We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61–65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique.

Background: Listeria monocytogenes can cause invasive diseases in humans and farm animals and is frequently isolated from dairy products and poultry. Listeriosis is uncommon in China but L. monocytogenes has been isolated from foods and food processing environments in China. However little is known of genetic diversity of Chinese L. monocytogenes isolates and their relationships with global isolates. Results: Two hundred and twelve isolates of L. monocytogenes from food sources from 12 provinces/cities in China were analysed by serotyping, Pulsed Field Gel Electrophoresis (PFGE) and Multi-locus Sequence Typing (MLST). The predominant serotypes are 1/2a, 1/2b and 1/2c accounting for 90.1% of the isolates. PFGE divided the isolates into 61 pulse types (PTs). Twenty nine PTs were represented by more than one isolates with PT GX6A16.0004 containing the most number of isolates. MLST differentiated the isolates into 36 STs, among which 15 were novel. The 3 most common STs were ST9 (29.1%), ST8 (10.7%) and ST87 (9.2%), accounting for 49.0% of the isolates. Conclusions: STs prevalent in other parts of the world are also prevalent in China including 7 STs (ST1-ST3, ST5, ST6, ST8, ST9) which caused maternal fetal infections or outbreaks, suggesting that these STs potentially can also cause severe human infections or outbreaks in China. Surveillance of these STs will provide important information for prevention of listeriosis. This study also enhances our understanding of genetic diversity of L. monocytogenes in China.

Background: The sequences of the 16S rRNA genes extracted from fecal samples provide insights into the dynamics of fecal microflora. This potentially gives valuable etiological information for patients whose conditions have been ascribed to unknown pathogens, which cannot be accomplished using routine culture methods. We studied 33 children with diarrhea who were admitted to the Children’s Hospital in Shanxi Province during 2006. Results: Nineteen of 33 children with diarrhea could not be etiologically diagnosed by routine culture and polymerase chain reaction methods. Eleven of 19 children with diarrhea of unknown etiology had Streptococcus as the most dominant fecal bacterial genus at admission. Eight of nine children whom three consecutive fecal samples were collected had Streptococcus as the dominant fecal bacterial genus, including three in the Streptococcus bovis group and three Streptococcus sp., which was reduced during and after recovery. We isolated strains that were possibly from the S. bovis group from feces sampled at admission, which were then identified as Streptococcus lutetiensis from one child and Streptococcus gallolyticus subsp. pasteurianus from two children. We sequenced the genome of S. lutetiensis and identified five antibiotic islands, two pathogenicity islands, and five unique genomic islands. The identified virulence genes included hemolytic toxin cylZ of Streptococcus agalactiae and sortase associated with colonization of pathogenic streptococci. Conclusions: We identified S. lutetiensis and S. gallolyticus subsp. pasteurianus from children with diarrhea of unknown etiology, and found pathogenic islands and virulence genes in the genome of S. lutetiensis.

The report describes a simple, sensitive and nearly instrument-free assay for visual and multiplex detection of Enterococcus faecalis and Staphylococcus aureus based on multiple loop-mediated isothermal amplification (mLAMP) and lateral flow biosensor (LFB). Detection and differentiation of the Ef0027 gene (E. faecalis-specific gene) and nuc gene (S. aureus-specific gene) were determined using fluorescein (FITC)-and digoxin-modified primers in the mLAMP process. In the presence of biotin- and FITC-/digoxin-modified primers, the mLAMP yielded numerous biotin- and FITC-/digoxin-attached duplex products, which were detected by LFB through biotin/streptavidin interaction (biotin on the duplex and streptavidin on the gold nanoparticle) and immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the LFB test line). The accumulation of gold nanoparticles generated a characteristic red line, enabling visual and multiplex detection of target pathogens without instrumentation. The analytical sensitivity, specificity and feasibility of LAMP-LFB technique were successfully examined in pure culture and blood samples. The entire procedure, including specimen (such blood samples) processing (30 min), isothermal reaction (40 min) and result reporting (within 2 min), could be completed within 75 min. Thus, this assay offers a simple, rapid, sensitive and specific test for multiplex detection of E. faecalis and S. aureus strains. Furthermore, the LAMP-LFB strategy is a universal technique, which can be extended to detect various target sequences by re-designing the specific LAMP primers.