1.A double antibody sandwich ELISA based assay for titration of severe fever with thrombocytopenia syndrome virus.
Lin LIU ; Quan-Fu ZHANG ; Chuan LI ; Jian-Dong LI ; Xiao-Lin JIANG ; Fu-Shun ZHANG ; Wei WU ; Mi-Fang LIANG ; De-Xin LI
Chinese Journal of Experimental and Clinical Virology 2013;27(3):215-217
OBJECTIVETo develop an assay for titration of severe fever with thrombocytopenia syndrome virus (SFTSV) based on double antibody sandwich ELISA.
METHODSA double antibody sandwich ELISA was developed for detection of SFTSV based on SFTSV nucleocapsid (N) protein specific poly- and monoclonal antibodies, procedures were optimized and evaluated. This ELISA based titration assay was compared with fluorescence assasy and plaque assay based titration method.
RESULTSThe results suggested that the titers obtained by ELISA based method are consistent with those obtained by IFA based method (R = 0.999) and the plaque assay titration method (R = 0.949).
CONCLUSIONThe novel ELISA based titration method with high sensitivity and specificity is easy to manage and perform, and can overcome the subjectivity associated with result determination of the fluorescence assay and plaque assay based methods. The novel ELISA based titration method can also be applied to high throughput detection.
Bunyaviridae ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; methods ; Fever ; virology ; Fluorescent Antibody Technique ; Humans ; Thrombocytopenia ; virology
2.The Advances in the Contamination and Detection of Foodborne Pathogen Noroviruses in Fresh Produce.
Chinese Journal of Virology 2015;31(6):685-697
This article reviewed the researches proceeding on the contamination and detection of the foodborne pathogen noroviruses (NoVs) in fresh produce, which involved the NoVs contaminations in fresh produce, the special attachment of NoVs in fresh produce, the NoVs outbreaks associated with fresh produce and the NoVs detection in fresh produce. There had been an increase in reported infectious disease risks associated with the consumptions of fresh produce for recent 30 years. Because the NoVs, as a primary cause of viral gastroenteritis thoughout the world, were highly contagious, had a low infectious dose, and were persistent in the environment. And also the methods for NoVs detection in food had significantly developed over the last 15 years. Currently NoVs were the most common pathogen accounting for 40% of outbreaks associated with fresh produce (i. e., fruits and vegetables). Data from outbreaks investigations verified fresh produce as the high risk food products for NoVs. The fresh produce were typically eaten raw with no thermal processing, can be contaminated at any step during production and processing from faecally polluted water and fertilizers, the poor hygiene practices by food handlers and the cross-contamination. The attachment of NoVs to the fresh produce was due to the physio-chemical factors of virus protein coat, the special attachment to different fresh produce, and the possibility for internalization of NoVs. It might provide answers to why those high risk foods were more frequently implicated (i. e., lettuce and raspberries). According to the data of foodborne NoVs outbreaks which were associated with fresh produce from EU countries and the USA, the outbreaks in EU countries were mainly associated with NoVs contaminated raspberries and lettuce, while in USA which were associated with NoVs contaminated lettuce. Unfortunately, there were no NoVs detection methods for fresh produce or the data of foodborne NoVs outbreaks which were associated with fresh produce in China. That made it difficult to analyze the NoVs contamination situation in China. The heterogeneous distributions of presumably low levels of virus, which presented in contaminated fresh produce, also made it difficult to detect NoVs. To solve this problem, different sampling methods, viral elution methods and RT-qPCR methods were chosen. For example, according to the isoelectric point of NoVs particles, high pH and high ionic strength solution could be used as means for releasing NoVs. For the elution from acidic fruit, the buffer capacity and the virus recovery could be increased by the addition of tris-HCl. When analyzing pectin containing raspberries or strawberries, the viral elution usually incubated with pectinase at neutral pH to avoid from foaming jelly. In this paper, the latest ISO standard for NoV detection in food and the new approaches for NoV detection were also reviewed to provide references for domestic researches. It was necessary to establish and develop domestic methods for NoV detection in fresh produce, especially the different NoV conventional molecular detection methods with corresponding NoV extraction methods, which targeted to the different adsorption characteristics of different fruits and vegetables, in order to strengthen the national food safety monitoring.
Food Analysis
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methods
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Food Contamination
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analysis
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Foodborne Diseases
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virology
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Fruit
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virology
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Gastroenteritis
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virology
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Humans
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Norovirus
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classification
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genetics
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isolation & purification
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physiology
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Vegetables
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virology
3.Progress on research of tissue culture of Siraitia grosvenorii.
Chang-Liang FU ; Xiao-Jun MA ; Long-Hua BAI ; Xin ZHAO
China Journal of Chinese Materia Medica 2005;30(5):325-328
In this paper we reviewed the development of tissue culture, current situations of virus-free plantlets industrialization and the way to deal with the situations, application prospects of Siraitia grosvenorii so as to give some advice for its further study and application.
Culture Media
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Momordica
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growth & development
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virology
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Plant Leaves
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growth & development
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virology
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Plant Stems
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growth & development
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virology
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Plant Viruses
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Plants, Medicinal
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growth & development
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virology
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Tissue Culture Techniques
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methods
4.Establishment of human colorectal tissue model in HIV-1 mucosal infection.
Yu YANG ; Ai-ping LIU ; Qing-lai MENG ; Jian-qing XU ; Xiao-yan ZHANG
Chinese Journal of Preventive Medicine 2011;45(2):127-131
OBJECTIVETo establish human colorectal tissue model in HIV-1 mucosal infection and by using pseudotyped virus to simulate the biological process of HIV-1 mucosal infection from HIV-1 entering into mucosa to local infection establishment.
METHODSTumor adjacent normal colorectal tissues were obtained with informed consent. After excised the muscularis externa, the mucosa and submucosa were dissected into the same blocks and cultured in 12-well cell culture plates. The cultured tissue structure and morphology were observed from day 0 to day 13 by staining with the hematoxylin eosin (HE), and the tissue activity was detected by 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The established tissues explants were infected by a single cycle replicated pseudotyped virus and propagated for 6 - 7 days, then subjected to the detection of p24 production within supernatant to verify the applicability of the model for the studying of HIV-1 mucosal infection. The applicability of the established explants for safety and reactivity evaluation of mucosa topical drugs was conducted by the using of first generation antiseptic Nonoxynol-9 (N-9) as an example.
RESULTSHE staining showed the structure of colorectal tissue was remained well until 5(th) day and still evident until 13(th) day. The tissue activity of cultured mucosa was above 80% at day 4, and still remained over 50% at day 7 as detected by MTT assay. After infected by pseudo virus, the increased level of p24 was detected from supernatant collected on 1(st), 4(th), 8(th) day, which indicated a local infection was created. In addition, the dose changing of N-9 was reflected sensitively by the activity of this model.
CONCLUSIONEx vivo human colorectal tissue model mimic HIV-1 mucosal infection was established that can be used to replicate the bioprocess of human HIV-1 mucosal infection.
Colon ; pathology ; virology ; HIV Infections ; pathology ; virology ; HIV-1 ; Humans ; Intestinal Mucosa ; pathology ; virology ; Models, Biological ; Rectum ; pathology ; virology ; Tissue Culture Techniques ; methods ; Tumor Cells, Cultured
5.Investigation of the Bovine Leukemia Virus Proviral DNA in Human Leukemias and Lung cancers in Korea.
Jehoon LEE ; Yonggoo KIM ; Chang Suk KANG ; Dae Hyun CHO ; Dong Hwan SHIN ; Young Na YUM ; Jae Ho OH ; Sheen Hee KIM ; Myung Sil HWANG ; Chul Joo LIM ; Ki Hwa YANG ; Kyungja HAN
Journal of Korean Medical Science 2005;20(4):603-606
The bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis. This study investigated the presence of the BLV in leukemia (179 acute lymphoblastic leukemia, 292 acute myeloid leukemia and 46 chronic myelogenous leukemia cases) and 162 lung cancer patients (139 adenocarcinoma, 23 squamous cell carcinoma) to determine if the BLV is a causative organism of leukemia and lung cancer in Koreans. A BLV infection was confirmed in human cells by PCR using a BLV-8 primer combination. All 517 cases of human leukemia and 162 lung cancer were negative for a PCR of the BLV proviral DNA. In conclusion, although meat has been imported from BLV endemic areas, the BLV infection does not appear to be the cause of human leukemia or lung cancer in Koreans. These results can be used as a control for further studies on the BLV in Koreans.
Acute Disease
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Adenocarcinoma/virology
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Cell Line
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DNA, Viral/*genetics/isolation & purification
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Humans
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Korea
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Leukemia/*virology
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Leukemia Virus, Bovine/*genetics
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Leukemia, Lymphocytic, Acute/virology
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Leukemia, Myeloid/virology
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Leukemia, Myeloid, Chronic/virology
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Lung Neoplasms/*virology
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Polymerase Chain Reaction/methods
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Proviruses/*genetics
6.Colorimetric detection of norovirus genotype GII by reverse transcription loop-mediated isothermal amplification.
Jian-Ming LUO ; Xi-Yang WU ; Zi-Qian XU ; Le LUO ; Kai NIE ; Meng-Jie YANG ; Ya-Lan ZENG ; Zhao-Jun DUAN ; Xue-Jun MA
Chinese Journal of Virology 2012;28(2):165-171
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.
Caliciviridae Infections
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diagnosis
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virology
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Colorimetry
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methods
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Feces
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virology
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Genotype
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Humans
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Norovirus
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genetics
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isolation & purification
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Nucleic Acid Amplification Techniques
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methods
7.Evaluation of screening performance of HPV DNA test on specimens from different sites of the female genital tract.
Shaokai ZHANG ; Leni KANG ; Bin LIU ; Jianfeng CUI ; Feng CHEN ; Xinfu LIU ; Hong WANG ; Wen CHEN
Chinese Journal of Oncology 2014;36(5):389-393
OBJECTIVETo evaluate the diagnostic performance of different specimens for detecting CIN2(+), and to find the solution of the problem that why the performance of self-collected specimen is worse than cervical specimen collected by physician.
METHODSThe cervix, lower 1/3 vagina, upper 1/3 vagina and self-collected specimens from each of the 806 women who took part in this multi-center screening program from May 2006 to April 2007 were tested by hybrid capture 2 (HC2) technique. The diagnostic performance of HC2 on the four specimens for detecting CIN2(+) lesions was calculated. Linear array was performed on the four specimens from 489 out of the 806 women and the diagnostic performance of linear array on the four specimens for detecting CIN2(+) lesions was also calculated. Z test was used to compare the area under ROC and McNemar or χ(2) test was used to compare the sensitivity and specificity of different specimens.
RESULTSThe area under ROC of the cervix, 1/3 upper vagina, 1/3 lower vagina and self-collected samples testing by HC2 for detecting CIN2(+) lesions were 0.902, 0.793, 0.769 and 0.773, respectively (P < 0.001). Using 1 RUL/CO as the cut-point of HC2, the sensitivity of the cervix, upper vagina, lower vagina and self-collected samples were 98.0%, 91.8%, 83.7% and 81.6%. Compared with the cervical specimen, the sensitivity of self-collected specimen for detecting CIN2(+) lesions was significantly lower (P = 0.008). Lowering the cutoff value for HC2 test could improve the sensitivity of self-collected specimen, but it significantly compromised the specificity. The sensitivity of self-collected specimen tested by linear array for detecting CIN2(+) lesions was 95.7% and it was not significantly different compared with the sensitivity of cervical specimen (97.9%) tested by HC2.
CONCLUSIONSThe performance of self-collected specimen tested by HC2 for detecting CIN2(+) lesions is lower than that of physician-collected cervical specimen, and lowering the cutoff value can't improve its diagnostic performance. Using linear array as the HPV DNA test can significantly improve the screening diagnostic performance of self-collected specimens.
Adolescent ; Cervical Intraepithelial Neoplasia ; diagnosis ; virology ; Cervix Uteri ; virology ; DNA, Viral ; analysis ; Female ; Human Papillomavirus DNA Tests ; Humans ; Mass Screening ; Papillomaviridae ; isolation & purification ; Papillomavirus Infections ; diagnosis ; virology ; Self-Examination ; Specimen Handling ; methods ; Uterine Cervical Neoplasms ; diagnosis ; virology ; Vagina ; virology
8.Development of an extraction and concentration method for the detection of hepatitis A virus in different samples.
Qing-Ling MENG ; Zi-Jie GUO ; Jing-Yuan CAO ; Sheng-Li BI
Chinese Journal of Experimental and Clinical Virology 2008;22(4):305-307
OBJECTIVETo develop an extraction and concentration method for the detection of hepatitis A virus (HAV) in shellfish, water, serum and saliva samples by nested RT-PCR.
METHODSHAV were artificially inoculated into the above samples, calm sample was extracted using glycine buffer pH9.5, PEG precipitation; water sample was PEG precipitated directly; then all the samples including serum and saliva samples were extracted using Trizol regent, followed by nested RT-PCR detection using primers from HAV VP1-2A region.
RESULTSThe detection limit for HAV in cultured cell lysis was 0.1TCID50; in water, serum or salva sample was 1TCID50 respectively, in calm sample was 1-10 TCID50. HAV RNA was detected in water and sera samples collected from the HAV outbreak region, sequenced and analysis.
CONCLUSIONThe method developed here is convenient, specific and capable of detecting low levels of HAV in different samples, would be useful for diagnostic laboratories in order to perform HAV analysis in cases of foodborne infections or for molecular epidemiology investigation of HAV outbreaks.
Animals ; Chemical Fractionation ; methods ; Fresh Water ; virology ; Genetic Techniques ; Hepatitis A ; diagnosis ; virology ; Hepatitis A virus ; genetics ; isolation & purification ; Humans ; RNA, Viral ; chemistry ; genetics ; isolation & purification ; Saliva ; virology ; Serum ; virology ; Shellfish ; virology
9.Distribution of hemorrhagic fever with renal syndrome virus in gamasid mites and chigger mites.
Yun ZHANG ; Jin ZHU ; Xiaozhao DENG ; Guanghua WU ; Jiaju ZHANG ; Yanping ZHOU
Chinese Journal of Preventive Medicine 2002;36(4):232-234
OBJECTIVETo study the distribution of hemorrhagic fever with renal syndrome virus (HFRSV) in mites.
METHODSIn situ reverse transcription-polymerase chain reaction (IS RT-PCR) was used for detecting the distribution of HFRSV in mites.
RESULTSHFRSV RNA was mainly located in ovary and mid-gut tissues of gamasid mites and chigger mites. The positive signal intensity in the third and fourth generations of gamasid mite was stronger than that in the first and second generations, and that in nymph of chigger mite more than larva.
CONCLUSIONBoth chigger mite and gamasid mite could play an important role in the transmission of HFRSV.
Animals ; Digestive System ; virology ; Female ; Hantaan virus ; genetics ; growth & development ; In Situ Hybridization ; methods ; Male ; Mites ; virology ; Nymph ; virology ; Ovary ; virology ; RNA, Viral ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; methods
10.Study on effect of enzyme linked immunosorbent assay kits of norovirus.
Qing ZHANG ; Miao JIN ; Shu-Xian CUI ; Na LIU ; Lei LI ; Zhao-Jun DUAN
Chinese Journal of Experimental and Clinical Virology 2009;23(3):227-228
OBJECTIVEEffects of RIDASCREEN Norovirus (C 1401) 3rd Generation kit (R-biopham AG, darmstadt, Germany) and IDEIA NLV kit (DAKOCytomation., Ely, UK) were compared for detecting human norovirus (HuNV) in fecal sample.
METHODSThe performance of the ELISA was compared with that of the reverse transcription polymerase chain reaction (RT-PCR) by testing a panel of 308 fecal samples collected from patients involved in outbreaks of gastroenteritis in Chang Chun and Guang Zhou. Gene sequencing was performed to positive samples tested by RT-PCR to determine genotype compared with standard sequences.
RESULTSRT-PCR is gold standard, RIDASCREEN Norovirus (C 1401) 3rd Generation kit had a high sensitivity of 96.10% but a specificity of 93.51%, and Dako kit had a low sensitivity of 95.83% but a high specificity of 95.76%.
CONCLUSIONRIDASCREEN Norovirus (C 1401) 3rd Generation kit is more Satisfactory for a preliminary screening.
Caliciviridae Infections ; diagnosis ; virology ; Disease Outbreaks ; Enzyme-Linked Immunosorbent Assay ; methods ; Feces ; chemistry ; virology ; Gastroenteritis ; diagnosis ; virology ; Humans ; Norovirus ; genetics ; immunology ; isolation & purification ; Reagent Kits, Diagnostic