Development of TaqMan-based Real-time PCR Assay for Detecting Transmissible Gastroenteritis Virus and Its Application in Vaccine Evaluation

[Objective] This study aimed to establish a TaqMan-based real-time PCR assay for detecting transmissible gastroenteritis virus （TGEV）. [Method] Primers and a probe were designed according to the conserved sequence of N gene in TGEV genome. After gradient dilution, the recombinant plasmid harboring the N gene was used as a standard for real-time PCR assay to establish the standard curve. [Re- sult] The results showed that the established real-time PCR assay exhibited a good linear relationship within the range of 102-10^10 copies/ul; the correlation coefficient was above 0.99 and the amplification efficiency ranged from 90% to 110%. The de- tection limit of real-time PCR assay for TGEV was 10 copies/μl, suggesting a high sensitivity; there was no cross reaction with other porcine viruses, indicating a good specificity; coefficients of variation within and among batches were lower than 3%, suggesting a good repeatability. The established real-time PCR method could be ap- plied in quantitative analysis and evaluation of the immune efficacy of TGEV vac- cines and detection of TGEV in clinical samples. [Conclusion] The TaqMan-based real-time PCR assay established in this study is highly sensitive and specific, which can provide technical means for the epidemiological survey of TGEV, development of TGEV vaccines and investigation of the pathogenesis of TGE.

A Method for Detecting Adhesive Related-Factors of Streptococcus suis Serotype 2 by Real-time PCR

[Objective] This study aimed to establish a method for quantitative detection of mRNA transcriptional level of SS2 adhesive related-factors of Streptococcus suis serotype 2 （SS2） by fluorescent quantitative PCR. []Vlethod] The gene fragments en- coding SS2 adhesive related-factors MRP, FBPS and CPS2J and a housekeeping gene aroA were amplified by reverse transcription PCR from the total RNA of SS2, cloned, and sequenced. The recombinant plasmids containing the target genes were constructed, and used as templates in Real-time PCR. [Result] Dynamic curves, stan- dard curves and melting curves of the adhesive related-factors and aroA were ob- tained by the optimized Real-time PCR system. The standard curves showed a good linear relationship between template copy number and circulation number, and the correlation coefficients （FF） of the standard curves were over 0.995. Also, these as- says were highly specific a^d there was single specific melting peak for every gene. Moreover, the assays were highly sensitive and had a detection limit of 1.0×102 copies in 1 μl of initial templates. Finally, it was highly repeatable and had a coeffi- cient of variation less than 2% for intra-assay. [Conclusion] This study will provide a way to reveal the adhesion mechanism of SS2 to different host cells at molecular level.

浅析双金属温度计的特点及其检定方法

双金属温度计可以直接测量各种工业生产过程中的中低温度下液体蒸汽和气体介质的温度,是一种测量中低温度的现场检测仪表。双金属温度计的检定需要相关部门根据中国计量出版社出版的《中华人民共和国国家计量检定规程JJG226-2001》中的有关方法进行标准检定,通过对规定项目的测量检验,得出相应数据,然后再通过对数据计算和对比,填入标准记录格式表中。

Quantitative evaluation of long-term liver repopulation and the reconstitution of bile ductules after hepatocellular transplantation

AIM： The treatment of liver disease is severely limited by a shortage of donor livers. In trying to address this growing problem, hepatocellular transplantation （HTx） has received much attention as an alternative to whole organ transplant. However, the expansion of transplanted cells is at low level, and the reconstitution of functional liver tissue is limited by this cellular property. We set up an animal model to better understand cell dose effect and the kinetics of liver repopulation following HTx. METHODS： Dipeptidyl peptidase Ⅳ （DPPⅣ）-deficient rats treated with retrorsine and subjected to partial hepatectomy were infused with DPPⅣ-positive hepatocytes. Rats were injected with varying numbers of donor hepatocytes down to 100 cells low, and liver repopulation was examined at different time points up to 20 mo long. Repopulation was assessed by computer-aided quantitative detection. RESULTS： Transplanted hepatocytes underwent multiple rounds of proliferation and stably repopulated the injured livers after 20 mo and at all cell doses. Transplanted cells divided 14 times within the 3-mo time period following infusion, and the liver repopulation reached a plateau between 3 and 20 too. Approximately 90% replacement occurred. Donor-derived cells also reconstituted the bile ductules of the recipients. CONCLUSION： The ability of transplanted hepatocytes to fully reconstitute injured livers strongly supports further investigation into the clinical potential of HTx. Additionally, the observation that transplanted hepatocytes also form components of the biliary system suggests that these cells may have bi-potential property of the stem cells.

Development and evaluation of immunoassay for zeranol in bovine urine

A high affinity polyclonal antibody-based enzyme linked immunosorbent assay （ELISA） was developed for the quantification of zeranol in bovine urine. On the basis of urine matrix studies, the optimized dilution factors producing insignificant matrix interference were selected as 1：5 in pretreatment. In the improved ELISA, the linear response range was between 0.02 and 1 μg/ml, and the detection limit was 0.02 μg/ml for the assay. The overall recoveries and the coefficients of variation （CVs） were in the range of 82%-127% and 3.5%-8.8%, respectively. Thirty-six bovine urine samples spiked with zeranol （ranging from 0.2 to 10 μg/ml） were detected by the ELISA and liquid chromatography （LC） method, and good correlations were obtained between the two methods （R^2=0.9643）. We conclude that this improved ELISA is suitable tool for a mass zeranol screening and can be an altemative for the conventional LC method for zeranol in bovine urine.

A quantitative analysis method for GPR signals based on optimal biorthogonal wavelet

Due to the disturbances arising from the coherence of reflected waves and from echo noise,problems such as limitations,instability and poor accuracy exist with the current quantitative analysis methods.According to the intrinsic features of GPR signals and wavelet time–frequency analysis,an optimal wavelet basis named GPR3.3 wavelet is constructed via an improved biorthogonal wavelet construction method to quantitatively analyse the GPR signal.A new quantitative analysis method based on the biorthogonal wavelet(the QAGBW method)is proposed and applied in the analysis of analogue and measured signals.The results show that compared with the Bayesian frequency-domain blind deconvolution and with existing wavelet bases,the QAGBW method based on optimal wavelet can limit the disturbance from factors such as the coherence of reflected waves and echo noise,improve the quantitative analytical precision of the GPR signal,and match the minimum thickness for quantitative analysis with the vertical resolution of GPR detection.

A Rapid and Sensitive One Step-SYBR Green Based Semi Quantitative Real Time RT-PCR for the Detection of peste des petits ruminants Virus in the Clinical Samples

A sensitive and rapid single step real time （rt） RT-PCR was standardized using one-step Brilliant SYBR Green kit for detection and semi-quantitation ofpeste des petitis ruminants virus （PPRV） using the virus RNA and matrix （M） protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with Tm of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit （sensitivity） of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was -0.0001 cell culture infectious dose 50% units （TCID50）. Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection （dpi） and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.

Development of a concentration method for detection of tobacco mosaic virus in irrigation water

Tobacco mosaic virus(TMV) causes significant yield loss in susceptible crops irrigated with contaminated water. However, detection of TMV in water is difficult owing to extremely low concentrations of the virus. Here, we developed a simple method for the detection and quantification of TMV in irrigation water. TMV was reliably detected at concentrations as low as 10 viral copies/μL with real-time PCR. The sensitivity of detection was further improved using polyethylene glycol 6000(PEG6000, MW 6000) to concentrate TMV from water samples. Among the 28 samples from Shaanxi Province examined with our method, 17 were tested positive after virus concentration. Infectivity of TMV in the original water sample as well as after concentration was confirmed using PCR. The limiting concentration of TMV in water to re-infect plants was determined as 102 viral copies/mL. The method developed in this study offers a novel approach to detect TMV in irrigation water, and may provide an effective tool to control crop infection.

Development of a quantum-dot-labelled magnetic immunoassay method for circulating colorectal cancer cell detection

AIM:To detect of colorectal cancer(CRC) circulating tumour cells(CTCs) surface antigens,we present an assay incorporating cadmium selenide quantum dots(QDs) in these paper.METHODS:The principle of the assay is the immunomagnetic separation of CTCs from body fluids in conjunction with QDs,using specific antibody biomarkers:epithelial cell adhesion molecule antibody,and monoclonal cytokeratin 19 antibody.The detection signal was acquired from the fluorescence signal of QDs.For the evaluation of the performance,the method under study was used to isolate the human colon adenocarcinoma cell line(DLD-1) and CTCs from CRC patients' peripheral blood.RESULTS:The minimum detection limit of the assay was defined to 10 DLD-1 CRC cells/mL as fluorescence was measured with a spectrofluorometer.Fluorescenceactivated cell sorting analysis and Real Time RT-PCR,they both have also been used to evaluate the performance of the described method.In conclusion,we developed a simple,sensitive,efficient and of lower cost(than the existing ones) method for the detection of CRC CTCs in human samples.We have accomplished these results by using magnetic bead isolation and subsequent QD fluorescence detection.CONCLUSION:The method described here can be easily adjusted for any other protein target of either the CTC or the host.

A real-time PCR targeted to the upstream regions of HlyB for specific detection of Edwardsiella tarda

Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry. Therefore, a rapid, reproducible, and sensitive method for detection and quantification of this pathogen is needed urgently. To achieve this purpose, we developed a TaqMan-based real-time PCR assay for detection and quantification orE. tarda. The assay targets the hemolysin activator HlyB domain protein of E. tarda. Our optimized TaqMan assay is capable of detecting as little as 40 fg of genomic DNA per reaction. A standard curve was generated from the threshold cycle values （y） against log10 （E. tarda genomic DNA concentration） as x. The intra- and inter-assay coefficient of variation （CV） values were less than 2.06% and 1.05% respectively, indicating that the assay had good reproducibility. This method is highly specific to E. tarda strains, as it shows no cross-reactivity to Edwardsiella ictaluri, a member of the same genus, or to nine other fish-pathogenic bacteria species belonging to three other genera. This sensitive and specific real-time PCR assay provides a valuable tool for diagnostic quantitation of E. tarda in clinical samples.

Cloning,tissue distribution and effects of fasting on pituitary adenylate cyclase-activating polypeptide in largemouth bass

Pituitary adenylate cyclase activating polypeptide （PACAP） has a wide range of biological functions. We cloned the full-length cDNAs encoding PACAP and PACAP-related peptide （PRP） from the brain of largemouth bass （Micropterus salmoides） and used real-time quantitative PCR to detect PRP- PACAP mRNA expression. The PRP-PACAP cDNA has two variants expressed via alternative splicing： a long form, which encodes both PRP and PACAP, and a short form, which encodes only PACAR Sequence analysis results are consistent with a higher conservation of PACAP than PRP peptide sequences. The expression of PACAP-Iong and PACAP-short transcripts was highest in the forebrain, followed by the medulla, midbrain, pituitary, stomach, cerebellum, intestine, and kidney; however, these transcripts were either absent or were weakly expressed in the muscle, spleen, gill, heart, fatty tissue, and liver. The level of PACAP-short transcript expression was significantly higher than expression of the long transcript in the forebrain, cerebella, pituitary and intestine, but lower than that of the long transcript in the stomach. PA CAP- long and PACAP-short transcripts were first detected at the blastula stage of embryogenesis, and the level of expression increased markedly between the muscular contraction stage and 3 d post hatch （dph）. The expression of PACAP-long and PACAP-short transcripts decreased significantly in the brain following 4 d fasting compared with the control diet group. The down-regulation effect was enhanced as fasting continued. Conversely, expression levels increased significantly after 3 d of re-feeding. Our results suggest that PRP- PA CAP acts as an important factor in appetite regulation in largemouth bass.

Using a non-radioisotopic, quantitative TRAP-based method detecting telomerase activities in human hepatoma cells

A non-radioisotopic, quantitative TRAP-based telomerase activity assay was established mainly by using SYBR Green-I staining instead of radioisotope. Comparing with conventional radioisotope based method, it was better in reproducibility and accuracy. Using this method, we found telomerase activities were absent in normal human liver cells, while detected in ail of four human hepatoma cell lines (BEL-7404, SMMC-7721, QGY-7903 and HCCM) without significant differences.

Study on the concentrated SF_6 quantitative intelligent ultrasonic detection system

In order to realize on-line quantitative detection on SF6 and effective control of nlnning state of SF6 high voltage power supply system, a concentrated SF6 quantitative ultrasonic on-line deteetion system has been developed based on the actual demand of electric power system consumers. There are four major characteristics in this system. Firstly, the gas of maximum 64 detection points is transferred through the specific air path to the detection devices to he detected and analyzed, thereby the electrical lines and the complicated installation of the collectors can be avoided; secondly, the differential technique is used to shield the influence of environmental factors, which effectively improves the accuracy of the acoustic detection; thirdly, the SF6 coneentration is determined by the speed and phase in the ultrasonic wave trans- mission process, therefore there is no secondary pollution for the purely physical means; finally, the ma- ture embedded technique is applied in this system to improve its intelligence and stability.

A Semi-quantitative Serological Method to Assess the Potency of Inactivated Rabies Vaccine for Veterinary Use

Potency is one of the most important indexes of inactivated vaccines.A number of methods have been established to assay the potency,of which the NIH test and single-dose mouse protection test are the "prescribed methods".Here,we report a method to semi-quantitatively assay the potency of an inactivated rabies vaccine,which uses fewer animals and takes less time to complete.Depending on the quality requirements of a vaccine(e.g.minimum potency),a rabies reference vaccine is,for example,diluted to the minimum potency,and 50 μL of the dilution is taken to inoculate 10 mice.The same amount of the test rabies vaccine is inoculated into another 10 mice.After two weeks,all mice are bled and serum samples are assayed for viral neutralizing antibody by the fluorescent antibody virus neutralization(FAVN) test.By comparing the median and interquartile range of antibody titers of the reference vaccine with those of the test vaccine,the test vaccine potency can be semi-quantitatively judged as to whether it is in accord with the required quality.The reliability of this method was also confirmed in dogs.The procedure can be recommended for batch potency testing during inactivated rabies vaccine production.

Determination of saccharin in preserved fruits by High Performance Liquid Chromatography

Quantitative analysis of saccharin in preserved fruits was performed by High Performance Liquid Chromatography （HPLC）. The separation was observed on a reversed phase ODS C）8 column （4.6 × 250 mm）. Mobile phase system consisted of ammonium acetate buffer： Methanol （60：40 v/v） at a flow rate of 1.0 mL.min^-1, and UV detector was set at 235 nm. The calibration curve of saccharin was linear in the concentration range between 1 to 100 mg·L^-1）, while the detection limit of saccharin was found to be 0.08 mg.L^-1. The mean value of recovery was 98.24% with standard deviation of0.32% （n=12）. The proposed method was successfully applied to determine the amount of saccharin in 12 preserved fruits, commercially available in Chiang Mai local markets. The results showed that the saccharin contents were found in the range of 6.22-78.60 mg.kg^-1.

Quantitative Detection of Screening for Cervical Lesions with ThinPrep Cytology Test

OBJECTIVE To investigate the available parameters in gynecological screening for cervical lesions by liquid-based cytology technology （ThinPrep Cytology Test, TCT） and The Bethesda System （TBS）, also with computer image analysis. METHODS With application of the image analysis system, all grades of cervical lesion cells were detected quantitatively and sorted in atypical squamous cells of undetermined significance （ASCUS）, atypical squamous cells-cannot exclude HSIL （ASC-H）, low-grade squamous intraepithelial lesion （LSIL）, high-grade squamous intraepithelial lesion （HSIL） and cervical squamous cell carcinoma （SCC） with the mean optical density （MOD）, average grey （AG）, positive units （PU）, and nucleus to cytoplasmic ratio （N： C）. Differences between each group of cells were compared and analyzed statistically. RESULTS Apart from four stereologic parameters in LSIL and HSIL groups there were no differences among them, in the other groups, there was statistically significant in differences between MOD, AG and PU values. Differences between them in the ratio of nucleus to cytoplasm were highly statistically significant. CONCLUSION Stereological indexes may serve as a screening tool for cervical lesions. The image analysis system is expected to become a new means of cytological assisted diagnosis.

Gross Error Detection and Identification Based on Parameter Estimation for Dynamic Systems

The detection and identification of gross errors, especially measurement bias, plays a vital role in data reconciliation for nonlinear dynamic systems. Although parameter estimation method has been proved to be a pow-erful tool for bias identification, without a reliable and efficient bias detection strategy, the method is limited in ef-ficiency and cannot be applied widely. In this paper, a new bias detection strategy is constructed to detect the pres-ence of measurement bias and its occurrence time. With the help of this strategy, the number of parameters to be es-timated is greatly reduced, and sequential detections and iterations are also avoided. In addition, the number of de-cision variables of the optimization model is reduced, through which the influence of the parameters estimated is reduced. By incorporating the strategy into the parameter estimation model, a new methodology named IPEBD (Improved Parameter Estimation method with Bias Detection strategy) is constructed. Simulation studies on a con-tinuous stirred tank reactor (CSTR) and the Tennessee Eastman (TE) problem show that IPEBD is efficient for eliminating random errors, measurement biases and outliers contained in dynamic process data.

Taqman PCR-based Methods for Rapid Detection of Salmonella in Pet Food

[ Objective] To establish a Taqman real-time PCR for detection of Salmonella in pet food. [Method] A pair of primers and a probe were designed based on published nucleotide sequence of invA gene encoding the invasion protein of Salmonella enterica. [ Result] The assay detects Salmonella specifically. The detection limit of the real-time PCR was 17 CFU/test （25 uL/test） for the positive strain. This method was effective to detect artificially contaminated pet food. [ Conclusion] The results showed that Taqman PCR assay was rapid and accurate for detection of Salmonella from infected pet food.

Expression Level of a Transcription Factor Gene Mdhd-Zip Up-regulated during Apple Fruit Senescence

[Objective] This study aimed to explore the role of one apple transcription factor of homeodomain-leucine zipper(MdHD-Zip) during apple fruit senescence.[Method] Postharvest Red Fuji fruits(Malus domestica Borkh ‘Red Fuji') were stored at room temperature(18 ℃-20 ℃) and cold condition(0 ± 1 ℃) separately.Fruit firmness and ethylene production during storage process were analyzed.Transcript level of Md HD-Zip was detected by real-time quantitative PCR during apple fruit storage under room temperature and cold condition.[Result] Expression level of Md HD-Zip was found up-regulated in later stage of apple fruit senescence at room temperature,while it showed a peak level after one month of cold storage.[Conclusion] The results of the present study suggest that Md HD-Zip may play a role in regulating apple fruit senescence.

Evaluation of Sensitivities and Specificities of SARS-CoV Detection by Real-time Quantitative Reverse Transcription-PCR Assays

The etiological agent of severe acute respiratory syndrome(SARS) was identified as a new coronavirus,termed SARS-CoV. Establishment of an efficient and sensitive diagnostic system of SARS-CoV genetic materials is crucial for SARS control. In this study,we quantified SARS-CoV mRNAs in both infected cell culture lysate and in supernatant by using Real-time quantitative revere transcription-PCR based on EvaGreenTM dye and Taqman-MGB probes. For extensive evaluation of sensitivities and specificities,13 pairs of primers and 4 probes were designed based on different genes of SARS-CoV. Glyceraldehydes-3-phosphate dehydrogenase(GAPDH) was selected as the internal control gene. Results showed that S-gene-specific PCR was the most sensitive for detection,but because of its sequence variability in the different viral strains,primers and a probe based on the N gene were suitable substitutions. Meanwhile,we found the mRNA concentrations in cell culture lysates were much higher than in cell supernatant and facilited more sensitive detection of the SARS-CoV.