Detection of measles virus infection and proliferation by real-time quantitative PCR / 上海预防医学

Objective A SYBR-Green real-time quantitative PCR(RT-qPCR)method was set up to detect the infection and proliferation of measles virus, which could be useful in virus titer determination. Methods In this project, we used a 405 bp fragment of the N gene of measles virus as a target sequence and constructed a plasmid to establish the standard curve in absolute quantitative experiment. We then used this method to obtain the proliferation curve of measles virus and to detect the virus proliferation at different MOI. Results There was a linear relationship between the virus copy number and the titer of the measles virus reference at the range of 6 to 2 lgCCID50/mL, with a correlation coefficient (r) of 0.991(P < 0.01). Based on the analysis of virus proliferation curve, measles virus mainly proliferated intracellularly within 48 h after its entering the cell. There was no detected increase in viral RNA level in the first 24 h, suggesting the virus was in a silent period in the cell. After 24 h, the virus expanded in large numbers and entered the exponential growth phase. The intracellular viral RNA level reached the plateau phase after its peak at 96 h. The virus secreted to the outside of the cell entered the exponential growth phase starting from 48 h, peaked at 144 h, then followed by plateau phase. Conclusion A SYBR-Green RT-qPCR method is established and used to monitor virus proliferation. Our result is helpful in understanding of the proliferation and secretion of measles virus in cells and provides experimental basis for detection of live attenuated virus titers.

Establishment of detection method for Chlamydophila abortus by using SYBR green real-time PCR and determination of bacterial load in mice / 中国人兽共患病学报

We established a fluorescent quantitative PCR (qPCR)method for the detection of Chlamydophila abortus (C. abortus),and replaced the method of smear staining which has subjective influence on the detection of C.abortus inactivated vaccine titer.According to the conserved sequence of the large cysteine-rich periplasmic protein(envB)of C.abortus,a specific primer was designed and the EnvB-PMD19T positive plasmid was used as the reference standard,optimization condition,sensi-tivity assay,specificity assay,repeatability assay and the bacteria loads of organs from mouse have been done.The results showed that the standard curve established with positive plasmid had a liner response from 1×102copies/μL to 1×106copies/μL with the correlation coefficient of 97%,sensitive for detecting C.abortus with the detection limit of 10 copies/μL,and re-peatable and stable with the coefficients of variation less than 2%.According to the result,the established method can detect the bacteria loads in organ of mouse,which provide a reliable method for evaluation of inactivated C.abortus vaccine.

Development and application of a SYBR Green Ⅱ real-time RT-PCR for detection of swine hepatitis E virus / 中国人兽共患病学报

In order to establish a real-time RT-PCR based on SYBR Green Ⅱ for detection of hepatitis E virus (HEV),a pair of special primers was designed according to the conserved sequences of ORF2 in GenBank.Result showed that the standard curve of established SYBR Green Ⅱ real-time RT-PCR had a wide dynamic range from 4.10 × 102-4.10 × 108 copies/μL with a linear correlation(r2) of 0.996.The sensitivity could reach 1.00 × 102 copies/μL.The melting curve analysis using SYBR Green Ⅱ dye showed one specific peak with a melting temperature(Tm) of 84.0 C ±0.1 C.No amplification was detected from the RNA samples of porcine reproductive and respiratory syndrome virus,classial swine fever virus,transmissible gastroenteritis virus,porcine bocavirus,porcine epidemic dearrhoea virus porcine kobuvirus and porcine rotavirus by this PCR,respectively.Excellent reproducibility was obtained for detecting constructed positive plasmid DNA with intra-assay of 0.83 ％-0.94 ％ and inter-assay of 0.83％-0.94％.Further detection of 61 specimens showed that 9 of them were HEV positive,and the results of the quantitative RT-PCR were the same as that of the conventional RT-PCR.In conclusion,the real-time quantitative RT-PCR for HEV is feasible,the real-time RT-PCR established in this study will be useful for earlier rapid laboratory diagnosis and pathogenesis of HEV.

Adaptación y optimización de un método de lectura por fluorometría en el modelo farmacológico in vitro de cultivo de Plasmodium falciparum / Adaptation and optimization of a fluorometric reading method, in the in vitro pharmacological model of Plasmodium falciparum culture

El modelo farmacológico de cultivo in vitro de P. falciparum es crucial en el tamizaje inicial de sustancias o extractos de plantas con posible actividad antiplasmodial. La densidad parasitaria puede determinarse mediante variadas metodologías, sin embargo, se han descrito numerosas ventajas y desventajas asociadas a cada una de ellas. Se evaluaron el tiempo de incubación necesario para la tinción y el uso de cultivos asincrónicos o sincrónicos en busca de valores óptimos; evidenciando un tiempo óptimo de 2 h, y límites de detección y cuantificación, menores en cultivos asincrónicos. Empleando las cepas FCR3 y FCB2 se evidenció un ruido de fondo de 12% y 38% respectivamente; la linealidad mostró una buena correlación, r² de 0,9644 (FCR3) y 0,9841 (FCB2) y una pendiente de 1761,8 y 852,4 respectivamente. Además, se comprobó había concordancia entre los métodos, fluorométrico con SYBR Green I (SYBRG I) y microscópico con Giemsa, con diferencia media de 0,00002% y 0,09109% para FCR3 y FCB2 respectivamente. Los límites de detección y cuantificación fueron 0,5% y 1,5% de parasitemia. El factor Z con FCB2 fue 0,376, en tanto que con FCR3 alcanzó 0,702. La concentración inhibitoria 50 (CI50) frente a P. falciparum FCR3, generada por cloroquina (CQ) fue 0,37 mcg/mL por microscopía y 0,35 mcg/mL por fluorometría. Nuestros hallazgos sugieren que el ensayo de fluorescencia con SYBRG I, empleando fluorómetros comúnmente disponibles en muchos laboratorios, es preciso, robusto, rápido y exacto; para la evaluación in vitro de sustancias o extractos con posible actividad antiplasmodial.

The in vitro pharmacological model of P. falciparum culture is crucial in the initial screening for substances or plant extracts with possible antiplasmodial activity. The parasite density can be determined by varied methods, however have been described numerous advantages and disadvantages associated with each of them. The incubation time required for staining and the use of synchronous or asynchronous cultures were assessed for optimal settings; showing optimal time of 2 h, and lower limits of detection and quantification in asynchronous cultures. Employing the FCB2 and FCR3 strains, was evidenced a background noise of 12% and 38% respectively; linearity showed a good correlation, r² of 0.9644 (FCR3) and 0.9841 (FCB2) and a slope of 1761.8 and 852.4, respectively. It was evidenced agreement between the methods, fluorometric with SYBR Green I (SYBRG I) and microscopic with Giemsa, the mean difference was 0.00002% and 0.09109% respectively for FCR3 and FCB2, The limits of detection and quantification were 0.5% and 1.5% of parasitaemia. The Z factor was 0.376 with FCB2, whereas with FCR3 reached 0.702. The inhibitory concentration 50 (IC50) against P. falciparum FCR3, generated by chloroquine (CQ), was 0.37 mcg/mL by microscopy and 0.35 mcg/mL by fluorometry. Our findings suggest that the SYBRG I fluorescence based assay, by using fluorometers commonly available in many laboratories, is precise, robust, fast and accurate; for the in vitro evaluation of substances or extracts with possible antiplasmodial activity.

Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India.

Background & objectives: Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Methods: Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. Results: In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated (P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Interpretation & conclusions: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods.

Establishment and optimization of loop-mediated isothermal amplification for rapid detection of pseu-domonas aeruginosa / 实用医学杂志

Objective To establish a Loop-mediated Isothermal Amplification (LAMP) for Pseudomonas aeruginosa rapid detection. Method 152 P. aeruginosa strains isolated from nasal swabs and 30 reference strains were applied. P. aeruginosa ATCC15442 was used to develop LAMP amplification and evaluate sensitivity and specificity. Results Sensitivity of LAMP was 103 times higher than PCR, with DNA amount as 132 fg. When LAMP was applied to 30 reference strains and 152 P. aeruginosa strains , the specification was 100% while iden-tification rate reached 94.7%. Conclusion The establishment LAMP showed a promising prospect in P. aerugi-nosa rapid detection.

Establishment and Application of SYBR Green I Real-Time PCR Assay for Rapid Detection of Hepatitis B Virus DNA / 现代检验医学杂志

Objective To develop SYBR Green I real-time PCR assay for detection and identification of Hepatitis B virus. Methods Based on the sequences of Hepatitis B virus gp1 gene,primers were designed.The reaction assay and thermal cyc-ling profile were optimized.The positive standard was from recombinant clone.Both the developed assay and Zhejiang kuake biotechnology company’s assay were applied in 100 patients serum.Results The detection limit was between 5×102 copies/ml to 5×108 copies/ml with a good liner correlation and no cross reaction.The whole process just needed 2.5 h.Comparing with the company products,the sensitivity and specificity of the developed assay were 100% and 92.5% respectively.Con-clusion The established assay is rapid,simple,high sensitivity and specificity.It is not only valuable for the identification of Hepatitis B virus patients,but also provide accurate quantitative analysis for HBV patients.

Dynamics of porcine circovirus type 2 infection and neutralizing antibodies in subclinically infected gilts, and the effect on their litters / Dinámica de la infección por el circovirus porcino tipo 2 y títulos de anticuerpos neutralizantes en las cerdas de reemplazo subclinicamente infectadas, y el efecto en sus camadas / Dinâmica da infeção pelo circovirus porcino tipo 2 e títulos de anticorpos neutralizantes em porcas nulíparas subclínicamente infectadas e efeito em sua leitegada

Background: porcine circovirus type 2 (PCV2) is associated with reproductive disease in newly populated herds and in replacement breeding stock from new sources and is almost exclusively reported in gilts. Objective: the main purpose of this study was to assess the dynamics of porcine circovirus type 2 infection and neutralizing antibodies in subclinically infected gilts and the effect on their piglets. Methods: the study was conducted with 40 gilts selected at random from four breeding herds. Blood samples, nasal and vaginal swabs were obtained from the gilts at arrival, acclimatization, farrowing, and one day after farrowing. Colostrum samples were collected immediately after parturition and one day after farrowing. Blood, nasal swab, or tissue samples were collected from four piglets prior to suckling. All serums were analyzed by virus neutralization test (VNT) to establish the presence of antibodies. All samples were subjected to SYBER Green real-time PCR assay to detect PCV2 DNA. Results: high levels of viremia and viral load of PCV2 in nasal and vaginal swabs were found in healthy gilts at arriving, confirming the introduction of infected animals into the farms. In addition, most gilts were positive for PCV2 DNA in serum, nasal and vaginal swabs at farrowing. PCV2 shedding was also observed in nasal and vaginal fluids and colostrum even in presence of serum neutralizing antibodies (NA). Subclinically infected dams had detectable viremia, developed anti-PCV2 antibodies, and there was PCV2 DNA in tissue samples of their born alive and healthy piglets. PCV2a and PCV2b genotypes were confirmed in PCV2 subclinical infection in both dams and piglets in utero. Conclusion: replacement gilts can be infected with PCV2 before entering the farm and continuous exposure seems to occur horizontally in acclimatization and gestation units or before farrowing. Exposure and infection during gestation may result in infected but apparently healthy piglets.

Antecedentes: el circovirus porcino tipo 2 (PCV2) es asociado con casos de falla reproductivas en granjas recién pobladas, en granjas de cría para cerdas jóvenes y casi exclusivamente en cerdas de reemplazo. Los signos clínicos descritos son: incremento en los abortos durante la segunda y tercera etapa de la gestación, fetos momificados, mortinatos y el nacimiento de lechones débiles no viables. Objetivo: el principal propósito de este estudio fue evaluar la dinámica de la infección por el circovirus porcino tipo 2 y títulos de anticuerpos neutralizantes en las cerdas de reemplazo subclinicamente infectadas y el efecto en su camada. Métodos: este estudio se realizó con 40 cerdas de reemplazo seleccionadas al azar en cuatro granjas porcinas de cría. De cada animal se colectaron muestras de sangre, hisopados nasales y vaginales al ingresar a la explotación, durante la cuarentena, en el momento del parto y un día post-parto. Igualmente, se colectaron muestras de calostro al terminar el parto y un día post-parto. De cuatro lechones neonatos, se colectaron muestras de sangre, hisopado nasal y tejidos antes de consumir calostro. Todos los sueros fueron analizados mediante la técnica de sero-neutralización para detectar anticuerpos anti-PCV2 y todas las muestras se analizaron por una técnica SYBER Green en tiempo real para detectar el ADN viral. Resultados: la detección de un alto nivel de viremia y la demostración de la eliminación viral en hisopados nasales y vaginales permitió demostrar la introducción a las granjas de cerdas de reemplazo infectadas, aparentemente sanas. Igualmente, el suero y los hisopados nasales y vaginales fueron positivos por PCR SYBER Green en la mayoría de las hembras al parto. Se demostró eliminación viral en fluidos nasales, vaginales y en calostro en presencia de anticuerpos séricos neutralizantes. La infección de las cerdas se manifestó en viremia, en el desarrollo de anticuerpos frente al PCV2 y en la presencia del ADN viral en los tejidos de lechones neonatos aparentemente sanos. Los genotipos PCV2a y PCV2b fueron detectados en la infección in utero. Conclusiones: las cerdas de reemplazo pueden estar infectadas con el PCV2 antes de ingresar a las explotaciones de cría o pueden infectarse por transmisión horizontal durante la cuarentena y gestación. La exposición e infección viral de las cerdas durante la gestación puede resultar en infección subclínica de los lechones neonatos.

Antecedentes: o circovírus suíno tipo 2 (PCV2) está associado a casos de falha reprodutiva em granjas recém-assentadas e fazendas de criação de marrãs e afeta principalmente a porcas nulíparas. Clinicamente observam-se aumento dos fetos abortados no segundo e terceiro estágios da gravidez, fetos mumificados, natimortos e nascimento de leitões inviáveis. Objetivo: o principal objetivo deste estudo foi avaliar a dinâmica da infeção pelo circovirus porcino tipo 2 e os títulos de anticorpos neutralizantes em porcas nulíparas com infecção subclínica e o efeito em sua leitegada. Métodos: este estudo foi realizado com 40 porcas em quatro granjas selecionadas aleatoriamente. Foram coletados de cada animal amostras de sangue, esfregaços nasais e vaginais ao entrar na fazenda, durante a quarentena, no parto e um dia pós-parto. Também foram coletadas amostras de colostro no parto e um dia pós-parto. Amostras de sangue, esfregaços nasal e tecido foram tomadas de quatro leitões antes de consumir colostro. As amostras foram analisadas pelo PCR Sybr Green para detectar e quantificar o PCV2. A detecção de anticorpos contra o vírus do PCV2 em soro foi realizada pelo teste de soroneutralização e todas as amostras foram analisadas através da técnica de SYBR Green PCR para a detecção do ADN viral. Resultados: a detecção de um nível elevado de viremia e a demonstração da excreção viral em esfregaços nasais e vaginais nas fêmeas permitiram demonstrar a introdução de porcas nulíparas aparentemente saudávels. Igualmente, o soro e as secreções vaginais e nasais foram positivos por PCR SYBR Green em tempo real na maioria das porcas no parto. Observou-se excreção viral em secreções vaginais e nasais e colostro na presença de anticorpos neutralizantes. A infecção das porcas manifestou-se no desenvolvimento de anticorpos neutralizantes e detecção de infecção fetal em leitões recém-nascidos aparentemente saudáveis , confirmando a transmissão vertical do PCV2. Os genótipos PCV2a e PCV2b foram detectados na infecção in utero. Conclusões: as porcas nulíparas podem estar infectadas com PCV2 antes de entrar nas granjas de criação e pode ser infectadas por transmissão horizontal durante a quarentena e gravidez. Exposição e infecção viral durante a gestação pode resultar em infecção subclínica de leitões recém-nascidos.

Serum miR-203 expression level detected by fluorescence quantitative PCR in cervical cancer patients and its significance / 国际检验医学杂志

Objective To establish the method of the SYBR Green Ⅰ real‐time fluorescent quantitative PCR for detecting the se‐rum miR‐203 expression level ,and to detect the serum miR‐203 expression levels in the patients with cervical cancer ,cervical benign diseases and healthy controls .Methods The miR‐203 ,U6 stem loop RT primers and the PCR amplification primers were designed for conducting fluorescence quantitative PCR ,with U6 as the internal relative quantification ,the serum miR‐203 levels were com‐pared among different cervical diseases .Results The established method could specifically detect the amplification signal of serum miR‐203 ,the melting curve was single and PCR products were specific .The serum miR‐203 level in the patients with cervical cancer was significantly higher than that in the patients with benign cervical diseases such as hysteromyoma and cervicitis ,the difference was statistically significant (P<0 .05) .Conclusion The SYBR Green Ⅰ real‐time fluorescent quantitative PCR is a quick ,simple detection method with high sensitivity and good specificity ,which may have a better application prospect in cervical cancer auxiliary diagnosis .

Rapid molecular identification of saffron (Crocus sativus L.) based on specific PCR / 中国药学杂志

OBJECTIVE: To establish a rapid molecular method for identifying saffron (Crocus sativus L.) and its adulterants by PCR: amplification using specific primers and fluorescent dyes detection. METHODS: The chloroplast barcode was sequenced and analyzed to find the SNPs between saffron and its adulterants. Specific primers were designed for the SNPs, the PCR reaction systems were built and optimized, and fluorescent dyes method was used to identify PCR products. RESULTS: A 421 bp saffron identification band based on the psbA-trnH barcode sequence was screened when 100 × SYBR Green I was added into the optimized PCR product under the following condition; initial denaturation at 90℃ for 1 min, denaturation at 90℃ for 5 s, annealing at 58℃ for 5 s, 26 cycles; the saffron (Crocus sativus L.) showed strong green fluorescence under 365 nm UV lamp whereas adulterants did not. CONCLUSION: Fast site-specific PCR can rapidly identify saffron and its adulteration.

Development of both multiple PCR and real-time SYBR green PCR for the detection of Vibrio cholerae non-O1/O139 serogroups / 中华流行病学杂志

Objective To develop methodology of both multiple PCR and real-time SYBR green PCR for the detection of Vibrio cholerae (V.cholerae) serogroups non-O1 and non-O139.Methods The outer membrane protein gene (ompW) specific for V.cholerae,as well as O antigen rfb genes specific for both O1 and O139,were used for the design of the PCR primers.Both multiple PCR and real-time SYBR green PCR systems were used to detect both O1 and O139.Specific rfb genes and ompW were developed to evaluate their specificity,limit of detection,reproducibility and consistency.Results We established multiple PCR and real-time SYBR green PCR methods.According to the specific electrophoretic bands (multiple PCR) and the specific melt curve temperature (real-time SYBR green PCR),both methods could specifically detect the non-O1,non-O139 V.cholerae,and to differentiate them from O1,O139 V.cholerae,other five Vibrios and 3 intestinal bacteria.The detection limits were 7 × 104 cfu/ml (multiple PCR) and 7 × 102 cfu/ml (real-time SYBR green PCR),with statistically significant difference seen (P＜0.05).For the reproducibility of real-time SYBR green PCR,the external coefficient variation ranging from 0.22％ to 0.92％ while the internal coefficient variation ranging from 0.27％ to 1.41％.370 strains of non-O1,non-O139 V.cholerae,were detected,with both consistency rates as 100％.Conclusion Both multiple PCR and real-time SYBR green PCR could detect non-O1,non-O139 V.cholerae,rapidly,specifically,and reproducibly,that could all be used for the detection and identification of non-O 1,non-O 139 under different conditions.

Establishment of Analysis Method for Detection of Petroleum Degrading Genes AlkB and Nah in Contaminated Soil and Its Application / 分析化学

SYBR Green I Real Time-qPCR method was developed to quantify the numbers of copyies of AlkB ( alkanes degradation gene) and Nah ( naphthalene dioxygenase degradation gene) functional degradation gene corresponding to alkanes and aromatic hydrocarbons degradation. Two pairs of primers AlkBf/AlkBr and Nahf/Nahr were designed for AlkB and Nah amplification respectively, according to the nucleotide sequences of related degradation microorganisms published in GenBank. The purified recovery products of traditional PCR were combined with pEASY-T1 vectors and transformed in competent cells to amplify. The recombinant plasmids were extracted and used as positive templates to create standard curve through gradient dilution. The conditions for the real time PCR were as the follows: the final concentration of forward and reverse primers were 0. 2 μmol/L, 2×TransStart Top Green qPCR SuperMix, and the annealing temperatures of AlkB and Nah PCR were 50℃ and 57℃, respectively. The method showed a sensitivity of 100 times higher than that of the traditional PCR method and good repeatability. The numbers of copies of AlkB in three functional regions of an oilfield indicated that oil producing zone with serious oil pollution had the highest AlkB copy numbers, and residential zone with lighter oil pollution had the lowest AlkB copy numbers. Nah degradation gene distribution was more uniform.

Simple and Rapid Detection of Melamine in Milk Based on T-Melamine-T Duplex Structure and SYBR Green Ⅰ / 分析化学

A novel fluorescence homogeneous biosensing strategy was developed for simple, rapid and sensitive detection of melamine in milk by using the polythymine oligonucleotide T24 and SYBR Green Ⅰ. In the absence of melamine, the fluorescence of SYBR Green Ⅰ was weak. The interactions between the single strand oligonucleotide T24 and SYBR Green Ⅰ were weak. In contrast, the presence of melamine drove the formation of T-melamine-T folded structure and enabled the SYBR Green Ⅰ to intercalate into double-strand DNA, resulting in the enhancement of fluorescence intensity. The results revealed that the method allowed a sensitive, simple, and rapid assay of melamine with a linear response range from 0. 1 μmol/L to 10 μmol/L and a detection limit of 35 nmol/L.

Establishment and application of SYBR real time fluorescence quantitative PCR for detecting Annexin Ⅱ mRNA of human breast cancer cells / 重庆医学

Objective To establish a SYBR Green based real-time fluorescence quantitative PCR method for detecting human Annexin Ⅱ mRNA expression,and to detect the level of Annexin Ⅱ mRNA in human breast cancer cells MCF-7 and MDA-MB-231.Methods The specific primers were designed according to the conserved sequence of human Annexin Ⅱ gene.Total RNAs were extracted from human breast cancer cells(MCF-7,MDA-MB-231),then RNAs were transcribed reversely into cDNAs.The plasmid standards were constructed.The relative expression levels of human Annexin Ⅱ mRNA in human breast cancer cells were detected by this method.Results The square(r2 )of correlation coefficient of the standard curve in this method was 0.997,the melting curve analysis showed the single peak.The the intra-batch and inter-batch variable coefficients in the pGM-T Annexin Ⅱplasmid standard substance were 6.2%,7.8% and 9.1%,12.3% respectively.The further study indicated that AnnexinⅡ mRNA in MDA-MB-231 was higher than that in MCF-7(P<0.01).Conclusion The established SYBR Green real time fluorescence quan-titative PCR method for detecting human AnnexinⅡ is of good specificity and repeatability and can be used for quantitatively detec-ting AnnexinⅡ mRNA in breast cancer cells.

Development of real-time fluorescence quantitative RT-PCR assay for β-actin gene of Panax ginseng / 中草药

Objective: To construct a real-time fluorescence quantitative RT-PCR method for the β-actin gene of Panax ginseng. Methods: According to the β-actin gene of other higher plants available in Genbank, A pair of primers weredesigned and the amplified fragment of β-actin gene was linked with pMD20-T vector to construct recombinant plasmids. Then the positive plasmids were diluted and the standard curve was established. The sensitivity, specificity, and repeatability were detected. Results: The results showed that the lowest copy number for detection of β-actin gene with this method was 43.0 copies/μL, and there was a good linear relationship in a wide range from 43 to 4.3 × 107 copies/μL (R2 = 0.995 3). The melting curve showed a single peak with the temperature of (84.51 ± 0.01) ℃. The coefficient of variation (CV) of five different concentration of positive plasmids was 0.58% to 2.79% and 2.61% to 4.41% in intra-assay and inter-assay, respectively. Conclusion: The method established in this paper has the advantage of rapidity, sensitivity, specificity, high throughput, and good repeatability, which provides a methodological basis for the quantitative analysis on the functional genes of P. ginseng when β-actin gene is taken as a reference gene.

A robust HIV-1 viral load detection assay optimized for Indian sub type C specific strains and resource limiting setting

BACKGROUND: Human Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is therefore an assay that is not only economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India. RESULTS: Linear regression of the observed and reference concentration of standards used in this study generated a correlation coefficient of 0.998 (p<0.001). Lower limit of detection of the test protocol was 50 copies/ml of plasma. The assay demonstrated 100% specificity when tested with negative control sera. The Spearman coefficient of the reported assay with an US-FDA approved, Taqman probe-based commercial kit was found to be 0.997. No significant difference in viral load was detected when the SYBR Green based assay was used to test infected plasma stored at -20°C and room temperature for 7 days respectively (Wilcoxon signed rank test, p=0.105). In a comparative study on 90 pretested HIV-1 positive samples with viral loads ranging from 5,000 - 25,000 HIV-1 RNA copies/ml and between two commercial assays it was found that the later failed to amplify in 13.33% and 10% samples respectively while in 7.77% and 4.44% samples the copy number values were reduced by >0.5 log value, a figure that is considered clinically significant by physicians. CONCLUSION: The HIV-1 viral load assay reported in this study was found to be robust, reliable, economical and effective in resource limited settings such as those existing in India. PCR probes specially designed from HIV-1 Subtype C-specific nucleotide sequences originating from India imparted specificity towards such isolates and demonstrated superior results when compared to two similar commercial assays widely used in India.

Application of SYBR Green real-time PCR assay for the specific detection of Salmonella spp / 대한수의학회지

The aim of this study was to applicate and evaluate a SYBR Green real-time PCR for the specific detection of Salmonella spp. Specificity of the PCR method was confirmed with 48 Salmonella spp. and 5 non-Salmonella strains using invA gene primer. The average threshold cycle (C(T)) of Salmonella spp. was 11.83 +/- 0.78 while non-Salmonella spp. was 30.86 +/- 1.19. Correlation coefficients of standard curves constructed using C(T) versus copy number of Salmonella Enteritidis ATCC 13076 showed good linearity (R2 = 0.993; slope = 3.563). Minimum level of detection with the method was > 10(2) colony forming units (CFU)/mL. These results suggested that the SYBR Green real-time PCR might be applicable for the specific detection of Salmonella spp. isolates.

Effects of Lipo-PGE1 on the expression of T-cell specific transcription factors T-bet/Gata-3 in acutelung injury / 中华急诊医学杂志

Objective To investigate the effects of Lipo-PGE1 on the expression of T-bet and Gata-3,and its potential mechanisms causing the shift of T cells from Th1 to Th2 on Acute lung injury(ALI)induced by Lipopolysaccharide(LPS)in mice.Methods Sixty male BALB-C mice were randomly divided into three groups(n =20 in each group):(1)control group,mice were treated with intravenous injection of NS in dose of 10 ml/kg,(2)LPS group,mice were exposed to LPS with dosage of 5 mg/kg(0.5 g/ml diluted in saline),and(3)LPS + PGE1 group,mice were treated with Lipo-PGE1 in dose of 15μg/kg.Sixhours after injection,the lungs were removed for observing the histopathological changes and determination of wet/dry lung weight(W/D)ratio.The levels of Th1 and Th2 were determined by flow cytometry,and the expressions of T-bet and Gata-3 mRNA were detected by using RT-PCR.One-way ANOVA was used for comparing differences between groups,and all data were presented in((x)± s).Results The histological changes of lung injury were lessened by PGEC ompared with the W/D ratio(5.74 ± 0.31)in LPS group,the one(4.92 ±0.27)in LPS +PGE1 group was lower significantly(P ＜0.01).The levels of Th1 and Th2 and their ratio Were higher in LPS +PGE1 group[(20.31 ±2.20)％,(10.50±0.80)％,(1.93±0.05)]than in LPS group[(16.65 ±1.70)％,(9.40 ±1.25)％,(1.73 ±0.03)](P＜0.01).Compared with control group,the expressions ofT-bet mRNA(1.183 ±0.495),and Gata-3 mRNA(0.693±0.285),and their ratio(1.713 ± 0.131)were lower(P ＜0.01); compared with LPS group,PGE1 significantly increased the expressions of T-bet mRNA(1.827 ± 0.705)and the ratio of T-bet/Gata-3 (2.502 ±0.352)(P ＜0.01),while didn(t)increased the expressions of Gata-3 mRNA(0.7191 ±0.186)significantly(P ＞ 0.05).Conclusions Lipo-PGE1 may up-regulate transcription factor T-bet which participates in the Th1 differentiation ratio,and then improve the inflammatorv svmntom.

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 / 中国病毒学·英文版

A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit(R) for detection and semi-quantitation of peste 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 conventional and real-time multiplex PCR assays for the detection of nosocomial pathogens

Nosocomial infections are major clinical threats to hospitalised patients and represent an important source of morbidity and mortality. It is necessary to develop rapid detection assays of nosocomial pathogens for better prognosis and initiation of antimicrobial therapy in patients. In this study, we present the development of molecular methods for the detection of six common nosocomial pathogens including Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. Conventional multiplex PCR and SYBR Green based real time PCR assays were performed using genus and species specific primers. Blind testing with 300 clinical samples was also carried out. The two assays were found to be sensitive and specific. Eubacterial PCR assay exhibited positive results for 46 clinical isolates from which 43 samples were detected by real time PCR assay. The sensitivity of the assay is about 93.7 percent in blind test isolates. The PCR results were reconfirmed using the conventional culture method. This assay has the potential to be a rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous detection of Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. This assay has the potential to detect nosocomial pathogens within 5 to 6 hours, helping to initiate infection control measures and appropriate treatment in paediatric and elderly (old aged) patients, pre-and post surgery patients and organ transplant patients and thus reduces their hospitalization duration .