further support the use of this assay for rapid in-field detection of ASFV.

Development of a rapid screening test for Karenia mikimotoi by using loop-mediated isothermal amplification and lateral flow dipstick

As a bloom-forming dinoflagellate, Karenia mikimotoi is toxic and globally distributed. Frequent outbreaks of K. mikimotoi blooms usually lead to the mass mortality of pelagic and benthic marine life, resulting in large economic losses. Therefore, the simple, rapid, and highly effective monitoring of K. mikimotoi is crucial to minimize the damage caused by this alga. This study aimed to establish a convenient, cost-effective, and efficient molecular detection technique through loop-mediated isothermal amplification (LAMP) combined with chromatographic lateral flow dipstick (LFD) for the daily monitoring and real-time detection of K. mikimotoi. The internal transcribed spacer (ITS) and large subunit (LSU) rDNA sequences of K. mikimotoi were used to design and screen an optimal primer (KmLF3) and a detection probe (KmLF3HP). The optimized LAMP conditions were as follows: dNTP concentration, 1.2 mM; betaine concentration, 1 M; ratio of the inner primer to the outer primer, 8:1; magnesium ion concentration, 8 mM; amplification temperature, 59 °C; and amplification time, 60 min. Cross-reactivity tests confirmed the specificity of LAMP–LFD. The detection limit of LAMP–LFD for K. mikimotoi genomic DNA was 1.70 × 10−4 ng μL−1, which is 100 times lower than that of conventional PCR. The detection limit of LAMP–LFD for the recombinant plasmid containing the LSU rDNA of K. mikimotoi was 6.21 × 103 copies μL−1, which was 10 times more sensitive than that of LAMP followed by agarose gel electrophoresis and SYBR Green I dye staining and 100 times lower than that of conventional PCR. The practicability of LAMP–LFD was validated by testing with simulated field-water samples, displaying a detection limit of 10−1 cells mL−1. In conclusion, the developed LAMP–LFD is a specific, sensitive, and accurate detection method and may be competent for the field detection of K. mikimotoi.

Specific and rapid identification of the Pheretima aspergillum by loop-mediated isothermal amplification.

Guang-dilong (Pheretima aspergillum) is a traditional Chinese animal medicine that has been used for thousands of years in China. In the present study, we purposed to establish a new rapid identification method for Guang-dilong. We provided a useful technique, loop-mediated isothermal amplification (LAMP), to differentiate Guang-dilong from other species. Four specific LAMP primers were designed based on mitochondrial cytochrome c oxidase I (COI) gene sequences of Guang-dilong. LAMP reaction, containing DNA template, four primers, 10× Bst DNA polymerase reaction buffer, dNTPs, MgSO4, and Bst DNA polymerase, was completed within 60 min at 63°C. The LAMP product can be visualized by adding SYBR Green I or detected by 2% gel electrophoresis. LAMP technology was successfully established for rapid identification of Guang-dilong. In addition, DNA template concentration of 675 fg/μl was the detection limit of LAMP in Guang-dilong, which was 1000-times higher than conventional PCR. The simple, sensitive, and convenient LAMP technique is really suited for on-site identification of Guang-dilong in herbal markets.

Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Hypothetical Protein Gene in Escherichia Coli Clinical Isolates.

Escherichia coli is the most common pathogenic bacteria that frequently causes life-threatening opportunistic human infections, diarrhea, and septicemia in immunocompromised hosts. This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of a hypothetical protein from an E. coli-specific gene (GenBank ID: 13702648). The gene was obtained through local and online BLAST, and specific primers were designed for this gene. Reaction conditions were optimized at 65ºC for 30 minutes and 80ºC for 2 minutes, whereas the reaction system contained 5.2 mM Mg2+, 8 U of Bst 2.0 DNA polymerase, 1.4 mM deoxyribonucleotide, and 0.2 and 1.6 µM of the outer and inner primers, respectively. The LAMP method was evaluated using 240 strains of E. coli and 150 strains of non-E. coli. Positive reactions were observed on all 240 strains of E. coli while all non-E. coli strains were negative. Plasmids with the specific gene and mice blood with E. coli were used for sensitivity analysis. The detection limit of LAMP was 100 bacterium/reaction. Results showed that the LAMP targeted to the hypothetical protein (GenBank ID: 13702648) is a fast, specific, sensitive, inexpensive, and suitable method for the detection of E. coli.

Establishment of loop-mediated isothermal amplification for rapid detection of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the three most pathogenic bacteria that frequently cause life-threatening opportunistic human infections, pneumonia, and lower respiratory tract infections in immunocompromised hosts, particularly in the burns ward. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of P. aeruginosa-specific gene hypothetical protein (GenBank ID: 882161). The gene was obtained through local and online BLAST, and specific primers were designed for this gene. Reaction conditions were optimized at 65°C for 30 min and 80°C for 2 min, whereas the reaction system contained 5.2 mM Mg2+, 8 U Bst 2.0 DNA polymerase, 1.4 mM deoxyribonucleotide and 0.2 and 1.6 µM of the outer and inner primers, respectively. The LAMP method was evaluated using 150 P. aeruginosa and 170 non-P. aeruginosa strains. Positive reactions were observed on 150 P. aeruginosa strains, whereas all non-P. aeruginosa strains exhibited negative results. Plasmids with the specific gene and mouse blood with P. aeruginosa were used for sensitivity assay. The detection limit of LAMP was 1 bacterium/reaction. Results indicated that the LAMP method targeted to hypothetical protein is a fast, specific, sensitive, inexpensive and suitable method for detection of P. aeruginosa.

Development and evaluation of isothermal amplification assay for the rapid and sensitive detection of Clostridium perfringens from chevon

Loop-mediated isothermal amplification (LAMP) is a promising, simple, rapid and sensitive molecular detection method. In the present study, LAMP assay was developed for detecting Clostridium perfringens in chevon. Primers were designed to detect the cpa gene of C. perfringens. A panel of 19 bacterial strains, including 3 C. perfringens and 16 other strains, were included in this study to standardize and evaluate the LAMP assay. No false positive amplification was observed indicating 100% specificity of the assay. The detection limit of LAMP and conventional PCR in the DNA extracted from pure C. perfringens was 0.34 pg and 3.4 pg, respectively. This revealed that LAMP assay is 10 times more sensitive than conventional PCR. The sensitivity of the LAMP assay for the detection of C. perfringens in raw chevon was found to be 1.2 × 102 CFU/g after 6-h enrichment and 1.2 × 105 CFU/g without enrichment in artificial spiking studies. Improved C. perfringens detection of 12 CFU/g within 12 h was obtained proving that LAMP assay is significantly faster than traditional methods that take >2 d. The developed LAMP assay also detected the targeted organism in clinical and environmental samples with the sensitivity and specificity of 97% and 84%, respectively with Kappa agreement of 0.824 respects to PCR assay. This method shows immense potential for routine diagnosis and monitoring of C. perfringens in food, environment and clinical samples. This is the first report in which the LAMP assay was optimized for the detection of C. perfringens in chevon.

Rapid detection of milk vetch dwarf virus by loop-mediated isothermal amplification

Milk vetch dwarf virus (MDV) is a member of the genus Nanovirus, and its genome is composed of multiple circular 1-kb ssDNA components. In this study, we first determined that the diseased tobacco samples obtained in Zhucheng, Shandong Province were naturally infected with MDV using a polymerase chain reaction (PCR) assay. Subsequently, loop-mediated isothermal amplification (LAMP) was developed for the detection of MDV for the first time. The Mg2+ and dNTP concentrations and the reaction temperature and time of the LAMP were optimized to 8 mM, 1.8 mM, 65 °C, and 60 min, respectively. The best ratio of the inner primers (FIP and BIP) to the outer primers (F3 and B3) was 2:1. The LAMP detection limit was 100 times greater than that of PCR. The nucleotide amplification could be clearly observed by adding SYBR Green I. The positive and negative reactions exhibit distinctly different colors in daylight; however, the positive reactions exhibit green fluorescence under a UV lamp. Therefore, the method is stable, sensitive and specific.

Surveillance of Dihydropteroate Synthase Genes in Stenotrophomonas maltophilia by LAMP: Implications for Infection Control and Initial Therapy.

Stenotrophomonas maltophilia is a common nosocomial pathogen that causes high morbidity and mortality. Because of its inherent extended antibiotic resistance, therapeutic options for S. maltophilia are limited, and sulfamethoxazole/trimethoprim (SXT) is the only first-line antimicrobial recommended. However, with the spread of dihydropteroate synthase (sul1 and sul2) genes, global emergence of SXT resistance has been reported. There is an urgent need to develop a rapid and sensitive but cost-efficient method to monitor the dissemination of sul genes. In this study, we developed loop-mediated isothermal amplification (LAMP) assays for sul1 and sul2 using real-time turbidity and hydroxy naphthol blue coloration methods. The assays could quickly detect sul genes with high sensitivity and specificity. The LAMP detection limit was 0.74 pg/reaction of extracted genomic DNA for sul1 and 2.6 pg/reaction for sul2, which were both 10-fold more sensitive than the corresponding traditional PCR assays. Additionally, the LAMP assays could positively amplify DNA from sul1-producing strains, but not from the negative controls. We then used the LAMP assays to investigate the dissemination of sul genes among S. maltophilia isolates from patients in three hospitals in Beijing, China. Among 450 non-duplicated samples collected during 2012–2014, 56 (12.4%) strains were SXT-resistant. All these SXT-resistant strains were positive for sul genes, with 35 (62.5%) carrying sul1, 17 (30.4%) carrying sul2, and 4 (7.1%) carrying both sul1 and sul2, which indicated that sul genes were the predominant resistance mechanism. Of 394 SXT-susceptible strains, 16 were also sul-positive. To provide epidemiological data for the appropriate choice of antimicrobials for treatment of sul-positive S. maltophilia, we further tested the susceptibility to 18 antimicrobials. Among these, sul-positive strains showed the highest susceptibility to tetracycline derivatives, especially minocycline (MIC50/MIC90, 0.5/4; susceptibility rate, 95.4%). Ticarcillin-clavulanate and new fluoroquinolones (moxifloxacin and levofloxacin) also showed some in vitro activity. Apart from these three kinds of antimicrobials, other agents showed poor activity against sul-positive strains.

Loop-Mediated Isothermal Amplification-Based Detection of Canine Parvoviral DNA in Faecal Samples of Diarrhoeic Dogs

The present study was undertaken to validate loop-mediated isothermal amplification (LAMP) assay based on PCR (Polymerase Chain Reaction)for the detection of canine parvovirus (CPV) and to compare the diagnostic potential of LAMP. A total of 54 faecal samples collected from diarrheic dogs suspected for CPV infection were subjected to PCR, using the primer amplifying the gene coding for the VP2 protein. These samples were also tested by LAMP assay using primer sets, and the results were statistically analysed using McNemar test. The sensitivity and specificity of LAMP assay based on PCR assay was analysed. The specificity of the LAMP primers was determined by cross-examination of templates extracted from canine adenovirus 1, canine coronavirus and canine distemper virus and Escherichia coli. Sensitivity of both assays was identified by subjecting a serial ten-fold dilution of the DNA (Deoxyribo Nucleic Acid) of CPV-2 vaccine strain (Megavac-P) to LAMP and PCR assays for the determination of their detection limits. A total of 32 samples (59.25 per cent) were detected as positive by PCR, which yielded a specific amplicon of 699 bp. In LAMP assay, 39 (72.2 per cent) samples were found to be positive. The sensitivity of the test as compared with PCR was found to be 100 per cent and specificity was 68.2. Statistical analysis of the results of PCR and LAMP assay using McNemar test revealed that there exists significant difference between these two tests (P < 0.05). The specificity determination of the LAMP primers showed that no amplification products were detected in the template DNA extracted from canine adenovirus 1, canine coronavirus and canine distemper virus vaccines and E. coli, whereas only CPV-2 vaccine strain showed positive reactions. The detection limit of LAMP was up to 10-7 in the place of 10-3 for PCR. This study showed that LAMP assay could be employed as a rapid field level diagnostic tool for the diagnosis of canine parvoviral diarrhoea.

A novel visual loop-mediated isothermal amplification assay targeting gene62181533 for the detection of Salmonella spp. in foods

In this study, we developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay for Salmonella detection targeting the gene62181533. The addition of manganous ions and calcein to the reaction system allowed the visualization of an apparent color change during the amplification reaction. The detection limit of LAMP using a six-primer combination was 1.057 copies/μl (standard plasmid), 4.1 CFU/ml (cell), and 7.68 fg/μl (genome). The LAMP assay was 100% specific based on 32 Salmonella strains and 25 non-Salmonella bacteria. In inoculated milk, the LAMP assay detected Salmonella at an initial inoculation concentration of 0.81 CFU/ml following 12 h of pre-enrichment in buffered peptone water. One hundred food samples (beef, chicken, and pork) were tested by the LAMP assay. The positive-sample ratios were 11/100 by LAMP, 11/100 by conventional PCR, and 11/100 by a culture-based method. The results revealed that the gene62181533-based LAMP assay is a rapid, specific, and sensitive method for the detection of Salmonella in foods.

Visual detection of Escherichia coli contamination in milk and fruit juice using loop-mediated isothermal amplification

We developed a dye-based visual detection technique for Escherichia coli, a common contaminant in raw milk and fruit juice. We used hydroxylnapthol blue (HNB) dye directly to E. coli-contaminated milk and juice samples, prior to loop-mediated isothermal amplification (LAMP) of tuf gene. The samples detected using HNB dye was further validated by running them in gel electrophoresis. LAMP was used for amplification of tuf gene as it is more specific and sensitive over conventional PCR. The detection limit of LAMP was estimated to be of 101 to 4 cfu ml−1 of E. coli, as against 102 to 5 cfu ml−1 in PCR. However, the detection limit of E. coli in milk (101 cfu ml−1) was lower compared to fruit juice (104 cfu ml−1) suggesting that the presence of fruit pulps in juice sample interfered with the detection. The developed HNB-based visual detection is superior over other dye based detection methods as HNB is added prior to LAMP reaction, and therefore the samples need not be opened for mixing dyes; thereby reducing the chances of cross contamination of the samples. The present study established a specific, easy-to-use, efficient, rapid and naked-eye based detection technique for E. coli contamination in milk and pulpy fruit juice.

Rapid and sensitive detection of Bartonella bacilliformis in experimentally infected sand flies by loop-mediated isothermal amplification (LAMP) of the Pap31 gene.

BackgroundCarrion' disease, caused by Bartonella bacilliformis, remains truly neglected due to its focal geographical nature. A wide spectrum of clinical manifestations, including asymptomatic bacteremia, and lack of a sensitive diagnostic test can potentially lead to a spread of the disease into non-endemic regions where competent sand fly vectors may be present. A reliable test capable of detecting B. bacilliformis is urgently needed. Our objective is to develop a loop-mediated isothermal amplification (LAMP) assay targeting the pap31 gene to detect B. bacilliformis. Methods and FindingsThe sensitivity of the LAMP was evaluated in comparison to qPCR using plasmid DNA containing the target gene and genomic DNA in the absence and presence of human or sand fly DNA. The detection limit of LAMP was 1 to 10 copies/µL, depending on the sample metrics. No cross-reaction was observed when testing against a panel of various closely related bacteria. The utility of the LAMP was further compared to qPCR by the examination of 74 Lutzomyia longipalpis sand flies artificially fed on blood spiked with B. bacilliformis and harvested at days (D) 1, 3, 5, 7 and 9 post feeding. Only 86% of sand flies at D1 and 63% of flies at D3 were positive by qPCR. LAMP was able to detect B. bacilliformis in all those flies confirmed positive by qPCR. However, none of the flies after D3 were positive by either LAMP or qPCR. In addition to demonstrating the sensitivity of the LAMP assay, these results suggest that B. bacilliformis cannot propagate in artificially fed L. longipalpis.ConclusionsThe LAMP assay is as sensitive as qPCR for the detection of B. bacilliformis and could be useful to support diagnosis of patients in low-resource settings and also to identify B. bacilliformis in the sand fly vector.

Prevalence and detection of Stenotrophomonas maltophilia carrying metallo-β-lactamase blaL1 in Beijing, China.

Intrinsic β-lactam resistance in Stenotrophomonas maltophilia is caused by blaL1 and/or blaL2, a kind of metallo-β-lactamase with a broad substrate spectrum including carbapenems. A rapid and sensitive molecular method for the detection of blaL1 in clinical samples is needed to guide therapeutic treatment. In present study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of blaL1 in clinical samples by using two methods including a chromogenic method using calcein/Mn2+ complex and the real-time turbidity monitoring to assess the reaction. Then dissemination of L1-producing S. maltophilia was investigated from ICU patients in three top hospital in Beijing, China. The results showed that both methods detected the target DNA within 60 min under isothermal conditions (65°C). The detection limit of LAMP was 3.79 pg/μl DNA, and its sensitivity 100-fold greater than that of conventional PCR. All 21 test strains except for S. maltophilia were negative for blaL1, indicative of the high-specificity of the primers for the blaL1. A total of 22 L1-positive isolates were identified for LAMP-based surveillance of blaL1 from 105 ICU patients with clinically suspected multi-resistant infections. The sequences of these blaL1 genes were conservative with only a few sites mutated, and the strains had highly resistant to β-lactam antibiotics. The MLST recovered that 22 strains belonged to seven different S. maltophilia sequence types (STs). Furthermore, co-occurrence of blaL1 and blaL2 genes were detected in all of isolates. Strikingly, S. maltophilia DCPS-01 was recovered to contain blaL1, blaL2, and blaNDM-1 genes, possessing an ability to hydrolyse all β-lactams antibiotics. Our data showed the diversity types of S. maltophilia carrying blaL1 and co-occurrence of many resistant genes in the clinical strains signal an ongoing and fast evolution of S. maltophilia resulting from their wide spread in the respiratory infections, and therefore will be difficult to control.