A multiplex liquid-chip assay based on Luminex xMAP technology for simultaneous detection of six common respiratory viruses.

We utilized one-step multiplex reverse transcription-PCR (RT-PCR) and Luminex xMAP technology to develop a respiratory multiplex liquid-chip assay (rMLA) for simultaneous detection of 6 common respiratory viruses, including influenza virus type A (FluA) and type B (FluB), para-influenza virus type 3 (PIV-3), respiratory syncytial virus (RSV), human metapneumovirus (MPV) and a threatening virus to China, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Performance of rMLA was evaluated by comparing with real-time RT-PCR. Detection data from clinical specimens showed that the rMLA had diagnostic sensitivities of 97.10% for FluA, 94.59% for FluB, 98.68% for PIV-3, 94.87% for RSV and 95.92% for MPV (No Data for MERS-CoV due to the lack of positive specimens). Data of analytical sensitivities showed that the detection limits of the rMLA assay were 5-25 viral RNA copies per µl for FluA, FluB, PIV-3 and MERS-CoV, approximate to the real-time RT-PCR assay; while the values were 8 and 22copies/µl for MPV and RSV, lower than the real-time RT-PCR(78 and 114 copies/µl respectively). The results indicated that the rMLA is a sensitive, specific detection tool and comparable to real-time RT-PCR, especially suitable for high-throughput detection of respiratory specimens.

Dynamic quantification of avian influenza H7N9(A) virus in a human infection during clinical treatment using droplet digital PCR.

This study involved a human infection with avian influenza H7N9(A) virus in Zhejiang province, the first one after implementing the closure measures of living poultry markets in China. The clinical symptoms, epidemiological and virological characteristics of the case were described briefly, and as the emphasis, H7N9 virus was detected quantitatively and continuously from the collected samples in 10 different periods of the patient's treatment in order to reveal changes of viral load in patient's body during the treatment. This study first used reverse-transcription droplet digital PCR (RT-ddPCR) assays to monitor viral load dynamically for human H7N9 infection, synchronously performing real-time RT-PCR as a reference technology to obtain more comprehensive data for comparison. Our results indicated that RT-ddPCR compared to real-time RT-PCR is more sensitive and accurate for quantifying H7N9 viral load without the use of standard curves. Furthermore it can provide reference data for clinical policies including infectivity judgement, ward transferring and therapy adjustment for the patient during treatment.

A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV.

A one-step multiplex real-time reverse transcription-PCR (RT-PCR) assay was developed for one-tube and simultaneous detection of three genogroups of human norovirus, genogroup I, II and IV (GI, GII and GIV). The specificity and sensitivity of the assay were evaluated and 50 samples were tested by using this assay. The results showed that the multiplex assay had high sensitivity and specificity. The amplification efficiencies of the assay were 91.3%, 90.1%, 88.9% and the detection limits were up to 16.9, 6.3, 43.0 copies/reaction respectively for norovirus GI, GII and GIV detection. No cross-reaction with the other examined RNA viruses was observed, and the qualitative analysis of samples showed that the multiplex assay had a good consistency with its corresponding monoplex assays for the detection of norovirus GI, GII and GIV (Kappa values were 0.848, 0.876 and 0.812 respectively).

[Protective effects of uric acid on nigrostriatal system injury induced by 6-hydroxydopamine in rats].

OBJECTIVE: To investigate the protective effects of uric acid on nigrostriatal system injury induced by 6-hydroxydopamine in rats. METHODS: Thirty male SD rats were divided into four groups. Uric acid of 100 mg/kg, 200 mg/kg, 250 mg/kg were injected intraperitoneally (ip) into 5, 10, 5 rats twice daily at a 2-hour interval for five days and saline was injected ip into 10 rats as controls. At Day 6, 6-hydroxydopamine was injected into striatum to establish Parkinson's disease (PD) model in rats. Then uric acid was injected ip into three groups and saline into controls for five days. Locomotion test, amphetamine-induced rotation and forepaw adjusting step test were performed at Weeks 3 and 4 respectively after injection of 6-hydroxydopamine. HPLC-MS/MS was performed to detect the contents of dopamine and its metabolite homovanillic acid (HVA) in striatum at Week 5. RESULTS: The scores of locomotion in 2 minutes of 200 mg/kg uric acid group (14 +/- 4/2 min) was higher significantly than saline group (4 +/- 5/2 min, P < 0.01). The amphetamine-induced rotation number in the 200 mg/kg uric acid group (10.8 +/- 7.5) was lower significantly that in the saline group (19.3 +/- 5.2, P < 0.01). Forepaw adjusting step test scores of 200 mg/kg uric acid group were higher significantly than those in the saline group (9.89 +/- 3.41 vs 4.36 +/- 3.72, P < 0.01). HPLC-MS/MS showed that the contents of DA (0.29 +/- 0.19) and HVA (1.22 +/- 0.5) in injured striatum of 200 mg/kg uric acid group were higher significantly than those in the saline group (0.05 +/- 0.03, P < 0.01; 0.24 +/- 0.13, P < 0.05). CONCLUSION: An appropriately elevated level of uric acid may protect the dopamine neuron of nigrostriatal system from injury of 6-hydroxydopamine in rats.