Identification of human DNA by loop-mediated isothermal amplification (LAMP) technique combined with white ring precipitation of Cu(OH)2

Loop-mediated isothermal amplification (LAMP) analysis has been developed for human identification. DNA templates of 8 sample species including Homo sapiens (human) were subjected to LAMP for apolipoprotein L1 gene (ApoL1) amplification. After DNA amplification, gel electrophoresis was performed. Furthermore, CuSO4 was added in to the LAMP product for staining visible to the naked eye. The LAMP products were only present with human DNA. The limit of detection was reproducibly as low as 10pg of genomic DNA. Moreover, by adding CuSO4 to the LAMP product, the Cu(OH)2 precipitate, which formed a ring-shaped deposit, was not seen with the human DNA, but was seen with the LAMP negative samples. Our findings show the LAMP technique to be a powerful method for identifying the presence of human DNA. The addition of CuSO4 to the LAMP product is an alternative that can be used in field studies, and does not require access to gel-electrophoresis

Mud crab susceptibility to disease from white spot syndrome virus is species-dependent

<p>Abstract</p> <p>Background</p> <p>Based on a report for one species (<it>Scylla serrata</it>), it is widely believed that mud crabs are relatively resistant to disease caused by white spot syndrome virus (WSSV). We tested this hypothesis by determining the degree of susceptibility in two species of mud crabs, <it>Scylla olivacea </it>and <it>Scylla paramamosain</it>, both of which were identified by mitochondrial 16 S ribosomal gene analysis. We compared single-dose and serial-dose WSSV challenges on <it>S. olivacea </it>and <it>S. paramamosain</it>.</p> <p>Findings</p> <p>In a preliminary test using <it>S. olivacea </it>alone, a dose of 1 × 10⁶WSSV copies/g gave 100% mortality within 7 days. In a subsequent test, 17 <it>S. olivacea </it>and 13 <it>S. paramamosain </it>were divided into test and control groups for challenge with WSSV at 5 incremental, biweekly doses starting from 1 × 10⁴and ending at 5 × 10⁶copies/g. For 11 <it>S. olivacea </it>challenged, 3 specimens died at doses between 1 × 10⁵and 5 × 10⁵copies/g and none died for 2 weeks after the subsequent dose (1 × 10⁶copies/g) that was lethal within 7 days in the preliminary test. However, after the final challenge on day 56 (5 × 10⁶copies/g), the remaining 7 of 11 <it>S. olivacea </it>(63.64%) died within 2 weeks. There was no mortality in the buffer-injected control crabs. For 9 <it>S. paramamosain </it>challenged in the same way, 5 (55.56%) died after challenge doses between 1 × 10⁴and 5 × 10⁵copies/g, and none died for 2 weeks after the challenge dose of 1 × 10⁶copies/g. After the final challenge (5 × 10⁶copies/g) on day 56, no <it>S. paramamosain </it>died during 2 weeks after the challenge, and 2 of 9 WSSV-infected <it>S. paramamosain </it>(22.22%) remained alive together with the control crabs until the end of the test on day 106. Viral loads in these survivors were low when compared to those in the moribund crabs.</p> <p>Conclusions</p> <p><it>S. olivacea </it>and <it>S. paramamosain </it>show wide variation in response to challenge with WSSV. <it>S. olivacea </it>and <it>S. paramamosain </it>are susceptible to white spot disease, and <it>S. olivacea </it>is more susceptible than <it>S. paramamosain</it>. Based on our single-challenge and serial challenge results, and on previous published work showing that <it>S. serrata </it>is relatively unaffected by WSSV infection, we propose that susceptibility to white spot disease in the genus <it>Scylla </it>is species-dependent and may also be dose-history dependent. In practical terms for shrimp farmers, it means that <it>S. olivacea </it>and <it>S. paramamosain </it>may pose less threat as WSSV carriers than <it>S. serrata</it>. For crab farmers, our results suggest that rearing of <it>S. serrata </it>would be a better choice than <it>S. paramamosain </it>or <it>S. olivacea </it>in terms of avoiding losses from seasonal outbreaks of white spot disease.</p

White spot syndrome virus: an overview on an emergent concern

Viruses are ubiquitous and extremely abundant in the marine environment. One of such marine viruses, the white spot syndrome virus (WSSV), has emerged globally as one of the most prevalent, widespread and lethal for shrimp populations. However, at present there is no treatment available to interfere with the unrestrained occurrence and spread of the disease. The recent progress in molecular biology techniques has made it possible to obtain information on the factors, mechanisms and strategies used by this virus to infect and replicate in susceptible host cells. Yet, further research is still required to fully understand the basic nature of WSSV, its exact life cycle and mode of infection. This information will expand our knowledge and may contribute to developing effective prophylactic or therapeutic measures. This review provides a state-of-the-art overview of the topic, and emphasizes the current progress and future direction for the development of WSSV control strategies

Time Course of Detection of Human Male DNA from Stained Blood Sample on Various Surfaces by Loop Mediated Isothermal Amplification and Polymerase Chain Reaction

This study explores determining the sex of humans from blood stains taken from different surfaces and compares the time course of detection with the conventional PCR, Conventional Loop Mediated Isothermal Amplification (LAMP), and LAMP-Lateral Flow Dipstick (LFD). For the DNA templates, 7 male and 7 female blood stained samples were extracted and added to LAMP and PCR reaction solution to amplify the SRY gene. The DNA samples were extracted from the following blood stained materials: cloth, wood, clay, and tile. Then, the samples were stored at room temperature for 1, 7, 30, and 60 day(s). After the DNA amplification, the gel electrophoresis process was applied to detect LAMP product. The LFD was combined with the LAMP to detect LAMP product on the male cloth samples. For the male samples, the time course of detection on the first and seventh days indicated positive for both LAMP and PCR products on all the surfaces while no DNA amplification was found on any of the female samples. On day 30, positive LAMP product was still found on all the male samples. However, it had faded on the tiles. Moreover, all the male samples, which had tested positive for PCR product, were blurred and unclear. On day 60, LAMP product was still found on all the male samples. Conversely, the PCR method resulted in no bands showing for any of the male samples. However, the LAMP-LFD method detected product on all the male samples of cloth. The results show that the LAMP is an effective, practical, and reliable molecular-biological method. Moreover, the LFD can increase the efficiency and sensitivity of the LAMP, making it more suitable for field studies because gel electrophoresis apparatus is not required

A Comparison of Four Molecular Methods for Detection of Aflatoxin-Producing Aspergillus in Peanut and Dried Shrimp Samples Collected from Local Markets around Pathum Thani Province, Thailand

Aspergillus flavus is an aflatoxin-producing fungus which is poisonous to humans and animals when consumed. Detecting the fungus can help to prevent this danger. The four molecular methods, namely, conventional isothermal amplification (LAMP), PCR, quantitative LAMP (qLAMP), and qPCR, were compared to determine their efficiency for A. flavus detection. Thirty samples of peanut and dried shrimp were collected from 15 markets around Pathum Thani Province in Thailand. The samples were artificially infected with 108 conidia/ml of A. flavus for 1 hr and enriched for one day to represent real contamination. The results show that the sensitivity detection for A. flavus in PCR, LAMP, qPCR, and qLAMP was 50 ng, 5 ng, 5 pg, and 5 pg, respectively. Aspergillus in 30 peanut and dried shrimp from the market was detected by all four methods. The detection rate was about 20%, 60%, 100%, and 100% with PCR, LAMP, qPCR, and qLAMP, respectively. The molecular detection technique, especially LAMP, qPCR, and qLAMP, can detect this pathogenic fungi very rapidly with high sensitivity and reliability in comparison to conventional PCR

Quantitative LAMP and PCR Detection of Salmonella in Chicken Samples Collected from Local Markets around Pathum Thani Province, Thailand

Salmonella is a bacterium that infects people when they consume contaminated food or liquids. To prevent humans from becoming ill, it is useful to have an efficient method of detecting Salmonella before the disease is passed on through the food chain. In this research, the efficiency of Salmonella detection was compared using the following four methods: conventional loop-mediated isothermal amplification (LAMP), PCR, quantitative LAMP (qLAMP), and qPCR. The artificial infection of chicken samples started with incubating of 10 mL of 108 CFU of S. typhimurium for 6 hr. and enriching for 2 hr. to represent real contamination of the samples. The results show that the sensitivity of Salmonella DNA detection in PCR, qPCR, LAMP, and qLAMP were 50 ng, 5 ng, 50 pg, and and 500 fg, respectively. Thirty samples of 10 g chicken were collected from 10 markets in Pathum Thani, Thailand; then, the infection was detected. The conventional LAMP, qLAMP, and qPCR methods detected Salmonella in all the chicken samples. However, the conventional PCR method detected Salmonella infection in only eight of the samples. Overall, the qLAMP method had the highest sensitivity of Salmonella DNA detection

Interleukin-8 in Hyperlipidemia and Coronary Heart Disease in Thai Patients Taking Statin Cholesterol-Lowering Medication While Undergoing Coronary Artery Bypass Grafting Treatment

The role of interleukin-8 (IL-8), a pivotal chemokine in atherogenesis and coronary heart disease (CHD) development, is diverse and remains unclear. This cross-sectional study investigates the association of the IL-8 expression in hyperlipidemia (H) and CHD patients who have been treated with statin cholesterol-lowering drugs while undergoing coronary artery bypass grafting treatment. Fifty-five Thai volunteers including 13 normal (N), 24 H, and 18 CHD patients were enrolled for the investigation. All the CHD patients had been treated continuously with statin cholesterol-lowering medications since the disease was diagnosed and were undergoing coronary bypass grafting approximately one month later. Therefore, the CHD group was representative of a pathogenesis improvement in CHD. The IL8 mRNA expression was determined by real-time quantitative PCR in the peripheral blood mononuclear cells from heparinized blood. The plasma IL-8 levels were assessed by enzyme-linked immunosorbent assay. The result shows that the IL8 mRNA expression in the H group tended to increase; however, in the CHD group, there was a significant decrease (p=0.0111) compared to the N group. The IL8 mRNA expression and the plasma levels in the CHD group were significantly lower than those in the H group (p<0.05). A significant negative correlation between the IL8 mRNA (r = −0.499) or plasma IL-8 (r = −0.3875) expression and CHD progression was observed (p<0.05). In conclusion, the transcriptomic and the phenotypic IL-8 expression decreased significantly in the Thai CHD patients who had continuously received statin-group medications compared to the H and N group participants. Therefore, IL-8 should serve as a feasible marker and could be used to evaluate the therapeutic effects of statins and illustrate the pathology of CHD treatment