Infectious diseases are clinically relevant, transmissible illnesses caused by microorganisms, such as bacteria, viruses, fungi, and parasites. According to a recent report, between 1980 and 2014 more than four million people in the USA have died from various infectious diseases.1 Accurate and timely detection of infection and identification of the causative pathogen are crucial in disease prevention, treatment and monitoring.2,3 Although reliable methods of pathogen detection are well and long established, many of such detection and diagnostics techniques are still limited in use to clinical laboratories due to the need for trained and skilled personnel, and sophisticated diagnostic instruments. Accordingly, there is a pressing need for simple, affordable, and easy-to-use diagnostic tools for the specific detection of pathogens at the point of care, e.g., doctors' offices, clinics, infirmaries, and particularly in resource-limited areas where medical infrastructure is lacking.

Interfacing Pathogen Detection with Smartphones for Point-of-Care Applications

Rapid detection and quantification of Enterovirus 71 by a portable surface plasmon resonance biosensor

Vibrio parahemolyticus (VP) mostly isolated from aquatic products is one of the major causes of bacterial food-poisoning events worldwide, which could be reduced using a promising on-site detection method. Herein, a rapid and quantitative method for VP detection was developed by applying a mixed-dye-loaded loop-mediated isothermal amplification (LAMP) assay on a self-priming compartmentalization (SPC) microfluidic chip, termed on-chip mixed-dye-based LAMP (CMD-LAMP). In comparison to conventional approaches, CMD-LAMP was advantageous on the limit of detection, which reached down to 1 × 103 CFU/mL in food-contaminated samples without the pre-enrichment of bacteria. Additionally, as a result of the use of a mixed dye and SPC chip, the quantitative result could be easily acquired, avoiding the requirement of sophisticated instruments and tedious operation. Also, CMD-LAMP was rapid and cost-effective. Conclusively, CMD-LAMP has great potential in realizing the on-site quantitative analysis of VP for food safety.

Rapid and Quantitative Detection of Vibrio parahemolyticus by the Mixed-Dye-Based Loop-Mediated Isothermal Amplification Assay on a Self-Priming Compartmentalization Microfluidic Chip

Visual detections based on fluorescence and the color changes under natural light are two promising product detections for isothermal nucleic acid amplifications (INAAs) such as the isothermal multiple-self-matching-initiated amplification (IMSA) as point-of-care testing techniques However, the currently used approaches have shortcomings in application For the former, fluorescence changes recognized by naked eye may be indistinguishable because of single fluorescence emitted and strong background noise, which requires empirical preset of cutoff intensity values For the latter, visual detection sensitivity under natural light is not comparable to that based on fluorescence Herein, hydroxyl naphthol blue (HNB) and SYBR Green I (SG) were coupled to acquire a label-free dual fluorescence for the visual product detection of IMSA The mixed-dye-loaded off-chip (tube-based) and on-chip (microfluidic chip-based) IMSAs for the detection of hepatitis B virus were conducted The results demonstrated that this dual fluorescence could realize distinguishable fluorescent color changes to improve visual detection sensitivity and avoid the preset of cutoff values Moreover, the mixed dye is stable when kept at room temperature and compatible with the IMSA's reagents without a contamination-prone step of opening tubes after amplification Also, this coupled dye inherits the advantages of achieving color changes under natural light from HNB and real-time detection from SG In conclusion, the mixed-dye-based dual fluorescence has a potential in the point-of-care testing application for realizing off-chip and on-chip product detection of IMSA, loop-mediated isothermal amplification (LAMP), or other INAAs

Mixed-Dye-Based Label-Free and Sensitive Dual Fluorescence for the Product Detection of Nucleic Acid Isothermal Multiple-Self-Matching-Initiated Amplification.

Coxsackievirus B3 (CV-B3) is usually associated with aseptic meningitis and myocarditis; however, the association between CV-B3 and hand, foot, and mouth disease (HFMD) has not been clearly demonstrated, and the phylogenetic dynamics and transmission history of CV-B3 have not been well summarized. Two HFMD outbreaks caused by CV-B3 were described in Hebei Province in 2012 and in Shandong Province in 2016 in China. To analyze the epidemiological features of two CV-B3 outbreaks, a retrospective analysis was conducted. All clinical specimens from CV-B3 outbreaks were collected and disposed according to the standard procedures supported by the WHO Global Poliovirus Specialized Laboratory. EV genotyping and phylogenetic analysis were performed to illustrate the genetic characteristics of CV-B3 in China and worldwide. Two transmissible lineages (lineage 2 and 3) were observed in Northern China, which acted as an important “reservoir” for the transmission of CV-B3. Sporadic exporting and importing of cases were observed in almost all regions. In addition, the global sequences of CV-B3 showed a tendency of geographic-specific clustering, indicating that geographic-driven adaptation plays a major role in the diversification and evolution of CV-B3. Overall, our study indicated that CV-B3 is a causative agent of HFMD outbreak and revealed the phylogenetic dynamics of CV-B3 worldwide, as well as provided an insight on CV-B3 outbreaks for effective intervention and countermeasures.

/pdf/two-coxsackievirus-b3-outbreaks-associated-with-hand-foot-15aj6g2nvj.pdf

Two Coxsackievirus B3 outbreaks associated with hand, foot, and mouth disease in China and the evolutionary history worldwide.

The invention belongs to the application field of biology technology and relates to various level mechanisms of disease prevention and control and sentinel hospitals, and the like which carry out simultaneous detection and typing of multiple enteroviruses (including HEV71 (human enterovirus17), CVA (coxsackievirus A) 16, CVA4, CVA5, CVA9, CVA10, CVB (coxsackievirus B) 1, CVB3 and CVB) of samples such as pharynx swab, night soil, and the like of hand-foot-and-mouth disease patients. Specifically, data of human enterovirus VP1 region nucleotide sequences of various serotypes and all enterovirus 5'UTR conservative fragments are downloaded from http://ncbi.nlm.nih.gov/nucleotide/; various serotype specific primers and intestinal universal primers are designed; single-tube multiplex (tenfold PCR) PCR detection on 9 enteroviruses and all the enterovirus 5'UTR conservative fragments is carried out; and a whole reaction process lasts less than 2 hours. According to the invention, not only can a problem that typing can not be carried out in conventional single-tube multiplex fluorescence qualitative PCR detection be solved, but also disadvantages such as complex operation, long operation time and relatively high cost with the conventional chip detection method are overcome; fresh ideas are provided for enterovirus typing technology; strong technical support is provided for rapidly accurate screening and typing of the enteroviruses associated with hand-foot-and-mouth diseases by virtue of high specificity, high sensitivity and speediness; and the application of the GeXP multiplex gene expression genetic analysis system has important significance for pathogenic spectrum researches on the hand-foot-and-mouth disease patients and investigation of molecular epidemiology.

Application of GeXP multiplex gene expression genetic analysis system in hand-foot-and-mouth disease pathogenic typing and detection

The invention relates to a method for detecting CA16 virus gene by color-based reverse transcriptase loop-mediated isothermal amplification, which belongs to the filed of biotechnology application and relates to a method for detecting and monitoring the human enterovirus CA16 type VP1 gene of such specimens as throat swabs, serum and excrement of fever patients with suspected hand-foot-mouth diseases which takes rash or herpes on hand, foot and oral cavity as main symptoms in all-level disease prevention and control institutions, enterovirus detection internet laboratories, sentinel hospitals and the like The advantages of the invention are specifically embodied as follows: analyzing the conserved sequence of the human enterovirus CA16 type VP1 gene with bioinformatics professional software by applying gene color based reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), designing six primers which are completely matched with eight binding domains in the recognized VP1 target sequence, omitting the steps of single reverse transcription and secondary amplification of nested PCR, adding HNB (hydroxynaphthol blue) dyes before reaction, being capable of completing amplification for 1h in the isothermal environment at 65 DEG C only by one step and finally observing the color variation and white precipitates at the tube bottom with naked eyes to determine the detection result Compared with the traditional RT-PCR method, the method of the invention has the advantages of safety, specificity, sensitivity and convenience

Method for detecting CA16 virus gene by color-based reverse transcriptase loop-mediated isothermal amplification

The invention belongs to the field of biological technology application and relates to detection and monitoring of human enterovirus EV71 type VP1 gene in nasopharyngeal swab, serum and night soil specimens of suspected hand-foot-and-mouth disease patients, who have main symptoms of fever, rash or herpes at hand, foot and oral cavity, etc, at various disease prevention and control mechanism, enterovirus network laboratory laboratory and sentinel point hospitals. Specifically, color based reverse transcription loop-mediated isothermal amplification technology (RT-LAMP) is employed, and a conserved sequence of human enterovirus EV71-type VP1 gene is analyzed by professional software of bioinformatics, so as to design six primers that are completely coupled with eight bonding areas in an identified VP1 target sequence and omit steps of individual reverse transcription and a secondary amplification of nest-type PCR. HNB (hydroxy naphthol blue) is added into a dye before a reaction, and only one step is needed to complete amplification in one hour at 65 DEG C; and finally colour variable and white precipitate at pipe bottom are observed by naked eyes to determine a detection result. Compared with a traditional RT-PCR method, the method of the invention has advantages of safety, specificity, sensitivity and convenience.

Color based reverse transcription loop-mediated isothermal amplification technology for detecting EV71 virogene

The invention relates to a novel isothermal nucleic acid amplification technology, namely an isothermal multiple self-pairing triggering amplification technology (IMSA) According to the technology, amplification of a pathogeny gene can be realized under the condition of isothermality and under the action of a single functional enzyme without a thermal cycle instrument The result determination of the isothermal multiple self-pairing triggering amplification technology (IMSA) can be realized by simple measures such as observing white precipitate of by-product magnesium pyrophosphate, adding a chromogenic agent and observing the color change before or after amplification The most unique characteristic of the technology is that multiple oligonucleotide structures which can perform self-pairing and then trigger circular amplification will be generated during the process of amplification so that the triggering probability of following circular amplification is markedly increased, the amplification efficiency and detection sensitivity are promoted On the basis of the isothermal multiple self-pairing triggering amplification technology (IMSA) we establish detection method of hand-foot-and-mouth disease pathogen coxsackievirus type A16 (CVA16) and human enterovirus type EV71 (EV71) VP1 gene, and rapid detection method of human influenza a virus H7N9 HA gene By the application of the invention, simple, rapid, highly sensitive and highly specific detection can be realized

Novel isothermal multiple self-pairing triggering amplification technology

Improved detection limit in rapid detection of human

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