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1.
Article in English | WPRIM | ID: wpr-690669

ABSTRACT

<p><b>OBJECTIVE</b>To detect Japanese encephalitis virus (JEV) rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction (RT-PCR) detection system was developed.</p><p><b>METHODS</b>By aligning the full-length sequences of JEV (G1-G5), six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay.</p><p><b>RESULTS</b>With the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/µL. The coefficients of variation of this real-time RT-PCR were all < 2.8%. The amplification efficiency of this method was between 90% and 103%.</p><p><b>CONCLUSION</b>A TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.</p>


Subject(s)
Animals , Culicidae , Virology , Encephalitis Virus, Japanese , Genetics , Polymerase Chain Reaction , Methods , Reproducibility of Results , Sensitivity and Specificity
2.
Article in Chinese | WPRIM | ID: wpr-607776

ABSTRACT

Small cell lung cancer is the most common primary neuroendocrine malignancy of the lung and is characterized by high malignant degree,rapid doubling time,easy metastasis in early stage and poor prognosis.Accurate staging of small cell lung cancer can formulate personalized therapeutic plans and improve the prognosis of patients.PET/CT can obtain metabolism and anatomical images of the whole body in one scan and improve the diagnostic accuracy and integrity.PET/CT has been widely applied to clinical practice now.PET/CT will play a more and more important role in diagnosis,staging,treatment and prognosis assessment of patients with small cell lung cancer.The value of PET/CT in staging and treatment of small cell lung cancer was reviewed in this article.

3.
Journal of Practical Radiology ; (12): 513-516,532, 2017.
Article in Chinese | WPRIM | ID: wpr-606778

ABSTRACT

Objective To investigate CT signs of peripheral small cell lung cancer (SCLC).Methods The CT signs of 78 patients with SCLC confirmed by pathology were retrospectively reviewed.According to the presence of mediastinal lymph node metastasis and its size, 78 cases of peripheral SCLC were divided into two types: typeⅠ(isolated lesion) and typeⅡ(lung lesion + lymph nodes).Type Ⅱwere divided into two subtypes:type Ⅱa (short diameter of lymph nodes of pulmonary hilar and mediastinum less than 10 mm) and type Ⅱ b (short diameter of lymph nodes of pulmonary hilar and mediastinum greater than or equal to 10 mm).Results Of the 78 SCLCs, typeⅠwas 7 cases, and typeⅡwas 71 cases,including 8 cases of typeⅡa and 63 cases of typeⅡb.All of the lesions were soild density.The shape were round or oval in 52 cases, vermicular or spindlein 9 cases, and other shapes in 17 cases.Among 71 cases performed CT enhancement, there were 9 cases with homogeneous enhancement, 58 cases with heterogeneous enhancement, 4 cases with non-enhancement large necrosis area.These cases showed the following CT signs: smooth edge in 65 cases, coarse edge in 12 cases, blurred edge in 1 case;air bronchogram in 3 cases, vacuole sign in 4 cases, calcification in 4 cases;lobulation sign in 46 cases, spiculated sign in 5 cases;thickening of the bronchovascular bundle in 41 cases, pleural indentation in 6 cases, marginal ground-glass opacity in 5 cases, vascular convergence sign in 1 case;emphysema in 42 cases;obstructive pneumonia in 4 cases;bronchus abruptly interruption on the edge of the nodules in 18 cases;enlargement of mediastinal lymph nodes in 63 cases, the diameter of mediastinal lymph nodes larger than the primary lesions in 42 cases;and a little pleural effusion in 9 cases.Conclusion Solid density, smooth margin with lobulation,and significantly enlarged mediastinal lymph nodes are common signs in peripheral SCLC.Thickening of the bronchovascular bundle indicates reletively advanced stage.

4.
Article in Chinese | WPRIM | ID: wpr-664045

ABSTRACT

Objective To investigate the early diagnosis and surgery timing of strangulated intestinal obstruction.Methods The clinical data of 90 patients with strangulated intestinal obstruction who were admitted into our hospital from January 2013 and January 2016 were retrospectively analyzed.And these patients were divided into the early stage group (28 cases) and the later stage group (62 cases).The rate of mortality and the rate of necrotic bowel resection of the two groups were analyzed.Results Among the 90 patients underwent emergency surgery,there were 88 cases cured and 2 cases died,and there were 10 cases (11.11%) of necrotic bowel resection among the survivor.In the early stage group,there were 8 cases of necrotic bowel resection and 1 case of death.In the later stage group,there were 2 cases of necrotic bowel resection and 1 case of death.Conclusion Early diagnosis and prompt surgical treatment can reduce the mortality of strangulation obstruction and necrosis of bowel resection.

5.
Journal of Practical Radiology ; (12): 1671-1674, 2017.
Article in Chinese | WPRIM | ID: wpr-696708

ABSTRACT

Objective To investigate CT findings of abnormal bronchovascular bundle in patients with peripheral small cell lung cancer (SCLC).Methods The CT findings of abnormal bronchovascular bundle in 78 peripheral SCLC patients confirmed by pathology were retrospectively reviewed.Abnormal bronchovascular bundle of peripheral SCLC was divided into three types:type Ⅰ (thickening of the bronchovascular bundle),type Ⅱ (string beads of bronchovascular bundle) and type Ⅲ (bronchial cast with bronchus cut-off).Results 41 of 78 patients had abnormal bronchovascular bundle,in which 26 cases were in type Ⅰ,10 in type Ⅱ,5 in type Ⅲ.Except for 1 case with no mediastinal lymph node metastasis among 41 cases with abnormal bronchovascular bundle,all other 40 cases had mediastinal lymph node metastasis.Conclusion The abnormal bronchovascular bundle could reflect the biologic character of SCLC.Abnormal bronchovascular bundle is associated with advanced patients.

6.
Article in English | WPRIM | ID: wpr-296519

ABSTRACT

<p><b>OBJECTIVE</b>To provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples.</p><p><b>METHODS</b>Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline.</p><p><b>RESULTS</b>NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease (HFMD) and identified other potential viruses or differentiated misdiagnosis events.</p><p><b>CONCLUSION</b>The improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.</p>


Subject(s)
Clinical Laboratory Techniques , DNA Barcoding, Taxonomic , DNA Primers , Enterovirus , Classification , Genetics , Herpesvirus 4, Human , Genetics , Humans , Influenza B virus , Genetics , Real-Time Polymerase Chain Reaction , Sequence Analysis, DNA , Methods , Sequence Analysis, RNA , Methods
7.
Article in Chinese | WPRIM | ID: wpr-510313

ABSTRACT

Objective To investigate the formation mechanism of string beads sign in peripheral small cell lung cancer (SCLC) and evaluate the significance of it in differential diagnosis among SCLC,peripheral lung adenocarcinoma and peripheral lung squa-mous cell carcinoma.Methods 78 cases of SCLC,69 cases of peripheral lung adenocarcinoma and 33 cases of peripheral lung squa-mous cell carcinoma,confirmed pathologically were included in this study.The positive rates of string beads sign,mediastinal lymph node metastasis and mediastinal lymph nodes larger than primary lung lesions were calculated and analyzed in these three groups.Results 10 out of SCLC cases (12.8%)have string beads sign,in which all mediastinal lymph nodes were larger than lung lesions.Mediasti-nal lymph node metastases were observed in 63(80.8%)of 78 cases,and 42 (53.8%)cases had larger mediastinal lymph nodes than lung lesions.No string beads sign was observed in patients with peripheral solid lung adenocarcinomas,but 25 of 69 cases (36.2%) have mediastinal lymph node metastasis and 2 cases (2.9%)had larger mediastinal lymph nodes than lung lesions.13 cases(39.4%) of 33 patients with peripheral lung squamous cell carcinomas had mediastinal lymph node metastasis,and 6 cases (16.7%)had larger mediastinal lymph nodes than lung lesions.The statistical results showed the positive rate of string beads sign was not significantly different between peripheral SCLC group and peripheral lung squamous cell carcinoma group,but that of mediastinal lymph node and larger mediastinal lymph nodes than lung lesions were statistically different among these three groups.Conclusion To some extent, string beads sign on CT could reflect the biologic character of SCLC.It played an important role in differential diagnosis of peripheral SCLC,peripheral lung adenocarcinoma and periph-eral lung squamous cell carcinoma,but it should be combined with mediastinal lymph node size.

8.
China Journal of Endoscopy ; (12): 46-50, 2016.
Article in Chinese | WPRIM | ID: wpr-621205

ABSTRACT

Objective To compare the differences of surgical site infection (SSI) between laparoscopic distal gas-trectomy (LDG) and open distal gastrectomy (ODG) for gastric cancer. Methods We set up strict inclusion and ex-clusion criteria. All the randomized controlled trials (RCT) on LDG and ODG for gastric cancer were collected. Meta-analysis was performed according to the recommendation by the Cochrane handbook. Results Six RCTs in-cluding 767 patients were analyzed, who were divided into LDG group (n =394) and ODG group ( n=373). Postop-erative wound infection and SSI were significantly lower in LDG than in ODG [RR=0.32, 95 %Cl (0.11, 0.91).P =0.03; RR= 0.28, 95 %Cl (0.12, 0.70),P =0.006]. There was no significant difference in intra-abdominal abscess between the two groups [RR=0.35, 95 % Cl (0.09, 1.31), P=0.12]. Conclusions LDG was associated with a lower incidence of SSI, especially wound infection, as compared with ODG in the meta-analysis.

9.
Article in Chinese | WPRIM | ID: wpr-296225

ABSTRACT

Given the Ebola outbreak in West Africa and the risks of spread to other regions, a rapid, sensitive and simple method for the detection of the Ebola virus (EBOV) is of great significance for the prevention and control of Ebola. We developed a simple colorimetric isothermal multiple self-matching initiated amplification (IMSA) for rapid detection of the Zaire subtype of the Ebola virus (EBOV-Z). This method employed six primers that recognized seven sites of the EBOV-Z nucleoprotein gene for amplification of nucleic acids under isothermal conditions at 63 degrees C for 1 h. Amplification products were detected through visual inspection of color change by pre-addition of hydroxyl naphthol blue dye. Relative sensitivity was validated by detection of serial tenfold dilutions of virus-like particles containing the partial EBOV-Z nucleoprotein gene and mock clinical sample. Specificity of IMSA was validated by detection of the plasma of 30 healthy volunteers, the dengue virus, and Japanese encephalitis virus. IMSA had comparable sensitivity to Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and cross-reaction with human plasma or other viruses was not observed. Reverse transcription-isothermal multiple self-matching initiated amplification (RT-IMSA) was also evaluated and compared in parallel with the commercial RT-qPCR kit for detection of EBOV-suspected samples of human blood in Sierra Leone. Sensitivity and specificity of the RT-IMSA was 91.4% and 100%, respectively. These data suggest that RT-IMSA is a valuable tool for the detection of the EBOV with the distinct advantages of simplicity and low cost compared with RT-qPCR.


Subject(s)
Colorimetry , Methods , DNA Primers , Genetics , Ebolavirus , Genetics , Hemorrhagic Fever, Ebola , Diagnosis , Virology , Humans , Nucleic Acid Amplification Techniques , Methods
10.
Chinese Journal of Virology ; (6): 210-214, 2016.
Article in Chinese | WPRIM | ID: wpr-296195

ABSTRACT

The quality control process throughout the Ebola virus nucleic acid detection in Sierra Leone-China Friendship Biological Safety Laboratory (SLE-CHN Biosafety Lab) was described in detail, in order to comprehensively display the scientific, rigorous, accurate and efficient practice in detection of Ebola virus of first batch detection team in SLE-CHN Biosafety Lab. Firstly, the key points of laboratory quality control system was described, including the managements and organizing, quality control documents and information management, instrument, reagents and supplies, assessment, facilities design and space allocation, laboratory maintenance and biosecurity. Secondly, the application of quality control methods in the whole process of the Ebola virus detection, including before the test, during the test and after the test, was analyzed. The excellent and professional laboratory staffs, the implementation of humanized management are the cornerstone of the success; High-level biological safety protection is the premise for effective quality control and completion of Ebola virus detection tasks. And professional logistics is prerequisite for launching the laboratory diagnosis of Ebola virus. The establishment and running of SLE-CHN Biosafety Lab has landmark significance for the friendship between Sierra Leone and China, and the lab becomes the most important base for Ebola virus laboratory testing in Sierra Leone.


Subject(s)
China , Ebolavirus , Classification , Genetics , Hemorrhagic Fever, Ebola , Diagnosis , Virology , Humans , Laboratories , Workforce , Reference Standards , Laboratory Infection , Quality Control , RNA, Viral , Genetics , Sierra Leone
11.
Chinese Journal of Zoonoses ; (12): 212-215, 2015.
Article in Chinese | WPRIM | ID: wpr-460505

ABSTRACT

The Flanders virus (FLAV) is a number of family Rhabdoviridae ,contains a single‐stranded ,negative‐sense vi‐ral RNA .Here we describe a molecular detection method developed for fast measurement of FLAV based on Taqman RT‐PCR method .In this study ,FLAV specific primers and probe were designed based on the FLAV L gene sequences published in GeneBank .Quantitative standard curve of FLAV TaqMan PCR was also successfully established .The specificity and stability test showed that the system is specific and the coefficient variables were all less than 1 .7% .Quantitative standard curve based on the genomic copy was drawn ,and the lowest detectable limit (LOD) of system was 100 copies/PCR ,with higher sensitivity and stability than that of the conventional RT‐PCR assay targeting the same gene .

12.
Article in Chinese | WPRIM | ID: wpr-296645

ABSTRACT

<p><b>OBJECTIVE</b>To develop a simple, rapid and sensitive colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of coxsackievirus A6 (CV-A6) based on the colour chang of hydroxy naphthol blue (HNB).</p><p><b>METHODS</b>The method employed a set of six primers that recognized sequences of VP1 gene for amplification of nucleic acid under isothermal conditions at 63 °C for 50 min. The products were detected through visual inspection of color change by the pre-addition of HNB dye. The specificity was validated by detecting a collection of different human enteroviruses. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of CV-A6 VP1 gene, and compared with real-time RT-PCR (rRT-PCR) in parallel. This assay was evaluated with 92 clinical specimens from patients with hand-foot-mouth disease.</p><p><b>RESULTS</b>A positive color (sky blue) was only observed in the preparation of CV-A6, whereas none of the other 23 kinds of human enteroviruses showed a color change. The HNB based RT-LAMP showed a sensitivity of 100 copies/reaction, which was at the same level as that of the rRT-PCR. The result of RT-LAMP in analysis of 92 clinical specimens was consistent with that of the rRT-PCR. The kappa correlation between the two methods was 1 and both of the sensitivity and specificity of the RT-LAMP assay were 100%.</p><p><b>CONCLUSION</b>The established RT-LAMP assay had good specificity and sensitivity and thus demonstrated to be a promising screening tool for CV-A6 infection. It also has the potential to be used in resource-limited clinical sites and field study.</p>


Subject(s)
Colorimetry , Coloring Agents , Chemistry , DNA Primers , Enterovirus , Hand, Foot and Mouth Disease , Virology , Humans , Indicators and Reagents , Chemistry , Naphthalenesulfonates , Chemistry , Nucleic Acid Amplification Techniques , Real-Time Polymerase Chain Reaction , Reverse Transcription , Sensitivity and Specificity
13.
Chinese Journal of Virology ; (6): 269-275, 2015.
Article in Chinese | WPRIM | ID: wpr-296289

ABSTRACT

A simple, rapid and sensitive colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) was developed for rapid detection of Middle East respiratory syndrome coronavirus (MERS-CoV). The method employed six primers that recognized sequences of a nucleocapsid gene for amplification of nucleic acids under isothermal conditions at 63 degrees C for 60 min. Products were detected through a LA-320c Loopamp Turbidimeter (real-time RT-LAMP) or visual inspection of color change by pre-addition of Hydroxynaphthol Blue dye (visual RT-LAMP). Specificity of RT-LAMP was validated by detection of several human coronaviruses and common respiratory viruses. MERS-CoV real-time RT-LAMP had a linear correlation (R2) of 0.995 at 10(3)-10(6) copies. The limit of detection of real-time RT-LAMP, visual RT-LAMP and quantitative real-time PCR was 500, 1000 and 100 copies/reaction, respectively. The established RT-LAMP assay was demonstrated to be a rapid screening tool for MERS-CoV infection, and could be suitable in resource-limited clinical sites and for field studies.


Subject(s)
Coronavirus Infections , Virology , DNA Primers , Genetics , Humans , Middle East Respiratory Syndrome Coronavirus , Classification , Genetics , Nucleic Acid Amplification Techniques , Methods , Reverse Transcription
14.
Chinese Journal of Virology ; (6): 137-142, 2013.
Article in Chinese | WPRIM | ID: wpr-339962

ABSTRACT

Resequencing Pathogen Microarray (RPM) is a new pathogen detection and identification technology based on DNA microarray. In order to apply RPM in the detection of unexplained infection and as a result, to improve the emergency response capacity, a new RPM-based respiratory pathogens detection assay was developed to simultaneously detect 19 common respiratory viruses, 9 influenza A viruses (Flu A),11 human rhinoviruses(HRV), 28 enteroviruses and 18 rare respiratory viruses. The specificity of multiplex system was examined by confirmed positive specimens for 16 common respiratory virus. The sensi-tivity was evaluated by serial ten-fold dilutions of plasmids or in vitro-transcribed RNA. RPM could detect and differentiate 16 virus types/subtypes at 10 - 1 000 copies/reaction level. Nucleic acids of 8 throat swabs with unexplained respiratory tract infections were pooled and detected by the new assay. The RPM result was verified by common PCR followed by sequencing as well as PLEX-ID (Abbott). Except for a false-positive of PIV1, no difference among the three assays was found. These results indicate the assay based on the new RPM is a highly sensitive, high throughput test for the detection of respiratory virus infections, which is significant for the management of emergent and epidemic infectious disease.


Subject(s)
Child , Child, Preschool , Female , Humans , Male , Oligonucleotide Array Sequence Analysis , Methods , Respiratory Tract Infections , Diagnosis , Virology , Sensitivity and Specificity , Viruses , Classification , Genetics
15.
Article in Chinese | WPRIM | ID: wpr-431726

ABSTRACT

Objective To investigate the risk factors for postoperative liver failure of patients with hepatocellular carcinoma (HCC) and bile duct tumor thrombus through a risk evaluation model.Methods The clinical data of 107 patients with HCC and bile duct tumor thrombus who received hepatic resection at the Eastern Hepatobiliary Surgery Hospital from March 2002 to February 2011 were retrospectively analyzed.All patients were divided into the non-liver failure group (98 patients) and liver failure group (9 patients).Risk factors associated with liver failure were analyzed and a risk evaluation model was established.All data were analyzed using the bivariate regression model,and factors with significance were further analyzed using the multivariate regression model.Results Of the 107 patients,105 received hepatic resection + choledochotomy + thrombectomy and 2 received hepatic resection + extrahepatic bile duct resection + cholangiojejunostomy.The operation time was 2.0-5.5 hours,and the intraoperative blood loss was 200-3500 ml.In the non-liver failure group,5 patients had pleural and peritoneal effusion,3 had biliary bleeding,2 had incisional infection,1 had biliary infection,1 had bile leakage,1 had stress-induced ulcer of upper digestive tract and 1 had thoracic epidural hematoma.The bleeding of the patients with thoracic epidural hematoma was stopped after thoracic spinal decompression,but subsequent paraplegia occurred.In the liver failure group,2 patients died of postoperative acute liver failure,and 7 patients died of postoperative subacute liver failure (death caused by tumor recurrence or medicine was excluded).The results of univariate analysis showed that preoperative total bilirubin,albumin,pre-albumin,albumin/globulin ratio,distribution of tumor thrombus,operative blood loss and ratio of postoperative residual liver volume to the total liver volume were correlated with the postoperative liver failure in patients with HCC and bile duct tumor thrombus (OR =3.017,0.191,0.248,2.681,9.048,4.759,13.714,P < 0.05).The results of multivariate analysis showed that preoperative total bilirubin > 256.5 μmol/L,albumin/globulin ratio ≤ 1.3 and postoperative residual liver volume < 50% were the independent risk factors of postoperative liver failure (OR =5.537,11.107,172.450,P < 0.05).The risk evaluation model was Z =1.77 × preoperative total bilirubin + 2.408 × preoperative albumin/globulin ratio + 5.150 × ratio of postoperative residual liver volume to the total liver volume-17.288.The risk of postoperative liver failure increased as the increase of Z value.The risk of postoperative liver failure > 50% when the Z value > 0.Conclusions Preoperative total bilirubin > 256.5μmol/L,albumin/globulin ratio ≤ 1.3 and postoperative residual liver volume < 50% were the independent risk factors of postoperative liver failure.Risk evaluation model is helpful in screening the risk factors so as to decrease the incidence of postoperative liver failure.

16.
Article in Chinese | WPRIM | ID: wpr-431706

ABSTRACT

Pathological types of gastric cancer in stage Ⅲ C include T4a-SEN3,T4b-SIN2,T4b-SIN3.Celiac artery metastatic lymphadenopathy fused into blocks,usually from bottom to top.Limited operation space revealed anatomical and pathological factors,the dissection of the celiac artery lymph nodes,processing the left gastric artery root difficulty.Application of novel celiac artery lymph nodes dissection path,avoiding the limitation of the celiac artery lymph node dissection space exposure factors,so that the dissection of the celiac artery lymph nodes is more complete,processing the left gastric artery root easily,reduce the amount of bleeding,shorten operation time,increase the average lymph node dissection and the Ⅲ C gastric cancer resection rate.

17.
Article in Chinese | WPRIM | ID: wpr-355742

ABSTRACT

<p><b>OBJECTIVE</b>To establish the reverse transcription loop-mediated isothermal amplification (RT-LAMP) methods for on-site HIV-1 detection.</p><p><b>METHODS</b>As for the real-time fluorescent RT-LAMP, we firstly tested the specificity and sensitivity, then explored its quantitative determination, and finally applied the method to the detection of 35 HIV-1 positive samples. For colorimetric judgment, after choosing different ameliorates to modify Hydroxynaphthol blue (HNB), we tested their real effects on coloration, and then picked out the modified dyes with obvious color change to test the sensitivity and the detection of the 35 HIV-1-positive samples.</p><p><b>RESULTS</b>The real-time fluorescent RT-LAMP showed great specificity of HIV-1, and the sensitivity to detect HIV-1 RNA was between 10 and 100 copies per reaction. On testing 35 HIV-1-positive samples, the method could reach 100 percent detection rate. However, for the quantitative determination, the quantitative relation was not observed regarding the HIV-1 RNA of below 10(3) copies per reaction. Three modified HNB dyes with clear color variation between the reaction tubes of the negative and the positive were got in the study, and their sensitivities equaled to the level of agarose gel electrophoresis. Similarly, 100% (35/35) detection rate was reached when the colorimetric RT-LAMP with the modified dyes was applied to detect 35 HIV-1-positive samples.</p><p><b>CONCLUSION</b>The established real-time fluorescence method and the modified color judgment of RT-LAMP could be helpful for truly achieving rapid, accurate, and sensitive on-site detection of HIV-1.</p>


Subject(s)
HIV-1 , Genetics , Nucleic Acid Amplification Techniques , Methods , Reverse Transcriptase Polymerase Chain Reaction
18.
Chinese Journal of Virology ; (6): 165-171, 2012.
Article in Chinese | WPRIM | ID: wpr-354753

ABSTRACT

A simple, rapid and sensitive colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was established to detect norovirus genotype GII. The method employed a set of six specially designed primers that recognized eight distinct sequences of RNA-dependant RNA polymerase and capsid protein gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for 60 minutes. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP was validated by detecting several different diarrhea viruses including norovirus genotype GII. The sensitivity was determined by serial dilutions of RNA molecules from in vitro transcription of norovirus genotype GII in parallel with conventional RT-PCR detection. The assay was further evaluated with 93 clinical specimens of diarrhea patients. The results showed that the sensitivity of RT-LAMP was 1 000 copies/microL with a high specificity and the relative sensitivity was at the same level as that of conventional RT-PCR. Positive rate of RT-LAMP in analysis of clinical specimens was approximately the same as that of conventional RT-PCR as well. This colorimetric RT-LAMP assay was potential for rapid detection of norovirus genotype GII on spot due to the observation of visual result with high specificity and sensitivity, time-saving and cost benefit.


Subject(s)
Caliciviridae Infections , Diagnosis , Virology , Colorimetry , Methods , Feces , Virology , Genotype , Humans , Norovirus , Genetics , Nucleic Acid Amplification Techniques , Methods
19.
Chinese Journal of Virology ; (6): 64-70, 2011.
Article in Chinese | WPRIM | ID: wpr-286077

ABSTRACT

A simple, rapid and sensitive colorimetric loop mediated isothermal amplification (LAMP) method was established to detect HPV6 and HPV 16 respectively. The method employed a set of four specially designed primers that recognized six distinct sequences of HPV6-E6 or HPV16-E7 for amplification of nucleic acid under isothermal conditions at 63 degrees C for one hour. The amplification process of LAMP was monitored by the addition of HNB (hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by real-time turbidimeter and agarose electrophoresis. Thirteen cervical swab samples having single infection with 13 different HPV genotypes were examined to evaluate the specificity. A serial dilution of a cloned plasmid containing HPV-E6 or HPV-E7 gene was examined to evaluate the sensitivity. The results showed that no cross-reaction with other HPV genotypes was observed. The colorimetric LAMP assay could achieve a sensitivity of 1000 copies, 10-20 times lower than that of real-time PCR. The assay was further evaluated with 62 clinical specimens and consistent results were obtained compared with the detection using Kai Pu HPV Genotyping Kit. We concluded that this colorimetric LAMP assay had potential usefulness for the rapid screening of the HPV6 or HPV16 infection in the laboratories and hospitals of provincial and municipal region in China.


Subject(s)
Colorimetry , Methods , DNA Primers , Chemistry , Genetics , Genotype , Human papillomavirus 16 , Genetics , Human papillomavirus 6 , Genetics , Humans , Nucleic Acid Amplification Techniques , Methods , Papillomavirus Infections , Virology
20.
Chinese Journal of Virology ; (6): 81-87, 2010.
Article in Chinese | WPRIM | ID: wpr-297902

ABSTRACT

A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.


Subject(s)
Animals , Colorimetry , Methods , DNA Primers , Genetics , Electrophoresis, Agar Gel , Hemagglutinin Glycoproteins, Influenza Virus , Genetics , Humans , Influenza A Virus, H1N1 Subtype , Genetics , Influenza A Virus, H3N2 Subtype , Genetics , Influenza, Human , Diagnosis , Virology , Naphthalenesulfonates , Chemistry , Nucleic Acid Amplification Techniques , Methods , Orthomyxoviridae Infections , Diagnosis , Virology , Reverse Transcriptase Polymerase Chain Reaction , Methods , Sensitivity and Specificity , Swine , Swine Diseases , Diagnosis , Virology , Temperature
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