Real-time RT-PCR Assay for the detection of Tahyna Virus

A real-time RT-PCR （RT-qPCR） assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.

Real-time RT-PCR Assay for the Detection of Culex flavivirus

Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed that CxFV could be detected using RT-qPCR with the specific CxFV primers and probes; other species of arboviruses were not detected. The stability test demonstrated a coefficient of variation of <1.5%. A quantitative standard curve for CxFV RT-qPCR was established. Quantitative standard curve analysis revealed that the lower detection limit of the RT-qPCR system is 100 copies/mu L. Moreover, RT-qPCR was used to detect CxFV viral RNA in mosquito pool samples. In conclusion, we established a real-time RT-PCR assay for CxFV detection, and this assay is more sensitive and efficient than general RT-PCR. This technology may be used to monitor changes in the environmental virus levels.

Combination of Loop-Mediated Isothermal Amplification Assay and Nested PCR for Detection of Borrelia burgdorferi sensu lato in Human Serum Samples

A set of universal loop-mediated isothermal amplification （LAMP） primers targeting the flo gene was designed to detect Borrelia burgdorferi sensu lato （B. burgdorferi s.I.） in human samples. The sensitivity of LAMP was 20 copies/reaction, and the assay did not detect false positives among 11 other related bacteria. A positive LAMP result was obtained for 9 of the 24 confirmed cases and for 12 of 94 suspected cases. The positive rate of LAMP was the same as that of nested PCR. The LAMP is a useful diagnostic method that can be developed for rapid detection of B. burgdorferi s.I. in human sera. Combination of the LAMP and nested PCR was more sensitive for detecting B. burgdorferi s.I. in human serum samples.

Rapid,sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification

Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming. The rapid detection and identification of V. anguillarum, and other pathogens that infect marine organisms, is crucial to effective disease management. In this study, we developed a loop-mediated amplification （LAMP） assay to detect V. anguillarum in an hour in a single tube without the need for thermal cycling. Conserved regions of the metalloproteinase （empA） gene of V. anguillarum served as the targets for primer design. A fragment of the empA gene was amplified at 65℃ in the presence of the primer mixture and Bst DNA polymerase. In the optimized LAMP assay, 6.7 pg of V. anguillarum DNA could be detected. Six strains of V. anguillarum and 17 strains of non-V, anguillarum bacteria were used in this study to evaluate the species specificity of the primers. The six V. anguillarum strains gave a positive result in the LAMP assay. This method was also validated in V. anguillarum-infected fish. This LAMP method is more sensitive than PCR in the detection of V. anguillarum and shows good species specificity. The LAMP assay is therefore an effective method for the quick detection of V. anguillarum both in the laboratory and in the field.

Affinity peptide developed by phage display selection for targeting gastric cancer

AIM:To develop an affinity peptide that binds to gastric cancer used for the detection of early gastric cancer.METHODS:A peptide screen was performed by biopanning the PhD-12 phage display library,clearing non-specific binders against tumor-adjacent normal appearing gastric mucosa and obtaining selective binding against freshly harvested gastric cancer tissues.Tumortargeted binding of selected peptides was confirmed by bound phage counts,enzyme-linked immunosorbent assay,competitive inhibition,fluorescence microscopy and semi-quantitative analysis on immunohistochemistry using different types of cancer tissues.RESULTS:Approximately 92.8% of the non-specific phage clones were subtracted from the original phage library after two rounds of biopanning against normal-appearing gastric mucosa.After the third round of positive screening,the peptide sequence AADNAKTKSFPV(AAD) appeared in 25%(12/48) of the analyzed phages.For the control peptide,these values were 6.8 ± 2.3,5.1 ± 1.7,3.5 ± 2.1,4.6 ± 1.9 and 1.1 ± 0.5,respectively.The values for AAD peptide were statistically signif icant(P < 0.01) for gastric cancer as compared with other histological classif ications and control peptide.CONCLUSION:A novel peptide is discovered to have a specific binding activity to gastric cancer,and can be used to distinguish neoplastic from normal gastric mucosa,demonstrating the potential for early cancer detection on endoscopy.

A quantitative RT-PCR assay for rapid detection of Eurasianlineage H10 subtype influenza A virus

Dear Editor,Influenza A viruses(IAVs)are single-stranded,negative sense RNA viruses.IAV subtype is determined on the basis of the viral surface glycoproteins,hemagglutinin(HA),and neuraminidase(NA).To date,18 HA and 11NA subtypes have been reported(Tong et al.,2012).

Evaluation of a Rapid Immunochromatographic Middle East Respiratory Syndrome Coronavirus Antigen Detection Assay

Middle East respiratory syndrome coronavirus(MERS-CoV)infection in humans has a high mortality of>30%.Dromedaries are the reservoir of MERS-CoV and the main source of human infections.However,MERS-CoV infections in dromedaries are usually subclinical.Rapid diagnosis of MERS-CoV infection in these animals is important in preventing camel-to-human transmission of the virus.The possible cross-reactivity of a previously reported rapid nucleocapsid protein-based antigen detection assay for MERS-CoV was examined with different CoVs,including Tylonycteris bat CoV HKU4,dromedary camel CoV UAE-HKU23,human CoV-229E,human CoV-OC43,severe acute respiratory syndrome CoV-2 and rabbit CoV HKU14,where none of them showed false-positive results.The assay was further validated using quantitative real-time reverse transcription-polymerase chain reaction-confirmed MERS-CoV-positive and MERS-CoV-negative dromedary nasal samples collected in Dubai,the United Arab Emirates,which showed that the rapid antigen detection assay has a specificity of 100%and sensitivity of 91.7%.

A sensitive and convenient enzyme-linked immunosorbent assay method in serum MG7 antigen detection in gastric cancer

Objective To explore a highly sensitive and highly specific method to detect the serum MG7 antigen(Ag)level for early gastric cancer diagnosis.Methods The serum MG7-Ag level was detected by enzyme-linked immunosorbent assay(ELISA)method in 116 preoperative gastric cancer patients,63 postoperative gastric cancer patients,41 patients with precancerous lesion,37 pa-

Colorimetric detection of glucose using a boronic acid derivative receptor attached to unmodified AuNPs

A simple, cheap and non-enzymatic colorimetric strategy for glucose detection has been designed based on the interactions between a phenylboronic acid （PBA） derivative, which is coupled with gold nanoparticles （AuNPs） as the colorimetric reporters, and glucose. The PBA-AuNPs hybrid system proposed here exhibits ordered photochemistry behaviors upon the addition of glucose at different pH values. There are two linear regions of glucose concentration for the glucose sensor at different pH values, i.e., between 0.1 mmol/L and 9.8 mmol/L at pH 6 with the detection limit of 64μmol/L and between 0 and 6.5 mmol/L with the detection limit of 48 μmol/L at pH 9, respectively. To test the practicality of the sensor system, we also applied this assay to detect a glucose sample in the artificial saliva.

Immune fluorescence test strips based on quantum dots for rapid and quantitative detection of carcino-embryonic antigen

At present,many researchers focused on the point-of-care testing（POCT）,a method of disease markers detection without large-scale instruments and specialized persons.However,most POCT diagnostic methods were suffered from poor detection sensitivity or inefficiency in quantitative detection.Herein,we developed a newly QD-immune fluorescence test strips（QD-IFTS） based on quantum dots（QDs） as the fluorescence nanocarrier to prepare the immune fluorescence probes in the classical immunochromatography detection system for sensing carcino-embryonic antigen（CEA）,a kind of glycoprotein produced by intestinal tissue and a broad spectrum of tumor marker for cancer diagnosis.And we designed a homemade strips fluorescence reader for detection of fluorescence intensity of QDs on the QD-IFTS.Under the optimized reaction conditions,chromatographic time of the newly QD-IFTS was only25 min,sample volume of the newly QD-IFTS was only 40 m L and the LOD of the newly QD-IFTS was 0.72 ng/m L.In addition,the efficiency and robustness of the newly QD-IFTS were confirmed by successfully application in 300 clinical serum samples,and the results revealed great potential in clinical POCT of other biomarkers.

Advances in single-particle detection for DNA sensing

Rapid, accurate and sensitive detection of particular DNA sequence is critical in fundamental biomedical research and clinical diagnostics. However, conventional approaches for DNA assay often suffer from cumbersome procedures, long analysis time and insufficient sensitivity. Recently, single-particle detection technology has emerged as a powerful tool in the biosensing area due to its significant advantages of ultrahigh sensitivity, low sample-consumption and rapid analysis time. Especially, the introduction of novel nanomaterials has greatly promoted the development of single-particle detection and its applications for DNA sensing. In this review, we summarize the recent advance in single-particle detection strategies for DNA sensing, and focus mainly on metallic nanoparticle-and semiconductor quantum dot-based single-particle detection. We highlight the emerging trends in this field as well.