Quantification of hepatitis E virus in raw pork livers using droplet digital RT-PCR.

Hepatitis E virus (HEV) is the causative agent of hepatitis E. Some of the rise in hepatitis E infection in China may be linked to undercooked pork. In this study, we established a reverse transcription droplet digital PCR (RT-ddPCR) method to detect HEV in raw pork livers. The detection limit of the assay for HEV RNA was as low as 1.81 copies/µL. The suggested approach was validated on 14 samples, demonstrating greater sensitivity, specificity, and anti-interference performance features than RT-qPCR. Furthermore, we amplified the partial ORF2 gene by nested RT-PCR and sequenced for the HEV RNA positive samples. The prevalence of HEV in all collected samples was 2.24% (14/626), and the viral load was between 8.0 copies/µL and 8975 copies/µL. Specifically, the virus was detected in 10.62% (12/113) of the samples collected from the bio-safety disposal centers for dead livestock and poultry, in 0.67% (2/300) of the samples collected from the slaughterhouses, and none of the samples collected from the retail markets was HEV RNA positive. The subsequent phylogenetic analysis revealed that all HEV isolates belonged to the subtype 4d, which is one of the most common subtypes in northern China.

Rapid detection of porcine encephalomyocarditis virus (EMCV) by isothermal reverse transcription recombinase polymerase amplification assays.

In this study, we combined reverse transcription recombinase polymerase amplification assay with the fluorescence detection platform (qRT-RPA) and lateral flow biosensor (LFB RT-RPA) to allow for rapid detection of porcine encephalomyocarditis virus (EMCV). Primers and probes were designed to target the highly conserved region of 3D gene of porcine EMCV. The optimal reaction condition of qRT-RPA and LFB RT-RPA was set as 42 °C for 20 min. The assays were highly specific to EMCV and no cross-reactions were observed with seven other porcine viruses. With a 10-fold serially diluted EMCV genomic RNA as template, the limit of detection was 1.0 × 102 and 1.0 × 101 copies for qRT-RPA assay and LFB RT-RPA assay, respectively. A total of 92 samples from different sources were examined using qRT-RPA, LFB RT-RPA and qRT-PCR. We found 100% diagnostic agreement between qRT-RPA (23/92) and qRT-PCR (23/92), and 97.83% diagnostic agreement between LFB RT-RPA (25/92) and qRT-PCR (23/92). There was no significant difference in performance between the RT-RPA assays developed in this study and a previously described qRT-PCR. However, RT-RPA assays were rapid and easy to perform while LFB RT-RPA exhibited higher sensitivity for EMCV than qRT-PCR. Therefore, the developed EMCV RT-RPA assays provide an attractive and promising tool for effective detection of EMCV in low-resource settings.

Development of a recombinase polymerase amplification assay for rapid detection of Haemophilus parasuis in tissue samples.

Haemophilus parasuis is the etiological agent of Glässer's disease in swine, which associates with severe economic losses in the swine industry worldwide. A real-time recombinase polymerase amplification assay (real-time RPA) was developed for direct and rapid detection of H. parasuis basing on the translation-initiation factor IF2 (infB) gene. The assay was performed successfully at 39°C for 20 min in Genie III, which is portable and chargeable by battery. The developed assay was highly specific for H. parasuis, and the limit of detection of the assay was 6.0 × 103  fg of H. parasuis genomic DNA, which was the same as that of a real-time PCR developed previously. The assay was further evaluated on 68 pig tissue samples, and 18 (26.5%), 20 (29.4%), and 8 (11.8%) samples were positive for H. parasuis by the real-time RPA, real-time PCR and bacterial isolation, respectively. With the bacteria isolation as the reference method, the real-time RPA showed a diagnostic specificity of 83.33% and a diagnostic sensitivity of 100%. The above data demonstrated the well-potentiality and usefulness of the developed real-time RPA assay in reliable diagnosis of swine Glässer's disease, especially in resource limited settings.

Monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for porcine hemoglobin quantification.

The major objective of this study was to establish a monoclonal antibody (mAb)-based sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of porcine hemoglobin (PHb) in raw meat products. Before assay development, two mAbs immunoreactive to PHb ß subunit with different epitopes were characterized. The optimized immunoassay was specific to PHb and had a wide PHb working range from 15.6 µg/mL to 3,000 µg/mL and high reproducibility with low coefficient of variations (CV < 20%). Through this assay, the estimated PHb residuals in pork loin and shoulder meats were 0.4 mg/g and 1.1 mg/g, respectively. In addition, this immunoassay could effectively quantify PHb in laboratory-spiked meats (pork loin, pork shoulder, and turkey breast) with acceptable recovery. Overall, this is the first mAb-based sandwich ELISA that is suitable for the government, food industry, and third-party authority to monitor PHb residuals or porcine blood adulteration in raw pork and pork-free meat products.

A Microfluidic Biosensor Based on Magnetic Nanoparticle Separation, Quantum Dots Labeling and MnO2 Nanoflower Amplification for Rapid and Sensitive Detection of Salmonella Typhimurium.

Screening of foodborne pathogens is an effective way to prevent microbial food poisoning. A microfluidic biosensor was developed for rapid and sensitive detection of Salmonella Typhimurium using quantum dots (QDs) as fluorescent probes for sensor readout and manganese dioxide nanoflowers (MnO2 NFs) and as QDs nanocarriers for signal amplification. Prior to testing, amino-modified MnO2 nanoflowers (MnO2-NH2 NFs) were conjugated with carboxyl-modified QDs through EDC/NHSS method to form MnO2-QD NFs, and MnO2-QD NFs were functionalized with polyclonal antibodies (pAbs) to form MnO2-QD-pAb NFs. First, the mixture of target Salmonella Typhimurium cells and magnetic nanoparticles (MNPs) modified with monoclonal antibodies (mAbs) was injected with MnO2-QD-pAb NFs into a microfluidic chip to form MNP-bacteria-QD-MnO2 complexes. Then, glutathione (GSH) was injected to dissolve MnO2 on the complexes into Mn2+, resulting in the release of QDs. Finally, fluorescent intensity of the released QDs was measured using the fluorescent detector to determine the amount of Salmonella. A linear relationship between fluorescent intensity and bacterial concentration from 1.0 × 102 to 1.0 × 107 CFU/mL was found with a low detection limit of 43 CFU/mL and mean recovery of 99.7% for Salmonella in spiked chicken meats, indicating the feasibility of this biosensor for practical applications.

An untargeted and pseudotargeted metabolomic combination approach to identify differential markers to distinguish live from dead pork meat by liquid chromatography-mass spectrometry.

An untargeted and pseudotargeted metabolomic combination approach was developed to identify reliable and stable differential markers which can distinguish between pork meat from live pigs conventionally butchered and pork meat from dead pigs butchered immediately after death from diseases or other abnormalities. In this study, 24 differential metabolites of interest were screened by the UHPLC-Triple-TOF-MS-based untargeted metabolomic method, and 14 differential markers were detected by the UHPLC-QTRAP-MS-based pseudotargeted metabolomic method after performing statistical analysis to remove false-positive differential metabolites. Among the possible differential markers identified using the Metlin database and references were carnosine, l-carnitine, l-histidine, N-acetylhistidine, acetylcholine, l-acetylcarnitine and two phosphatidylcholines. The results of the principal component analysis (PCA) and the hierarchical clustering analysis (HCA) indicate that 14 differential markers could be potentially used to distinguish live and dead pork meat. This reliable and stable approach not only could detect the unknown differential markers, but also accurately quantify them.

Direct detection of Actinobacillus pleuropneumoniae in swine lungs and tonsils by real-time recombinase polymerase amplification assay.

Actinobacillus pleuropneumoniae is the etiological agent of swine contagious pleuropneumoniae, which is distributed globally and associated with severe economic losses in the pig rearing industry. In this study, a real-time recombinase polymerase amplification assay (real-time RPA) based on the apxIVA gene was developed to rapid detect A. pleuropneumoniae. Real-time RPA was performed successfully in Genie III at the constant temperature of 39 °C for 20 min. The developed assay was highly specific for A. pleuropneumoniae, and the sensitivity at 95% probability was 536 fg of A. pleuropneumoniae genomic DNA. The real-time RPA for A. pleuropneumoniae was further evaluated on the 112 clinical swine lung and tonsil samples, and 25 (22.3%), 27 (24.1%), and 12 (10.7%) samples were positive for A. pleuropneumoniae by the real-time RPA, real-time PCR and bacterial isolation, respectively. With a real-time PCR as the reference method, the real-time RPA showed a diagnostic specificity of 98.8%, a diagnostic sensitivity of 88.9%, a positive predicative value of 96.0%, a negative predictive value of 96.5%, and a kappa value of 0.900. The above data demonstrated the well potentiality and usefulness of the developed real-time RPA assay in the reliable detection of A. pleuropneumoniae, especially in resource limited settings.

Rapid and sensitive detection of Mycoplasma hyopneumoniae by recombinase polymerase amplification assay.

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia, which is associated with high economic losses in swine production worldwide. In this study, recombinase polymerase amplification assays using real-time fluorescence detection (real-time RPA) and lateral flow strip detection (LFS RPA) were developed to detect M. hyopneumoniae based on the conserved region of the mhp165 gene. Real-time RPA was performed in Genie III at 39 °C for 20 min, while the LFS RPA was performed in an incubator block at 39 °C for 15 min, and the products were visible on the LFS inspected by the naked eyes within 2 min. Both assays were specific for M. hyopneumoniae, as there were no cross-reactions with other pathogens tested. The limit of detection of both RPA assay was 5.0 × 102 fg of M. hyopneumoniae DNA, which was the same as that of a real-time PCR assay. Of the 146 clinical samples, M. hyopneumoniae DNA was identified in 41, 42, and 47 samples by the real-time RPA, LFS RPA and real-time PCR, respectively. Compared to real-time PCR, the real-time RPA and LFS RPA assays showed diagnostic specificity of 100%, a diagnostic sensitivity of 87.23% and 89.36%, and a kappa value of 0.903 and 0.909, respectively. These results have demonstrated that the developed RPA assays are suitable for rapid and reliable detection of M. hyopneumoniae in diagnostic laboratory and at point-of-need facility.

Visual and equipment-free reverse transcription recombinase polymerase amplification method for rapid detection of foot-and-mouth disease virus.

BACKGROUND: Foot-and-mouth disease (FMD), which is caused by foot-and-mouth disease virus (FMDV), is a highly contagious tansboundary disease of cloven-hoofed animals and causes devastating economic damages. Accurate, rapid and simple detection of FMDV is critical to containing an FMD outbreak. Recombinase polymerase amplification (RPA) has been explored for detection of diverse pathogens because of its accuracy, rapidness and simplicity. A visible and equipment-free reverse-transcription recombinase polymerase amplification assay combined with lateral flow strip (LFS RT-RPA) was developed to detect the FMDV using primers and LF probe specific for the 3D gene. RESULTS: The FMDV LFS RT-RPA assay was performed successfully in a closed fist using body heat for 15 min, and the products were visible on the LFS inspected by the naked eyes within 2 min. The assay could detect FMDV serotypes O, A and Asia1, and there were no cross-reactions with vesicular stomatitis virus (VSV), encephalomyocarditis virus (EMCV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV2) and pseudorabies virus (PRV). The analytical sensitivity was 1.0 × 102 copies in vitro transcribed FMDV RNA per reaction, which was the same as a real-time RT-PCR. For the 55 samples, FMDV RNA positive rate was 45.5% (25/55) by LFS RT-RPA and 52.7% (29/55) by real-time RT-PCR. For the LFS RT-RPA assay, the positive and negative predicative values were 100% and 80%, respectively. CONCLUSIONS: The performance of the LFS RT-RPA assay was comparable to real-time RT-PCR, while the LFS RT-RPA assay was much faster and easier to be performed. The developed FMDV LFS RT-RPA assay provides an attractive and promising tool for rapid and reliable detection of FMDV in under-equipped laboratory and at point-of-need facility, which is of great significance in FMD control in low resource settings.

A real-time loop-mediated isothermal amplification method for rapid detection of Lawsonia intracellularis in porcine fecal samples.

Porcine proliferative enteritis is a common diarrheal disease characterized by thickening of the intestinal mucosa in swine due to enterocyte proliferation, which is caused by Lawsonia intracellularis. In this study, a real-time loop-mediated isothermal amplification (LAMP) assay was developed to detect L. intracellularis based on the conserved region of the 16S ribosomal RNA gene. The optimal reaction conditions of the real-time LAMP was 65 °C for 60 min. The LAMP products could be detected by both real-time turbidity and direct visual inspection. The assay was specific for L. intracellularis, as no cross-reaction was observed with other pathogens. The detection limit of the real-time LAMP assay was 1.4 × 10-1pg of L. intracellularis DNA, which was the same as that of real-time PCR and approximately 100 times more sensitive than that of conventional PCR. Of the 136 clinical samples, L. intracellularis DNA was identified in 60 samples by real-time LAMP, which was the same as real-time PCR and higher than conventional PCR (36.8%, 50/136). The specific, sensitive and rapid real-time LAMP assay developed in this study could be a useful alternative tool in point-of-care (POC) diagnosis of L. intracellularis infection.

Rapid detection of transmissible gastroenteritis virus in swine small intestine samples using real-time reverse transcription recombinase polymerase amplification.

A rapid and specific real-time reverse-transcription recombinase polymerase amplification assay (RT-RPA) was developed to detect the transmissible gastroenteritis virus (TGEV) in this study. The primers and exo probe were designed to be specific for a portion of spike (S) gene conserved in TGEV, but absent in the closely related porcine respiratory coronavirus (PRCV). The amplification was performed at 40 °C for 20 min. The assay could only detect the TGEV, and there was no cross-reaction with other pathogens tested. Using the in vitro transcribed TGEV RNA as template, the limit of detection of the developed RT-RPA was 100 copies per reaction. The assay performance was evaluated by testing 76 clinical samples by RT-RPA and a real-time RT-PCR. Fourteen samples were TGEV RNA positive in RT-RPA (18.4%, 14/76), which were also positive in the real-time RT-PCR. The diagnostic agreement between the two assays was 100% (76/76). The R2 value of RT-RPA and real-time RT-PCR was 0.959 by linear regression analysis. The developed RT-RPA assay provides a useful alternative tool for rapid, simple and reliable detection of TGEV in resource-limited diagnostic laboratories and on-site facilities.

Real-time reverse transcription recombinase polymerase amplification assay for rapid detection of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea (PED), which is caused by porcine epidemic diarrhea virus (PEDV), is an acute, highly contagious enteric disease characterized by severe watery diarrhea, vomiting, dehydration, and high mortality in suckling piglets. A real-time reverse-transcription recombinase polymerase amplification assay (RT-RPA) was developed based on the nucleocapsid gene of PEDV. RT-RPA assay was performed at 40 °C for 20 min. The assay could detect both the classical and variant PEDV strains, and there was no cross-reaction with other pathogens tested. Using the in vitro transcribed PEDV RNA as template, the analytical sensitivity was 23 copies per reaction. The assay performance was evaluated by testing 76 clinical samples. PEDV RNA positive rate was 55.3% (42/76) by RT-RPA and 59.2% (45/76) by real-time RT-PCR. The diagnostic agreement between the two assays was 96.1% (73/76), and the R2 value of the two assays was 0.903 by linear regression analysis. The developed RT-RPA assay provides a useful alternative tool for simple, rapid and reliable detection of PEDV in resource-limited diagnostic laboratories and on-site facilities.

Recombinase polymerase amplification assay for rapid detection of porcine circovirus 3.

The objective of this study was to develop a real-time recombinase polymerase amplification (rt-RPA) assay for the rapid detection of porcine circovirus 3 (PCV3). Specific RPA primers and exo probes were designed for the cap gene of PCV3 within the conserved region of viral genome. The amplification was performed at 38 °C for 20 min. The rt-RPA was specific for PCV3, as there was no cross-reaction with other pathogens tested. Using the recombinant plasmid pUC57-PCV3 as template, the analytical sensitivity was 23 copies. Of the 186 clinical samples, PCV3 DNA was identified in the 51 samples by the rt-RPA, and the positive rate was 27.4% (51/186). The diagnostic agreement between the rt-RPA and real-time PCR was 96.2%. The R2 value of rt-RPA and real-time PCR was 0.919 by linear regression analysis. The developed rt-RPA assay shows promise for rapid and sensitive detection of PCV3 in diagnostic laboratories and at point-of-need, thus facilitating the prevention and control of PCV3.

An exo probe-based recombinase polymerase amplification assay for the rapid detection of porcine parvovirus.

Recombinase polymerase amplification (RPA), an isothermal amplification technology, has been developed as an alternative to PCR in pathogen detection. A real-time RPA assay (rt-RPA) was developed to detect the porcine parvovirus (PPV) using primers and exo probe specific for the VP2 gene. The amplification was performed at 39°C for 20min. There was no cross-reaction with other pathogens tested. Using the recombinant plasmid pPPV-VP2 as template, the analytical sensitivity was 103 copies. The assay performance was evaluated by testing 115 field samples by rt-RPA and a real-time PCR assay. The diagnostic agreement between assays was 100%, and PPV DNA was detected in 94 samples. The R2 value of rt-RPA and real-time PCR was 0.909 by linear regression analysis. The developed rt-RPA assay provides a useful alternative tool for rapid, simple and reliable detection of PPV in diagnostic laboratories and at point-of-care, especially in remote and rural areas.

Online Detection of Peroxidase Using 3D Printing, Active Magnetic Mixing, and Spectra Analysis.

A new method for online detection of peroxidase (POD) using 3D printing, active magnetic mixing, fluidic control, and optical detection was developed and demonstrated in this study. The proposed POD detection system consisted of a 3D printing and active magnetic mixing based fluidic chip for online catalytic reaction, an optical detector with a fluidic flow cell for quantitative determination of the final catalysate, and a single-chip microcontroller based controller for automatic control of two rotating magnetic fields and four precise peristaltic pumps. Horseradish peroxidase (HRP) was used as research model and a linear relationship between the absorbance at the characteristic wavelength of 450 nm and the concentration of HRP of 1/4-1/128 µg mL-1 was obtained as A = 0.257ln⁡(C) + 1.425 (R2 = 0.976). For the HRP spiked pork tests, the recoveries of HRP ranged from 93.5% to 110.4%, indicating that this proposed system was capable of detecting HRP in real samples. It has the potential to be extended for online detection of the activity of other enzymes and integration with ELISA method for biological and chemical analysis.

Reverse transcription recombinase polymerase amplification assay for the rapid detection of type 2 porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in pigs, and has tremendous negative economic impact on the swine industry worldwide. PRRSV is classified into the two distinct genotypes: type 1 and type 2, and most of the described PRRSV isolates in China are type 2. Rapid and sensitive detection of PRRSV is of great importance for the disease control and regional eradication programs. Recombinase polymerase amplification (RPA) has emerged as a novel isothermal amplification technology for the molecular diagnosis of infectious diseases. In this study, a fluorescence reverse transcription RPA (RT-RPA) assay was developed to detect the type 2 PRRSV using primers and exo probe specific for the viral nucleocapsid gene. The reaction was performed at 40°C within 20min. The RT-RPA assay could detect both the classical (C-PRRSV) and highly pathogenic PRRSV (HP-PRRSV), but there was no cross-reaction to other pathogens. Using the in vitro transcribed PRRSV RNA as template, the analytical sensitivity of RT-RPA was 690 copies. The assay performance was evaluated by testing 60 field samples and compared to real-time RT-PCR. The detection rate of RT-RPA was 86.6% (52/60), while the detection rate of real-time RT-PCR was 83.3% (50/60). This simple, rapid and reliable method could be potentially applied for rapid detection of PRRSV in point-of-care and rural areas.