Gitika Panicker

Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays

ABSTRACT This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50°C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 102 to 103 CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.

Rapid Detection of Vibrio vulnificus in Shellfish and Gulf of Mexico Water by Real-Time PCR

ABSTRACT In this paper we describe optimization of SYBR Green I-based real-time PCR parameters and testing of a large number of microbial species with vvh-specific oligonucleotide primers to establish a rapid, specific, and sensitive method for detection of Vibrio vulnificus in oyster tissue homogenate and Gulf of Mexico water (gulf water). Selected oligonucleotide primers for the vvh gene were tested for PCR amplification of a 205-bp DNA fragment with a melting temperature of approximately 87°C for 84 clinical and environmental strains of V. vulnificus. No amplification was observed with other vibrios or nonvibrio strains with these primers. The minimum level of detection by the real-time PCR method was 1 pg of purified genomic DNA or 102V. vulnificus cells in 1 g of unenriched oyster tissue homogenate or 10 ml of gulf water. It was possible to improve the level of detection to one V. vulnificus cell in samples that were enriched for 5 h. The standard curves prepared from the real-time PCR cycle threshold values revealed that there was a strong correlation between the number of cells in unenriched samples and the number of cells in enriched samples. Detection of a single cell of V. vulnificus in 1 g of enriched oyster tissue homogenate is in compliance with the recent Interstate Shellfish Sanitation Conference guidelines. The entire detection method, including sample processing, enrichment, and real-time PCR amplification, was completed within 8 h, making it a rapid single-day assay. Rapid and sensitive detection of V. vulnificus would ensure a steady supply of postharvest treated oysters to consumers, which should help decrease the number of illnesses or outbreaks caused by this pathogen.

Real-Time PCR Detection of Vibrio vulnificus in Oysters: Comparison of Oligonucleotide Primers and Probes Targeting vvhA

ABSTRACT We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold (CT) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 103V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA. Detection of 3 × 103 CFU V. vulnificus, resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus-related illnesses and deaths.

PCR Detection of a Newly Emerged Pandemic Vibrio parahaemolyticus O3:K6 Pathogen in Pure Cultures and Seeded Waters from the Gulf of Mexico

ABSTRACT This study describes the optimization of PCR parameters and testing of a wide number of microbial species to establish a highly specific and sensitive PCR-based method of detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 strain in pure cultures and seeded waters from the Gulf of Mexico (gulf water). The selected open reading frame 8 (ORF8) DNA-specific oligonucleotide primers tested were found to specifically amplify all 35 pathogenic V. parahaemolyticus O3:K6 pandemic isolates, whereas these primers were not found to detectably amplify two strains of V. parahaemolyticus O3:K6 that were isolated prior to the 1996 outbreaks, 122 non-O3:K6 strains of V. parahaemolyticus, 198 non-V.parahaemolyticus spp., or 16 non-Vibrio bacterial spp. The minimum level of detection by the PCR method was 1 pg of purified genomic DNA or 102 ORF8-positive V. parahaemolyticus O3:K6 cells in 100 ml of water. The effectiveness of this method for the detection of ORF8-positive isolates in environmental samples was tested in gulf water seeded with 10-fold serial dilutions of this pathogen. A detection level of 103 cells per 100 ml of gulf water was achieved. Also, the applicability of this methodology was tested by the detection of this pathogen in gulf water incubated at various temperatures for 28 days. This PCR approach can potentially be used to monitor with high specificity and well within the required range of sensitivity the occurrence and distribution of this newly emerged pathogenic V. parahaemolyticus O3:K6 strain in coastal, marine, and ship ballast waters. Early detection of V. parahaemolyticus O3:K6 will help increase seafood safety and decrease the risk of infectious outbreaks caused by this pathogen.

Characterization of the Human Cervical Mucous Proteome

IntroductionCervical cancer is among the most common cancers in women worldwide. Discovery of biomarkers for the early detection of cervical cancer would improve current screening practices and reduce the burden of disease.ObjectiveIn this study, we report characterization of the human cervical mucous proteome as the first step towards protein biomarker discovery.MethodsThe protein composition was characterized using one- and two-dimensional gel electrophoresis, and liquid chromatography coupled with mass spectrometry. We chose to use this combination of traditional biochemical techniques and proteomics to allow a more comprehensive analysis.Results and ConclusionA total of 107 unique proteins were identified, with plasma proteins being most abundant. These proteins represented the major functional categories of metabolism, immune response, and cellular transport. Removal of high molecular weight abundant proteins by immunoaffinity purification did not significantly increase the number of protein spots resolved. We also analyzed phosphorylated and glycosylated proteins by fluorescent post-staining procedures. The profiling of cervical mucous proteins and their post-translational modifications can be used to further our understanding of the cervical mucous proteome.

Seroprevalence of 9 Human Papillomavirus Types in the United States, 2005–2006

BACKGROUND A 9-valent human papillomavirus (HPV) vaccine, licensed in 2014, prevents 4 HPV types targeted by the quadrivalent vaccine (6/11/16/18) and 5 additional high-risk (HR) types (31/33/45/52/58). Measuring seropositivity before vaccine introduction provides baseline data on exposure to types targeted by vaccines. METHODS We determined seroprevalence of HPV 6/11/16/18/31/33/45/52/58 among 4943 persons aged 14-59 years who participated in the National Health and Nutrition Examination Survey, 2005-2006. RESULTS Among females, seroprevalence was 40.5% for any of the 9 vaccine types, 30.0% for any 7 HR types (16/18/31/33/45/52/58), 19.0% for any 5 additional types (31/33/45/52/58), and 18.3% for 16/18. Compared with non-Hispanic whites, non-Hispanic blacks had higher seroprevalence of 31/33/45/52/58 (36.8% vs 15.9%) and 16/18 (30.1% vs 17.8%), while Mexican Americans had higher seroprevalence of 31/33/45/52/58 (23.6% vs 15.9%) (P < .05 for all). In multivariable analyses of data from females, race/ethnicity, number of sex partners, and age were associated with 16/18 and 31/33/45/52/58 seropositivity. Seropositivity was lower among males than among females (P < .001 for all type categories). CONCLUSIONS In 2005-2006, about 40% of females and 20% of males had serological evidence of exposure to ≥1 of 9 HPV types. Seroprevalence of all type categories, especially HPV 31/33/45/52/58 among females, varied by race/ethnicity.

Optimization of SELDI-TOF protein profiling for analysis of cervical mucous

Cervical mucous, produced in the region where cervical neoplasia occurs, is thought to be a good choice for discovery of biomarkers to improve cervical cancer screening. In this study, SELDI-TOF MS analysis was used to evaluate parameters for protein profiling of mucous. Proteins were extracted from mucous collected with Weck-Cel sponges. Several parameters like extraction reagent, loading protein concentration, matrix type, bind/wash conditions and sample fractionation, on different protein chip surfaces were evaluated. SELDI peak number and consistency in the resulting spectra were used to evaluate each condition. Analysis of spectra generated by different protein chips revealed an average of 30 peaks in the 2.5-30 kDa mass range using sinnapinic acid in the unfractionated sample. Sample concentration and buffer conditions evaluated did not lead to large alterations in the profiles. Quality control spectra were reproducible with intra- and inter-assay intensity CV for CM10, H50 and Q10 arrays being less than 20% and 30% respectively. IMAC30-Cu chips had higher intra- and inter-assay CVs at 25% and 35%. Current data showed that optimizing pre-analytical parameters can help in standardization and reproducibility of protein profiles produced by cervical mucous, and thus can be used for protein biomarker discovery with the SELDI platform.

Monitoring for Human Papillomavirus Vaccine Impact Among Gay, Bisexual, and Other Men Who Have Sex With Men-United States, 2012-2014.

BACKGROUND Gay, bisexual, and other men who have sex with men (MSM) are at high risk for human papillomavirus (HPV) infection; vaccination is recommended for US males, including MSM through age 26 years. We assessed evidence of HPV among vaccine-eligible MSM and transgender women to monitor vaccine impact. METHODS During 2012-2014, MSM aged 18-26 years at select clinics completed a computer-assisted self-interview regarding sexual behavior, human immunodeficiency virus (HIV) status, and vaccinations. Self-collected anal swab and oral rinse specimens were tested for HPV DNA (37 types) by L1 consensus polymerase chain reaction; serum was tested for HPV antibodies (4 types) by a multiplexed virus-like particle-based immunoglobulin G direct enzyme-linked immunosorbent assay. RESULTS Among 922 vaccine-eligible participants, the mean age was 23 years, and the mean number of lifetime sex partners was 37. Among 834 without HIV infection, any anal HPV was detected in 69.4% and any oral HPV in 8.4%, yet only 8.5% had evidence of exposure to all quadrivalent vaccine types. In multivariate analysis, HPV prevalence varied significantly (P < .05) by HIV status, sexual orientation, and lifetime number of sex partners, but not by race/ethnicity. DISCUSSIONS Most young MSM lacked evidence of current or past infection with all vaccine-type HPV types, suggesting that they could benefit from vaccination. The impact of vaccination among MSM may be assessed by monitoring HPV prevalence, including in self-collected specimens.

Development and evaluation of multiplexed immunoassay for detection of antibodies to HPV vaccine types

Reliable antibody based-assays are needed to evaluate the immunogenicity of current vaccines, impact of altered dosing schemes or of new vaccine formulations. An ideal assay platform would allow multiplex type-specific detection with minimal sample requirement. We used the Meso Scale Discovery (MSD) electrochemiluminescence based detection platform to develop a multiplex direct virus-like particle (VLP) ELISA to detect antibodies to HPV 6, 11, 16, and 18 with a protocol developed for detection using the SI 6000 imager (M4ELISA). MSD prepared the plates in the 7-spot/well format, using the purified VLPs (4 spots) and PBS+BSA pH7.4 (3 blank spots). Three-point titrations and the parallel line method were used to calculate antibody levels. Dynamic range, precision, and stability of pre-printed plates were determined using a panel of previously characterized sera. Cut-off values using childrens sera were established using 99% RLU limits based on the 4-parameter Johnson Su best fit curve. Results of the M4ELISA were compared to competitive Luminex Immunoassay (cLIA) on n = 4454 sera from a predominantly unvaccinated cohort. Using a VLP coating concentration of 80 μg/ml with BSA provided the most robust RLU signal for all types. The dynamic range of the assay was about 1000 fold, with assay variability under 25% for each of the four vaccine types. Long-term stability of the plates extended to about 7 months from the time plates was received in the laboratory after printing. There was moderate agreement (κ = 0.38-0.54) between M4ELISA and cLIA, with antibody detection for each of the 4 types more frequent with M4ELISA. Quantitative analysis however showed a good correlation between concordant samples by both assays (ρ ≥ 0.6). The MSD platform shows promise for simultaneous quantitation of the antibody responses to four HPV vaccine types in a high-throughput manner.

Occurrence and distribution of capB in Antarctic microorganisms and study of its structure and regulation in the Antarctic biodegradative Pseudomonas sp. 30/3

The analysis of the cold-shock domain (CSD)-encoding genes, capB and cspA, by PCR amplification showed presence of capB in all 18 Antarctic Pseudomonas isolates, but the absence of cspA. Nucleotide sequence analysis of capB ORF from a biodegradative Pseudomonas 30/3 and its regulatory sequences including the promoter and 5′-UTR was determined and compared with the other CSD-encoding genes. Expression analysis using translational gene fusion of the putative capB promoter and its flanking sequence from Pseudomonas sp. 30/3 with lacZ′ exhibited a significant increase in β-galactosidase activity at 15 and 6°C. Unlike the expression of E. coli CspA, Pseudomonas sp. 30/3 showed a slow but steady increase of the CapB expression at 6°C. Subcellular localization of CapB at 6°C showed accumulation in and around the nucleoid whereas at 22 or 30°C, it was identified around the nucleoid as well as in the cytosol. Our study attempts to elucidate the detailed structure of capB from Pseudomonas 30/3 and the role of 5′UTR in the transcriptional regulation along with the possible role of CapB in transcription and translation suited for the cold adaptation of this bacterium in Antarctic environment.