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.     

Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of canine parvovirus DNA directly in faecal specimens

Aims: To develop a specific and highly sensitive loop-mediated isothermal amplification (LAMP) technique for the rapid detection of canine parvovirus (CPV) DNA directly in suspected faecal samples of dogs by employing a simple method of template preparation. Methods and Results: LAMP reaction was developed by designing two sets of outer and inner primers, which target a total of six distinct regions on VP2 gene of CPV. The template DNA was prepared by a simple boiling and chilling method. Of the 140 faecal samples screened by the developed LAMP and the conventional PCR assays, 104 samples (74·28%) were found positive by LAMP, whereas 81 samples (57·85%) were found positive by PCR. The specificity of the LAMP assay was tested by cross-examination of common pathogens of dogs and further confirmed by sequencing. The detection limit of the LAMP was 0·0001 TCID(50) ml(-1) , whereas the detection limit of the PCR was 1000 TCID(50) ml(-1) . Conclusions: The developed LAMP assay detects CPV DNA in faecal specimens directly within an hour by following a simple and rapid boiling and chilling method of template preparation. The result also shows that the developed LAMP assay is specific and highly sensitive in detecting CPV. Significance and Impact of the Study: The result indicates the potential usefulness of LAMP which is a simple, rapid, specific, highly sensitive and cost-effective field-based method for direct detection of CPV from the suspected faecal samples of dogs.     

Simple and rapid detection of Salmonella serovar Enteritidis under field conditions by loop‐mediated isothermal amplification

Aim: The objective of this study is to develop a serovar‐specific loop‐mediated isothermal amplification (LAMP) method for sensitive, rapid, and inexpensive detection of Salmonella serovar Enteritidis under field conditions. Methods: A set of six specific primers was designed with Salmonella Enteritidis DNA as the target. LAMP conditions were optimized by incubating the target DNA with the Bst DNA polymerase large fragment in a simple water bath. The sensitivity and specificity of LAMP was then compared with those of fluorescent quantitative real‐time polymerase chain reaction (FQ‐PCR). Results: The results were as follows. (1) Serovar‐specific Salmonella Enteritidis DNA was amplified at 65°C in as early as 20 min in a water bath. (2) A colour change visible to the naked eye indicated a positive amplification reaction. (3) The detection limit of the LAMP assay was 4 copies μl^?1; thus, the sensitivity and specificity of this assay is similar to those of the FQ‐PCR. Conclusions: LAMP is a high‐throughput detection technique with high sensitivity, specificity, and simplicity; these factors make it suitable for specifically detecting Salmonella Enteritidis under field conditions and in laboratory settings. Thus, LAMP eliminates the need for complicated equipment and technical training in the detection of this specific serovar. Significance and impact of the study: This is the first study involving the use of LAMP to detect Salmonella serovar‐specific DNA sequences. It is also the first to report an ideal method of distinguishing between Salmonella Enteritidis and other Salmonella under field conditions.     

Loop-mediated isothermal amplification targeting the apxIVA gene for detection of Actinobacillus pleuropneumoniae

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions with high specificity and efficiency. We developed a diagnostic method based on LAMP for detection of Actinobacillus pleuropneumoniae . Using six specific primers targeting the apxIVA gene, the LAMP assay rapidly amplified the target gene within 30 min, requiring only a laboratory water bath for the reaction to occur. The resulting amplificon was visualized by adding SYBR Green I to the mixture. The results obtained from testing 15 A. pleuropneumoniae reference strains and other seven bacterial species strains showed that the LAMP was as specific as and 10 times more sensitive than nested PCR. Sixty-five tonsil samples were collected from 65 healthy pigs. All the samples were negative for A. pleuropneumoniae by immunomagnetic separation-based (IMS) bacterial isolation, nested PCR and LAMP, respectively. Meanwhile, 115 tonsil samples were also collected from 115 pigs with apparent respiratory problems. Twenty-two were positive by IMS bacterial isolation. All the samples that were positive by IMS bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated exceptionally higher sensitivity than nested PCR by picking up 14 additional positive cases (χ² test, P <0.0001); we concluded that LAMP was a highly sensitive and reliable method for detection of A. pleuropneumoniae infection.     

Development and evaluation of loop-mediated isothermal amplification for rapid detection of Haemophilus parasuis

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions and has a high specificity and efficiency. We developed a LAMP assay targeting the 16S rRNA gene for rapid detection of Haemophilus parasuis. The results obtained from testing 31 H. parasuis strains and 28 other bacterial species strains showed that LAMP was as specific as, and more sensitive than, nested PCR. Fifty-five lung samples were collected from 55 healthy pigs. All the samples were negative for H. parasuis by bacterial isolation, nested PCR and LAMP, respectively. In addition, 122 lung samples were collected from 122 pigs with apparent respiratory problems. Sixty-five were positive by bacterial isolation. All the samples that were positive by bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated higher sensitivity than nested PCR, picking up 16 additional cases. The LAMP assay also gave a same result compared with the nested PCR when the two assays were used, respectively, to detect H. parasuis from samples obtained from experimentally infected pigs. We concluded that LAMP is a highly sensitive and reliable method for detection of H. parasuis infection.     

NLF2 gene expression in the endometrium of patients with implantation failure after IVF treatment

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 35 min 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 1 pg 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.     

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.     

Sensitive and rapid detection of Flavobacterium columnare in channel catfish Ictalurus punctatus by a loop-mediated isothermal amplification method

AIMS: To evaluate the loop-mediated isothermal amplification method (LAMP) for rapid detection of Flavobacterium columnare and determine the suitability of LAMP for rapid diagnosis of columnaris infection in channel catfish, Ictalurus punctatus. METHODS AND RESULTS: A set of four primers, two outer and two inner, were designed specifically to recognize 16S ribosomal RNA gene of this pathogen. Bacterial genomic DNA templates were prepared by hot lysis in a lysis buffer. Amplification of the specific gene segments was carried out at 65 degrees C for 1 h. The amplified gene products were analysed by agarose gel electrophoresis and detected by staining gels with ethidium bromide. A PCR assay was also included in this study. Our results demonstrate that the ladder-like pattern of bands from 204 bp specific to the Fl. columnare 16S ribosomal RNA gene was amplified. The detection limit of the LAMP assay was comparable to that of PCR in prepared genomic DNA reactions. In addition, this optimized LAMP assay was able to detect the Fl. columnare 16S ribosomal RNA gene in experimentally infected channel catfish. CONCLUSIONS: The LAMP assay for Fl. columnare detection in channel catfish was established. SIGNIFICANCE AND IMPACT OF THE STUDY: Because LAMP assay is a rapid, sensitive, specific, simple and cost-effective assay for Fl. columnare detection in channel catfish, it is useful for rapid diagnosis of Fl. columnare in fish hatcheries and the field.     

Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by Loop-Mediated Isothermal Amplification

We developed a technique for detecting the heat-labile I (LTI) and heat-stable I (STI) genes of enterotoxigenic Escherichia coli (ETEC) using a novel DNA amplification procedure designated Loop-Mediated Isothermal Amplification (LAMP). The detection limit of accelerated LAMP utilizing loop primers was 4 CFU/test for LTI and was 40 CFU/test for STI, which are 10-fold higher than those of conventional PCR assay (detection limit, 40 CFU/test and 400 CFU/test, respectively). No DNA amplification was observed in LT and ST non-producing E. coli or other bacterial strains; thus, high specificity was verified. The specificity of LAMP assay was also confirmed by digestion of LAMP products using restriction enzymes and DNA sequence analysis. In the accelerated LAMP assay, DNA amplification was detected within 35 min, and thus LAMP is superior to conventional PCR in terms of rapidity. It was confirmed that increased concentrations of primers and Bst DNA polymerase could further facilitate the reaction. Furthermore, with the high amplification efficiency of the LAMP assay, amplification can be visually observed by the turbidity caused by magnesium pyrophosphate, a byproduct of the reaction. Detection of LTI and STI in ETEC by LAMP is thus an extremely rapid procedure with high sensitivity and specificity that requires no specialized equipment. This assay is expected to become a valuable tool for rapid diagnosis in ETEC infection.     

DNA环介导恒温扩增技术快速检测霍乱弧菌

霍乱弧菌是一种重要的食源性致病菌,主要引起急性肠道传染病,其快速检测具有重要意义。根据霍乱弧菌的mdh管家基因序列,设计2对特异性检测引物,利用DNA环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP),经反应体系优化,成功建立了霍乱弧菌的LAMP快速检测方法。该方法最佳反应温度为65℃,60min完成检测,对培养菌的检测限为25CFU/mL,污染食品中霍乱弧菌的检测限为32CFU/g。对33株同种或近源细菌进行LAMP检测,仅霍乱弧菌得到阳性扩增。LAMP方法实践应用结果表明,对1057份虾、蟹、牡蛎、肉类、人腹泻物等样本进行检测,共检出85份阳性,与国际标准(ISO TS21872-1-2007)检测结果的符合率为100%。结果表明,本研究建立的霍乱弧菌LAMP检测方法特异性强、灵敏度高、操作简便,有利于霍乱弧菌疫情的监测。     

A simple and sensitive method for detection of Bacillus anthracis by loop‐mediated isothermal amplification

Aims: To develop a rapid and simple system for detection of Bacillus anthracis using a loop‐mediated isothermal amplification (LAMP) method and determine the suitability of LAMP for rapid identification of B. anthracis infection. Methods and Results: A specific LAMP assay targeting unique gene sequences in the bacterial chromosome and two virulence plasmids, pXO1 and pXO2, was designed. With this assay, it was possible to detect more than 10 fg of bacterial DNA per reaction and obtain results within 30–40 min under isothermal conditions at 63°C. No cross‐reactivity was observed among Bacillus cereus group and other Bacillus species. Furthermore, in tests using blood specimens from mice inoculated intranasally with B. anthracis spores, the sensitivity of the LAMP assay following DNA extraction methods using a Qiagen DNeasy kit or boiling protocol was examined. Samples prepared by both methods showed almost equivalent sensitivities in LAMP assay. The detection limit was 3·6 CFU per test. Conclusions: The LAMP assay is a simple, rapid and sensitive method for detecting B. anthracis. Significance and Impact of the Study: The LAMP assay combined with boiling extraction could be used as a simple diagnostic method for identification of B. anthracis infection.     

Development and application of a simple,rapid and sensitive method for detecting moderately carbendazim‐resistant isolates in Botrytis cinerea

Sustainable disease management depends on the ability to monitor the development of fungicide resistance in pathogen populations. A point mutation resulting in an alteration (F200Y) at codon 200 of the target protein β‐tubulin leads to a moderate level of resistance to carbendazim in Botrytis cinerea. Although traditional methods remain a cornerstone in detection of fungicide resistance, molecular methods that do not require the isolation of pathogens, can detect the presence of resistance alleles at low frequencies, and require less time and labour than traditional methods. In this study, we present an efficient, rapid, and highly specific method for detecting the moderately carbendazim‐resistant isolates in B. cinerea based on loop‐mediated isothermal amplification (LAMP). By using specific LAMP primers, we detected the resistance‐conferring mutation underlying β‐tubulin F200Y. The concentrations of LAMP components and LAMP parameters were optimised, resulting in reaction temperatures and times of 61–65°C and 45 min, respectively. The feasibility of the LAMP assay was verified by assaying the diseased samples with artificial inoculation in the different hosts. The LAMP assay developed in the current study was specific, stable, repeatable and sensitive, and was successfully applied for detection of moderately carbendazim‐resistant isolates of B. cinerea in plant samples.     

Rapid detection of mecA and spa by the loop‐mediated isothermal amplification (LAMP) method

Aim: To develop a detection assay for staphylococcal mecA and spa by using loop‐mediated isothermal amplification (LAMP) method. Methods and Results: Staphylococcus aureus and other related species were subjected to the detection of mecA and spa by both PCR and LAMP methods. The LAMP successfully amplified the genes under isothermal conditions at 64°C within 60 min, and demonstrated identical results with the conventional PCR methods. The detection limits of the LAMP for mecA and spa, by gel electrophoresis, were 10² and 10 cells per tube, respectively. The naked‐eye inspections were possible with 10³ and 10 cells for detection of mecA and spa, respectively. The LAMP method was then applied to sputum and dental plaque samples. The LAMP and PCR demonstrated identical results for the plaque samples, although frequency in detection of mecA and spa by the LAMP was relatively lower for the sputum samples when compared to the PCR methods. Conclusion: Application of the LAMP enabled a rapid detection assay for mecA and spa. The assay may be applicable to clinical plaque samples. Significance and Impact of the Study: The LAMP offers an alternative detection assay for mecA and spa with a great advantage of the rapidity.