Isothermal Self-Primer EXPonential Amplification Reaction (SPEXPAR) for Highly Sensitive Detection of Single-Stranded Nucleic Acids and Proteins.

Development of versatile sensing methods for sensitive and specific detection of clinically relevant nucleic acids and proteins is of great value for disease monitoring and diagnosis. In this work, we propose a novel isothermal Self-primer EXPonential Amplification Reaction (SPEXPAR) strategy based on a rationally engineered structure-switchable Metastable Hairpin template (MH-template). The MH-template initially keeps inactive with its self-primer overhanging a part of target recognition region to inhibit polymerization. The present targets can specifically compel the MH-template to transform into an "activate" conformation that primes a target-recyclable EXPAR. The method is simple and sensitive, can accurately and facilely detect long-chain single-stranded nucleic acids or proteins without the need of exogenous primer probes, and has a high amplification efficiency theoretically more than 2n. For a proof-of-concept demonstration, the SPEXPAR method was used to sensitively detect the characteristic sequence of the typical swine fever virus (CSFV) RNA and thrombin, as nucleic acid and protein models, with limits of detection down to 43 aM and 39 fM, respectively, and even the CSFV RNA in attenuated vaccine samples and thrombin in diluted serum samples. The SPEXPAR method may serve as a powerful technique for the biological research of single-stranded nucleic acids and proteins.

A label-free fluorescent aptasensor based on a novel exponential rolling circle amplification for highly sensitive ochratoxin A detection.

Rapid and sensitive analysis of ochratoxin A (OTA) plays an important role in food safety. Here, an aptasensor based on novel exponential rolling circle amplification (ERCA) was proposed for ultrasensitive and label-free fluorescence detection of OTA. The attachment of OTA to its aptamer could release H and rapidly hybridize with CT to initiate rolling circle amplification (RCA). The amplicons could further displace H from APH to initiate recycled RCA, achieving exponential growth of amplification products that contained G4 dimers for lighting up ThT. Benefiting from the exponential amplification efficiency of the ERCA strategy and the high fluorescence quantum yield of G4 dimer/ThT, this strategy exhibited a wide linear range from 10 fg/mL to 10 ng/mL with a detection limit of 4.3 fg/mL. In addition, the aptasensor displayed satisfactory recoveries in real sample analysis. We believe that this novel aptasensor possesses promising application prospects in food safety and medicine detection.

Rapid detection of porcine reproductive and respiratory syndrome virus by reverse transcription loop-mediated isothermal amplification assay.

A rapid detection assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been developed for detecting porcine reproductive and respiratory syndrome virus (PRRSV). The RT-LAMP assay utilized a set of six primers to amplify the open reading frame 6 (ORF6) of the PRRSV. The amplified products were analyzed by agarose gel electrophoresis or visualized by colorimetric method. The results demonstrated that the RT-LAMP assay detected all 22 different PRRSV isolates, had no cross-reaction with four other swine viruses (i.e., PCV2, SIV, CSFV, and PEDV), and obtained a 91.3% sensitivity in 23 positive clinical samples in reference to the permissive cells-based virus isolation procedure. Therefore, the RT-LAMP assay provides a specific and sensitive means for detecting PRRSV in a simple, fast, and cost-effective manner. Furthermore, the RT-LAMP assay can be performed in less well-equipped laboratories as well as fields.

Development of a novel real-time RT-PCR assay with LUX primer for the detection of swine transmissible gastroenteritis virus.

Real-time RT-PCR assay, based on light upon extension (LUX) fluorogenic primer and LightCycle technology, was developed for rapid detection of transmissible gastroenteritis virus (TGEV). Viral RNA from different TGEV isolates and clinical specimens was detected. To evaluate the sensitivity of the assay, a gel-based RT-PCR method targeted at the same 101 bp sequence was also developed. Serial 10-fold dilutions of TGEV RNA were detected by the two methods. Although the real time method used only 2 microl RNA for each reaction, a 10-fold increase of sensitivity over that of the gel-based method, which used 10 microl RNA was demonstrated. The study indicates that the LUX assay reported below is rapid, reliable and sensitive and it has the potential for use as an alternative molecular method for TGEV diagnosis.

Combination of multiplex PCR with denaturing high-performance liquid chromatography for rapid detection of Mycobacterium genus and simultaneous identification of the Mycobacterium tuberculosis complex.

A new assay with the combination of multiplex polymerase chain reaction and denaturing high-performance liquid chromatography analysis was developed for simultaneous detection of Mycobacterium genus and identification of the Mycobacterium tuberculosis complex (MTC). Targeting at genus-specific 16S rRNA sequence of Mycobacterium and specific insertion elements IS6110 and IS1081 of MTC, the assay was validated with 84 strains covering 23 mycobacteria species and 30 strains of non-mycobacteria species. No cross reactivity was observed. Clinical application was carried out on 198 specimens (155 human sputum and 43 bovine tissue samples) and compared with culture. The multiplex assay detected all culture-positive (36 in number) and 35.2% (57/162) culture-negative specimens. The molecular assay was fast that could be completed within 1 h on purified DNA, with the limit of detection as 0.8-1.6 pg per reaction on DNA template. This work provided a useful laboratory tool for rapid identification of Mycobacterium and differentiation of MTC and nontuberculous mycobacteria.