Detection of Target Genes in Viable Bacteria and Extracellular DNA Using Loop-Mediated Isothermal Amplification Assay

When the loop-mediated isothermal amplification(LAMP)assay is used for detecting target genes,DNA extraction is unnecessary in many cases.Simple pretreatment(e.g.heating)is enough to obtain rather sensitive responses.Even test samples without any pretreatment can be used as template.This feature suggests that LAMP is superior to PCR in developing point-of-care test strategies.In this study,using Stx1 gene from E.coli as model,we verified that viable cells,dead cells and extracellular DNA could function as template in the LAMP assay.In the incubation at 63℃,viable bacteria in the LAMP reaction mixture lysed completely within 2 min,providing DNA template for nucleic acid amplification.The Stx1 gene in diluted culture medium,spiked tap water,spiked seawater and real seawater all could be detected,with or without the step of DNA extraction.We found that the complex substances in real sample(e.g.natural seawater)exhibited considerable inhibitory effect on the sensitivity of the LAMP assay.These outcomes are meaningful for building a point-of-care strategy by employing the LAMP assay for environmental monitoring,bio-resource surveys,food safety,etc.in particular those based on environmental DNA.

Isolated repeated anastomotic recurrence after sigmoidectomy

Repeated anastomotic recurrence(AR)of colonic cancer is uncommon.We report a case of a double-isolated AR after sigmoidectomy.In 2003,a 60-year-old woman underwent stapled sigmoid resection for a moderately differentiated adenocarcinoma.Further rectal bleeding occurred after six months,and colonoscopy detected an AR.Thus,an additional stapled colorectal anastomosis was performed.Ten months later,a colonoscopy detected a circumferential AR that prompted the completion of a second colorectal resection,with a doublestapled colorectal anastomosis.Twenty-four hours after surgery,a massive pulmonary embolism occurred,and the patient died within a few hours.At present,onlysix cases of repeated isolated AR have been described.Repeated segmental colorectal resections are generally associated with a favourable prognosis,with a median survival rate of 45 mo(range,13-132 mo).Repeated isolated ARs are rare,and segmental colorectal resections are generally associated with long-term diseasefree survival.

A resazurin-based,nondestructive assay for monitoring cell proliferation during a scaffold-based 3D culture process

Development of viable cell estimation method without sacrificing proliferation and functions of cells cultured on regenerative biomaterials is essential for regenerative engineering.Cytotoxicity and depletion of resazurin are critical but often overlooked limitations that hindered applications of resazurin in viable cell estimation.The present work found that cytotoxicity and depletion of resazurin depended on cell concentration,resazurin concentration and resazurin incubation time.A simple strategy which only allowed cells to incubate with resazurin during each measurement was developed to eliminate negative effects of resazurin.This strategy was verified by monitoring proliferation of MC3T3-E1 preosteoblasts on poly(D,L-lactic acid)scaffold during a continuous 3D culture process for up to 21 days,comparing the accuracy with MTT assay which is a destructive assay with high sensitivity and accuracy and commonly used in regenerative engineering and comparing viability,proliferation and differentiation functions of MC3T3-E1,which were treated with/without this strategy for nondestructive evaluation.This method showed comparable linearity of standard curve and characteristics of growth curve to MTT assay.No major negative effects of this method on MC3T3-E1 viability and functions were found.Our work highlighted the importance of the concentration and incubation time of resazurin in designing application-specific nondestructive viability assay and would be helpful in improving the implanted medical devices as well as in regenerative engineering.