VSITA, an Improved Approach of Target Amplification in the Identification of Viral Pathogens / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Unbiased next generation sequencing (NGS) is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences.</p><p><b>METHODS</b>A viral Sequence Independent Targeted Amplification (VSITA) approach using a set of non-ribosomal and virus-enriched octamers (V8) was developed and compared with traditionally used random hexamers (N6). Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR (qPCR). Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis.</p><p><b>RESULTS</b>A minimum 30 hexamers matching to viral reference sequences (sense and antisense) were selected from a dataset of random 4,096 (46) hexamers (N6). Two random nucleotides were added to the 5' end of the selected hexamers, and 480 (30 × 42) octamers (V8) were obtained. In general, VSITA approach showed higher enrichment of virus-targeted cDNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin.</p><p><b>CONCLUSION</b>The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples.</p>

Secular trend analysis of lung cancer incidence in Sihui city, China between 1987 and 2011 / 癌症

<p><b>BACKGROUND</b>With industrial and econom ic development in recent decades in South China, cancer incidence may have changed due to the changing lifestyle and environment. However, the trends of lung cancer and the roles of smoking and other environmental risk factors in the development of lung cancer in rural areas of South China remain unclear. The purpose of this study was to explore the lung cancer incidence trends and the possible causes of these trends.</p><p><b>METHODS</b>Joinpoint regression analysis and the age-period-cohort (APC) model were used to analyze the lung cancer incidence trends in Sihui, Guangdong province, China between 1987 and 2011, and explore the possible causes of these trends.</p><p><b>RESULTS</b>A total of 2,397 lung cancer patients were involved in this study. A 3-fold increase in the incidence of lung cancer in both sexes was observed over the 25-year period. Joinpoint regression analysis showed that while the incidence continued to increase steadily in females during the entire period, a sharp acceleration was observed in males starting in 2005. The full APC model was selected to describe age, period, and birth cohort effects on lung cancer incidence trends in Sihui. The age cohorts in both sexes showed a continuously significant increase in the relative risk (RR) of lung cancer, with a peak in the eldest age group (80-84 years). The RR of lung cancer showed a fluctuating curve in both sexes. The birth cohorts identified an increased trend in both males and females; however, males had a plateau in the youngest cohorts who were born during 1955-1969.</p><p><b>CONCLUSIONS</b>Increasing trends of the incidence of lung cancer in Sihui were dominated by the effects of age and birth cohorts. Social aging, smoking, and environmental changes may play important roles in such trends.</p>

Pathogenic spectrum of enteroviruses associated with hand, foot and mouth disease by a GeXP™-based multiplex reverse transcription-PCR assay in Jinan, China, 2009-2012 / 病毒学报

We investigated the pathogenic spectrum of enteroviruses associated with hand, foot and mouth disease (HFMD) in Jinan, China. A total of 274 specimens with a clinical diagnosis of HFMD in Jinan from 2009 to June 2012 were used. A GenomeLab™ (GeXP)-based multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay was employed to simultaneously detect 15 serotypes of human enteroviruses: human enterovirus (EV)71; coxsackievirus A (CVA)16, 4, 5, 6, 9, and 10; CVB1, 3 and 5; echovirus (Echo) 6, 7, 11, 13 and 19. Results showed that all samples were enterovirus-positive, with the most common serotypes being EV71 (25.18%) and CVA16 (16.06%), followed by CVA10 (14.23%), CVA6 (7.30%), CVB1 (1.09%), Echo6 (0.73%), CVA9 (0.36%), CVB3 (0.36%) and co-infections (5.11%). CVA10 and CVA6 had the third and fourth highest prevalence of pathogens for HFMD, respec- tively. The most prevalent season for CVA10 was from April to August, with a peak in April; for CVA6 it was from April to August, with a peak in June. This is the first report of the pathogenic spectrum of en- teroviruses associated with HFMD in Jinan using the GeXP-based multiplex RT-PCR assay. These data will provide the scientific evidence for the prevention and control of epidemics, as well as therapy for HFMD patients.

Visual detection of HIV-1 by reverse transcription loop-mediated isothermal amplification with the hydroxynaphthol blue dye / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of HIV-1.</p><p><b>METHODS</b>RT-LAMP primers were designed according to conservative sequences of HIV-1 gag gene, and their sensitivity and specificity were evaluated by the established RT-LAMP protocol with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The performance of RT-LAMP on clinical samples was compared with real-time reverse transcription PCR(qRT-PCR).</p><p><b>RESULTS</b>The RT-LAMP assay showed a high specificity, and its detection limit was 1000 copies RNA per tube. The sensitivity and specificity of this method using 43 clinical samples were 94.6% and 100%, respectively,in comparison with those of qRT-PCR.</p><p><b>CONCLUSION</b>RT-LAMP assay using hydroxynaphthol blue dye does not need expensive instruments, and offer an alternative for the rapid detection of HIV-1 with the potential to be applied in field diagnosis.</p>

The establishment of a new resequencing pathogen microarray-based assay for detecting unexplained respiratory tract infections / 病毒学报

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.

Colorimetric detection of norovirus genotype GII by reverse transcription loop-mediated isothermal amplification / 病毒学报

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.

Detection and typing of human papillomavirus by a GeXP based multiplex PCR assay / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a new and rapid GeXP based multiplex PCR assay for the detection and typing of human papillomavirus 6, 11, 31, 33 and 52.</p><p><b>METHODS</b>Nucleotide sequences of HPV6, HPV11, HPV31, HPV33 and HPV52 from NCBI were obtained and compared. Genotype-specific primers were then designed and the sensitivity and specificity of multiple PCR assay was evaluated. Optimized assay was further validated with 30 clinical specimens collected from the cervical secretions of patients.</p><p><b>RESULTS</b>A GeXP based multiplex PCR was developed for sensitive detection and reliable differentiation of five HPV genotypes (HPV6, 11, 31, 33 and 52),</p><p><b>CONCLUSION</b>A GeXP based multiplex PCR assay is demonstrated to be a new and rapid technique for simultaneous detection and typing of 5 different human papillomaviruses.</p>

A GeXP based multiplex RT-PCR assay for simultaneous detection of twelve human respiratory viruses / 病毒学报

A GeXP based multiplex RT-PCR assay was developed to simultaneously detect twelve different respiratory viruses types/subtypes including influenza A virus, influenza B virus, influenza A virus sH1N1, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, human rhinovirus, human metapneumovirus, adenovirus, respiratory syncytial virus A, respiratory syncytial virus B and human bocavirus. Twelve sets of specific primers were designed based on the conserved sequences of available respiratory-virus sequence database. The specificity of the multiplex system was examined by positive specimens confirmed previously. The sensitivity to detect twelve respiratory viruses simultaneously was 10(3) copies/microL. Twenty four clinical specimens were further detected by this novel assay and the results were compared with that of the real-time RT-PCR. These results showed that this novel assay based on GeXP is a fast, sensitive, and high throughput test for the detection of respiratory virus infections.

Colorimetric detection of HPV6 and HPV16 by loop mediated isothermal amplification / 病毒学报

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.

A novel method for multiplex detection of gastroenteritis-associated viruses / 病毒学报

To develop and optimize a simultaneous detection method of RotavirusA, Norovirus GI, GII, Sapovirus, human astrovirus, enteric adenoviruses and HBoV2 with GenomeLab GeXP analysis system. The sensitivity was verified to be 10(4) copies/microL with plasmids containing the viral targets in triplicate on different days, and no cross-reaction with enterovirus71, human Parechovirus and PicobirnavirusII was observed. Finally, we successfully developed a high throughout, rapid and maneuverable multiplex RT-PCR assay for simultaneous detection of seven viruses related with viral gastroenteritis, which provide a novel method for the molecular diagnosis of diarrhea-associated virus.

Development of a GeXP based multiplex RT-PCR assay for simultaneous differentiation of nine human hand food mouth disease pathogens / 病毒学报

A multiplex RT-PCR assay based on GeXP system was developed in order to detect simultaneously human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) and other coxsackieviruses (CVA4, 5, 9 and 10, CVB1, 3 and 5). Enterovirus detection was performed with a mixture of 12 pairs of oligonucleotide primers including one pair of published primers for amplifying all known pan-enterovirus genomes and eleven primer pairs specific for detection of the VP1 genes of EV71, C A16, CVA4, CVA5, CVA9, CVA10, CVB1, CVB3 and CVB5, respectively. The specificity of multiplex RT-PCR system was examined using enterovirus cell cultures and positive strains identified previously from hand-foot-and-mouth disease (HFMD) patients. Serial dilution of titrated EV71 and C A16 cell cultures and in vitro transcripted RNA of enterovirus VP1 regions were used to detect the sensitivity of the multiplex RT-PCR system. The limit of detection for this multiplex RT-PCR system was 10(0.5) TCID50/microL for EV71 and C A16 cell cultures and 1000 copies for in vitro transcripted RNA of nine viruses per assay. This multiplex RT-PCR assay is a rapid, sensitive and specific assay for the diagnosis of common enterovirus infection in cases of HFMD outbreak and is also potentially useful for molecular epidemiological investigation.