Loop-mediated DNA Amplification Research Articles

High-performance LAMP-based method for human sex identification using Y chromosome-specific genetic markers

Finding fast and cheap strategies for DNA typing and human sample identification is of interest in forensic science. We report a new versatile alternative for molecular sex determination using Y-specific targets (TSPY, TTTY, alphoid regions, and Y-Amelogenin). This system uses an isothermal loop-mediated DNA amplification (LAMP) with a set of 6 primers for each target, designed to improve sensibility and specificity, and reducing detection time to only 45 min. Furthermore, detecting the different targets on the Y chromosome either individually or in combination revealed accurate results. Assay sensitivity was determined with a mixture of human female and male DNA at different concentrations to mimic forensic samples. Single primer sets showed high sensitivity at DNA concentrations ranging from 58.6 to 3.7 pg/µL. When a combined primers set was used, sensitivity yielded a detection as low as 0.1 pg/µL of male DNA, making it 10 times more sensitive than qPCR-DNA quantification kits. Finally, high specificity was observed when tested against 6 domestic species.

One step nucleic acid amplification (OSNA) for detection of lymph node metastasis during robotic radical prostatectomy for prostate cancer: A pilot study

The OSNA technique is based on reverse transcription loop-mediated DNA amplification for the detection of cytokeratin 19 (CK19) messen-ger RNA (mRNA). The purpose of our paper, which represents the first study in the literature, is to test the accuracy of this method in the detection of lymph node metastases in patients undergoing robotic radical prostatectomy with lymph node dis-section. Our cohort consisted of patients that have undergone robotic radical prostatectomy with extended lymph node dissec-tion. Lymph nodes were evaluated with imprint technique and then with frozen section examination. The remaining tissue was evaluated by OSNA method. Lymph nodes were defined as 'neg-ative' or 'positive' according to mRNA copy number. 7 patients and 25 lymph nodes were included in our cohort. Two patients were found negative with all pathology methods. In one patient the standard stains revealed a suspi-cious outcome but it was positive for micrometastasis with OSNA. In another patient the outcome was positive for standard stains and negative for OSNA. Finally, 2 patients were found positive for OSNA and negative for imprint methods. One Step Nucleic Acid Amplification (OSNA) method using CK19 seems to fail in detection of lymph node metastases in prostate cancer patients undergoing radical prostatectomy and lymph node dissection.

Nile tilapia reared under full-strength seawater: Hemato-immunological changes and susceptibility to pathogens

Co-culture of Nile tilapia, Oreochromis niloticus, with marine shrimp has been an increasing farm practice as evidence has suggested that the co-culture is one of a few ways to mitigate shrimp diseases, especially the acute hepatopancreatic necrosis disease that has affected shrimp culture industry worldwide. Since Nile tilapia are basically freshwater species, having them in co-culture with marine shrimp therefore requires a certain degree of salinity adaptation, in which the fish are readily capable to. The present study was aimed at studying certain hemato-immunological changes of the fish reared under elevated salinity, as well as their susceptibility to pathogens. Nile tilapia (180g initial BW) were reared under gradually increasing salinity within one month from 0 to 30ppt at 3ppt/3d and maintained for another month in 30-ppt salinity. At the end of the 60-day experimental period, 50% of the fish survived with mortality occurred mainly during the second month when the salinity was at 30ppt. Their growth rate was retarded to 60% of that of the control fish maintained under freshwater (0ppt). The fish reared under elevated salinity also had significantly lower hematocrit, higher total white blood cell counts, higher absolute numbers of lymphocytes and thrombocytes and higher serum lysozyme activity, compared to those reared under freshwater. Presence of three representative pathogens: infectious spleen and kidney necrosis virus (ISKNV), Francisella noatunensis and Streptococcus agalactiae; were determined in the fish reared under freshwater and in those reared under elevated salinity, using polymerase chain reaction (PCR) method. Of the three pathogens, only ISKNV was detected in 1/10 of the freshwater and 6/10 of the elevated-salinity fish. In both groups, the virus was present in the gills, brain, liver, gonads, skin, kidney and spleen, with moderately and severely positive reaction observed in the kidney and spleen. The detection of ISKNV was confirmed by in situ hybridization of the gonads and kidney, using loop-mediated DNA amplification with digoxigenin, which clearly showed dark-brown stain of the positive signals in the PCR-positive samples. These results suggest that a substantial number of O. niltoicus could survive elevated salinity up to full-strength seawater provided that the salinity had been gradually elevated. And under that situation, changes in hemato-immunological functions of the fish did occur, which probably caused lethal proliferation of pre-existing ISKNV that otherwise would remain dormant.

In situ DIG-labeling, loop-mediated DNA Amplification (ISDL) for highly sensitive detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV)

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is also known as Penaeus stylirostris densovirus (PstDNV). Since it is listed as a reportable crustacean disease by the World Organization for Animal Health (OIE), many countries require testing for IHHNV in shrimp stocks imported for aquaculture, necessitating rapid and highly sensitive detection methods to guarantee and maintain absence of IHHNV in traded stocks. Here we describe a new, highly sensitive method for detection of IHHNV in shrimp tissues. It involves in situ loop-mediated DNA amplification (IS-LAMP) employing digoxigenin-11-dUTP (DIG-dUTP) as part of the deoxynucleotide triphosphate (dNTP) mix. This resulted in DIG-labeling of the DNA amplicons (i.e., in situ, DIG-labeling LAMP or ISDL) allowing direct in situ, immuno-histochemical detection of IHHNV using an anti-DIG antibody conjugated with alkaline phosphatase, and eliminating the lengthy hybridization step of conventional ISH. The target region for the method is at the 3'end of the IHHNV genome that has been reported least likely to give rise to endogenous viral elements (EVE) in the shrimp genome. When compared to the results for IHHN detection by conventional in situ hybridization (ISH) using a DIG-labeled oligonucleotide probe, signals from the ISDL method were stronger and also gave positive results with some tissues unreactive by the conventional ISH method. Additional comparison of the two methods revealed that the proteinase-K digestion step needed for normal ISH was unnecessary for ISDL. This further shortened the assay time and improved the tissue integrity necessary to clearly indicate the location of positive hybridization signals. The ISDL method gave no cross- reactions with tissues of shrimp infected with other common pathogens, and the total time needed to carry out ISDL was one-third of that needed for conventional ISH. Statement of relevanceThis work describes an in situ, DIG-labeling LAMP (ISDL) method, as a new, highly sensitive method for detection of IHHNV in shrimp tissues. The developed method can be used as a confirmatory test carried out with tissue sections to confirm the results from molecular assays, especially when immune-histochemical methods are not available to do so. ISDL is especially important for molecular detection from a new or unexpected location and for molecular detection in newly suspected carriers to determine whether they are actually infected or just mechanical carriers.

Loop‐mediated isothermal amplification of single pollen grains

The polymerase chain reaction (PCR) has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental requirements of PCR have limited its widespread application in field situations. The recent development of isothermal DNA amplification methods provides an alternative to PCR, which circumvents key limitations of PCR for direct amplification in the field. Being able to analyze DNA in the pollen cloud of an ecosystem would provide very useful ecological information, yet would require a field-enabled, high-throughput method for this potential to be realized. Here, we demonstrate the applicability of the loop-mediated DNA amplification method (LAMP), an isothermal DNA amplification technique, to be used in pollen analysis. We demonstrate that LAMP can provide a reliable method to identify species from the pollen cloud, and that it can amplify successfully with sensitivity down to single pollen grains, thus opening the possibility of field-based, high-throughput analysis.

A Simple Paper-Based Lab-on-a-Chip for the Detection of a Highly Pathogenic Strain of Porcine Reproductive and Respiratory Syndrome Virus

A paper-based laboratory-on-a-chip assay for the rapid detection of a highly pathogenic strain of porcine reproductive and respiratory syndrome virus (HP-PRRSV) was developed for the first time. The single-unit chip was simply fabricated using Whatman filter paper and plastic lamination. The chip measured 2.5 × 3.0 cm2 and was divided into two parts, one for nucleic acid amplification and the other for signal detection. The HP-PRRSV assay was performed by specific ORF I Nsp 2 gene amplification via an isothermal reverse transcription loop-mediated DNA amplification platform, whereas the cDNA signal detection was performed by visual observation of colorimetric changes in blue silver nanoplates (AgNPls). Positive results caused non-aggregation of the blue AgNPls on the detection pad, whereas negative results induced colorimetric changes in the AgNPls from blue to colourless on the pad. The assay had a limit of detection of 100 copies of the target Nsp 2 gene and high specificity for other types of infectious viruses. The assay required only one hour to complete. This work demonstrates a simple and rapid assay for viruses using a simple, low-cost, paper-based chip.

Digital LAMP in a sample self-digitization (SD) chip

This paper describes the realization of digital loop-mediated DNA amplification (dLAMP) in a sample self-digitization (SD) chip. Digital DNA amplification has become an attractive technique to quantify absolute concentrations of DNA in a sample. While digital polymerase chain reaction is still the most widespread implementation, its use in resource-limited settings is impeded by the need for thermal cycling and robust temperature control. In such situations, isothermal protocols that can amplify DNA or RNA without thermal cycling are of great interest. Here, we accomplished the successful amplification of single DNA molecules in a stationary droplet array using isothermal digital loop-mediated DNA amplification. Unlike most (if not all) existing methods for sample discretization, our design allows for automated, loss-less digitization of sample volumes on-chip. We demonstrated accurate quantification of relative and absolute DNA concentrations with sample volumes of less than 2 μl. We assessed the homogeneity of droplet size during sample self-digitization in our device, and verified that the size variation was small enough such that straightforward counting of LAMP-active droplets sufficed for data analysis. We anticipate that the simplicity and robustness of our SD chip make it attractive as an inexpensive and easy-to-operate device for DNA amplification, for example in point-of-care settings.