Rapid and visual Chlamydia trachomatis detection using loop-mediated isothermal amplification and hydroxynaphthol blue.

We developed an assay comprising crude DNA lysis by simple heat treatment coupled loop-mediated isothermal amplification with hydroxynaphthol blue for Chlamydia trachomatis detection (petty patent pending), and evaluated the developed assay for its feasibility as a one-step point-of-care detection on 284 endocervical swab specimens from clinically symptomatic C. trachomatis and healthy subjects. This assay is sensitive to 0·04 pg of ompA, specific with six primers targeting C. trachomatis ompA region, rapid (45 min total assay time), inexpensive (approx. 3 USD/reaction), does not require sophisticated instrumentation, and has comparable assay effectiveness (95% specificity, 90-100% sensitivity) to bacterial DNA isolation by a commercial kit coupled with polymerase chain reaction and gel electrophoresis (98-100% specificity, 87-100% sensitivity) based on the clinical samples test. The test result could be read by naked eye through the colour change from violet (negative) to sky blue (positive) for C. trachomatis-infected specimens. Further, this assay uses all safe chemical reagents and is hence safe to the users. SIGNIFICANCE AND IMPACT OF THE STUDY: Chlamydia trachomatis is the major bacterial sexually transmitted disease worldwide. The clinical symptoms are broad, and chronic C. trachomatis infections could lead to blindness, ectopic pregnancy, sterility in males and females, and a higher risk of the development of cervical cancer. The result indicates the potential usefulness of our crude DNA lysis coupled loop-mediated isothermal amplification with hydroxynaphthol blue for a simple, rapid, specific, sensitive and cost-effective assay for C. trachomatis detection from suspected specimens. This assay offers an alternative in the clinical diagnosis of C. trachomatis in resource-limited health-care facilities and clinical laboratories in developing countries, and in field tests.

Detection of natural infection of infectious spleen and kidney necrosis virus in farmed tilapia by hydroxynapthol blue-loop-mediated isothermal amplification assay.

AIMS: Infectious spleen and kidney necrosis virus (ISKNV) has recently been recognized as a causative agent of serious systemic disease in tilapia. Our objective was to establish a new colorimetric loop-mediated isothermal amplification (LAMP) assay with pre-addition of hydroxynapthol blue (blue-LAMP) to investigate ISKNV transmission in tilapia. METHODS AND RESULTS: The blue-LAMP, targeting a major capsid protein gene of ISKNV, was conducted at 65°C for 45 min, allowing unaided visual detection of the pathogen based on colour change without cross-amplification of other known fish pathogens tested. Comparison of blue-LAMP and PCR assays revealed a higher detection level for blue-LAMP assay (41·33%) in a population of farmed tilapia infected with ISKNV. The investigation of ISKNV transmission pattern in farmed red tilapia using the blue-LAMP revealed a possible matroclinical form. The presence of ISKNV in the gonad samples was confirmed by in situ LAMP assay. Positive signals only appeared in ovarian follicles, and not in oocytes. Moreover, tissue tropism assay revealed that the brain was the main target organ in both farmed red tilapia (40%) and Nile tilapia (20%). CONCLUSIONS: The developed blue-LAMP assay has the potential to be used as a viable tool for screening covert and natural infections of ISKNV in tilapia. The evidence of vertical transmission of ISKNV infection in tilapia indicates the seriousness of this disease and will require a close attention and collaboration between tilapia hatcheries and disease experts in order to find a solution. SIGNIFICANCE AND IMPACT OF THE STUDY: The new blue-LAMP assay is a time-saving and economically viable detection tool, which allows unaided visual detection for ISKNV in tilapia, and it could be applicable for field applications. Evidence on the vertical transmission of ISKNV in farmed tilapia suggests a need for developing farm management practices to control the spread of virus in aquaculture industries.

Graphene oxide based fluorescence resonance energy transfer and loop-mediated isothermal amplification for white spot syndrome virus detection.

Graphene oxide (GO) is attractived for biological or medical applications due to its unique electrical, physical, optical and biological properties. In particular, GO can adsorb DNA via π-π stacking or non-covalent interactions, leading to fluorescence quenching phenomenon applicable for bio-molecular detection. In this work, a new method for white spot syndrome virus (WSSV)-DNA detection is developed based on loop-mediated isothermal amplification (LAMP) combined with fluorescence resonance energy transfer (FRET) between GO and fluorescein isothiocyanate-labeled probe (FITC-probe). The fluorescence quenching efficiency of FITC-probe was found to increase with increasing GO concentration and reached 98.7% at a GO concentration of 50 µg/ml. The fluorescence intensity of FITC-probe was recovered after hybridization with WSSV LAMP product with an optimal hybridization time of 10 min and increased accordingly with increasing amount of LAMP products. The detection limit was estimated to be as low as 10 copies of WSSV plasmid DNA or 0.6 fg of the total DNA extracted from shrimp infected with WSSV. In addition, no cross reaction was observed with other common shrimp viral pathogens. Therefore, the GO-FRET-LAMP technique is promising for fast, sensitive and specific detection of DNAs.

Evaluation of colorimetric loop-mediated isothermal amplification assay for visual detection of Streptococcus agalactiae and Streptococcus iniae in tilapia.

UNLABELLED: Streptococcus agalactiae and Strep. iniae are bacterial pathogens that cause streptococcosis in many fish species. An accelerated colorimetric loop-mediated isothermal amplification (LAMP) assay with pre-addition of calcein was established, and the transmission and detection of Strep. agalactiae and Strep. iniae in tilapia under natural aquatic environment were investigated. A positive reaction was observed by a colour change from orange to green through the naked eyes after completion at 63°C for 30 min with 10 times higher sensitivity than that of nested PCR assays and without cross-amplification with other fish bacterial pathogens. All sample types of Nile and red tilapia (broodstock, fertilized egg, fry) were Strep. agalactiae- and Strep. iniae positive by this new method, implying that they could be vertically transmitted. With its application for screening broodstock and fry before stocking and for monitoring fish health in grow-out ponds, the method would become very useful in fish farming industry. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of colorimetric LAMP with pre-addition of calcein offers simple, rapid and sensitive technique with applicability for small field laboratories. This technique explored the possible vertical transmission mode of Strep. agalactiae and Strep. iniae under natural aquatic environment. It could be such preliminary data provided for the screening broodstock before breeding and/or the specific-pathogen-free production.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with colorimetric gold nanoparticle (AuNP) probe assay for visual detection of Penaeus vannamei nodavirus (PvNV).

UNLABELLED: Penaeus vannamei nodavirus (PvNV) is an emerging viral infection that has caused muscle necrosis or a white tail disease in cultivated whiteleg shrimp Penaeus (Litopenaeus) vannamei, leading to loss in the productions. Rapid detection of PvNV is essential for further control disease. A combination between reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay and colorimetric gold nanoparticle (AuNP) probe was developed for rapid, sensitive and inexpensive detection of PvNV in this study. RT-LAMP reaction successfully detected PvNV at 63°C for 45 and 5 min for hybridization of LAMP amplified product, followed by salt-induced AuNP-labelled ssDNA probe aggregation to visual colour development. This method showed identical results to LAMP followed by gel electrophoresis and spectrophotometric detection, and it was 10 times sensitive more than conventional nested RT-PCR. The new method revealed negative results with other shrimp pathogens. This study provides the direct visualization of LAMP reaction by AuNP probe hybridization, that significantly reduces the time and cost required for the molecular diagnostic of infectious diseases and offers to use in aquaculture health management. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents the first report of Penaeus vannamei nodavirus (PvNV) detection using the colorimetric RT-LAMP technique. The application of RT-LAMP assay combined with colorimetric gold nanoparticle (AuNP) probe to detect the PvNV offers simple, rapid and sensitive technique and does not require sophisticated equipment with applicable for small or field laboratories. This method can further employ to screen broodstock before breeding, to screen postlarvae before ponds stocking, to monitor shrimp in rearing ponds and to assess the occurrence of this virus in shrimp farming.

Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp.

AIMS: Enterocytozoon hepatopenaei is an emerging microsporidian parasite that has been linked to recent losses caused by white faeces syndrome (WFS) in cultivated giant or black tiger shrimp Penaeus (Penaeus) monodon and whiteleg shrimp Penaeus (Litopenaeus) vannamei in Asia. To more accurately assess its impact on shrimp production and to determine reservoir carriers for control measures, our objective was to establish a loop-mediated isothermal amplification (LAMP) assay combined with colorimetric nanogold (AuNP) for rapid, sensitive and inexpensive detection of this parasite. METHODS AND RESULTS: A set of six specific primers was designed to successfully detect the SSU rRNA gene of E. hepatopenaei by a LAMP reaction of 45 in at 65°C combined with visual detection of the amplification product via hybridization at 65°C for 5 min with a ssDNA-labelled nanogold probe, followed by salt-induced AuNP aggregation (total assay time, approximately 50 min). This method gave similar results to LAMP followed by electrophoresis or spectrophotometric detection, and it was more sensitive (0·02 fg total DNA) than a conventional nested PCR (0·2 fg total DNA). The new method gave negative results with shrimp DNA templates extracted from diseased shrimp containing other pathogens, indicating that the LAMP-AuNP assay was specific for E. hepatopenaei. CONCLUSIONS: Without sacrificing sensitivity or specificity, the new LAMP-AuNP assay significantly reduced the time, ease and cost for molecular detection of E. hepatopenaei in shrimp. SIGNIFICANCE AND IMPACT OF THE STUDY: The new method employs simple, inexpensive equipment and involves simple steps making it applicable for small field laboratories. Wider application of the method to screen broodstock before use in a hatchery, to screen postlarvae before stocking shrimp ponds, to test for natural carriers and to monitor shrimp in rearing ponds would help to assess and reduce the negative impact of this parasite in shrimp farming.

Detection of hepatopancreatic parvovirus (HPV) infection in Penaeus monodon using PCR-ELISA.

A rapid and sensitive PCR-ELISA has been developed for detection of hepatopancreatic parvovirus (HPV) in Penaeus monodon. The specific primer set amplified 156 bp fragment and could detect as a little as 0.01 fg of purified HPV DNA which equivalent to three viral particles. No cross-reactivity was observed when nucleic acid templates from white spot syndrome virus, yellow-head virus, monodon baculovirus and shrimp were tested. The crude DNA simple prepared from hepatopancreas can be used as DNA template and provide a favorable result. Using this technique for detection of HPV infection in 87 carrier shrimps revealed the higher sensitivity and efficiency of detection when compared to histological examination and conventional PCR. Sixty-two percent infection was detected by PCR-ELISA from samples with HPV negative diagnosed by histological examination. Therefore, this sensitive and specific method is promisingly useful for early detection of HPV infection in broodstock, carriers and for ex situ application where large numbers of samples can be analyzed simultaneously.

A non-stop, single-tube, semi-nested PCR technique for grading the severity of white spot syndrome virus infections in Penaeus monodon.

A single-tube, non-stop, semi-nested polymerase chain reaction (PCR) technique was developed for simultaneous detection and severity grading of white spot syndrome virus (WSSV) infections in the black tiger shrimp Penaeus monodon. The test uses 1 sense primer and 3 antisense primers that produce up to 3 PCR products (1100, 526 and 250 base pairs [bp]) depending upon the severity of infection. Specifically, heavy infections (> or = 2 x 10(4) viral particles) of WSSV produce all 3 fragments, while moderate infections (around 2 x 10(3) viral particles) produce 2 (526 and 250 bp) and light infections (20 to 200 viral particles) produce 1 (250 bp). In addition, the technique uses internal control primers that yield a shrimp characteristic fragment for non-infected samples and samples with a low quantity of viral target in order to assure integrity and reproducibility of the PCR assays. The non-stop, single-tube, semi-nested PCR technique is simple and convenient and can detect as little as 5 fg WSSV DNA (20 viral particles) in crude extracts of postlarval samples or extracts of pleopods and haemolymph from larger shrimp.