Detection Of Viral Genes Research Articles

Viral gene detection based on multifunctional microspheres and AgNPs coated substrate interface area SERS scheme

Surface-enhanced Raman spectroscopy (SERS) has emerged as an effective method for detecting viral genes in aqueous solutions. However, SERS has a low efficiency and poor reproducibility in multi-component aqueous solutions. Thus, developing a SERS scheme with stable hotspots generation, significant enhancement effects, and efficient viral gene enrichment remains a challenge. This study sought to proposes an enhanced hotspot generation scheme combining multifunctional microspheres and SERS substrates, which can achieve stable enhancement in the interface area. Multifunctional microspheres can be used to selectively enrich target biomolecules in aqueous solutions. Furthermore, simulation results show that these microspheres can synergistically form a stable enhancement area with SERS substrates. Finally, this scheme was used for immunocapture and Raman detection of hemagglutinin 7 (H7) and neuraminidase 9 (N9) specific gene in an aqueous solution containing multiple types of influenza viral genes. The relationship between the characteristic peaks and concentrations was analysed using two-dimensional correlation analysis. Spectral data analysis revealed an exceedingly high linear correlation between the selected characteristic peak intensity and concentrations. The calculation results show that the SERS enhancement factor of the proposed scheme for the H7 and N9 specific gene are 9.23×107 and 1.59×108, respectively. Notably, the detection limit of this scheme was 1 pM. Therefore, this scheme holds the promise to realize the detection of viral genes in heterogeneous, complex, and dynamic aqueous biological samples.

Surveillance of Wastewater to Monitor the Prevalence of Gastroenteritis Viruses in Chiba Prefecture (2014-2019).

In Japan, sentinel surveillance is used to monitor the trend of infectious gastroenteritis. Another method of pathogen surveillance, wastewater-based epidemiology, has been used recently because it can help to monitor infectious disease without relying on patient data. Here, we aimed to determine the viral trends reflected in the number of reported patients and number of gastroenteritis virus-positive samples. We focused on gastroenteritis viruses present in wastewater and investigated the usefulness of wastewater surveillance for the surveillance of infectious gastroenteritis. Real-time polymerase chain reaction was used for viral gene detection in wastewater. The number of reported patients per pediatric sentinel site and number of viral genome copies were compared for correlation potential. The number of gastroenteritis virus-positive samples reported by National Epidemiological Surveillance of Infectious Disease (NESID) and the status of gastroenteritis viruses detected in wastewater were also evaluated. Genes of norovirus genotype I, norovirus genotype II, sapovirus, astrovirus, rotavirus group A, and rotavirus group C were detected in wastewater samples. Viruses were detected in wastewater during periods when no gastroenteritis virus-positive samples were reported to NESID. Norovirus genotype II and other gastroenteritis viruses were detected in wastewater even during periods when no gastroenteritis virus-positive samples were found. Therefore, surveillance using wastewater can complement sentinel surveillance and is an effective tool for the surveillance of infectious gastroenteritis.

Advances and Challenges in SARS-CoV-2 Detection: A Review of Molecular and Serological Technologies.

The urgent need for accurate COVID-19 diagnostics has led to the development of various SARS-CoV-2 detection technologies. Real-time reverse transcriptase polymerase chain reaction (RT-qPCR) remains a reliable viral gene detection technique, while other molecular methods, including nucleic acid amplification techniques (NAATs) and isothermal amplification techniques, provide diverse and effective approaches. Serological assays, detecting antibodies in response to viral infection, are crucial for disease surveillance. Saliva-based immunoassays show promise for surveillance purposes. The efficiency of SARS-CoV-2 antibody detection varies, with IgM indicating recent exposure and IgG offering prolonged detectability. Various rapid tests, including lateral-flow immunoassays, present opportunities for quick diagnosis, but their clinical significance requires validation through further studies. Challenges include variations in specificity and sensitivity among testing platforms and evolving assay sensitivities over time. SARS-CoV-2 antigens, particularly the N and S proteins, play a crucial role in diagnostic methods. Innovative approaches, such as nanozyme-based assays and specific nucleotide aptamers, offer enhanced sensitivity and flexibility. In conclusion, ongoing advancements in SARS-CoV-2 detection methods contribute to the global effort in combating the COVID-19 pandemic.

Construction of a screening and evaluation system for anti-hepatitis B virus drugs based on quadratic exponential smoothing model

Abstract Exploring the construction of an anti-hepatitis B virus drug screening and evaluation system is to better develop anti-HBV virus drugs. In this paper, we analyzed the types of hepatitis B virus present in different hepatocytes, starting from the hepatitis B cell line model. Based on the quadratic, exponential smoothing model, a QES-LSTM viral gene detection model was constructed by introducing a long and short term memory neural network, and experimental analysis of the sensitivity and specificity of viral gene detection was conducted for this model. From the sensitivity experiments, the sensitivity of HBV DNA, DHBV DNA and DHBV cccDNA were 60 copies/ml, 60 copies/ml and 10 copies/ml, respectively. From the specificity experiments, the mean values of specificity of HBV DNA, DHBV DNA, and DHBV cccDNA were 0.489, 0.481, 0.429, respectively, 0.429, which showed positive amplification compared to other types. This indicates that effective discrimination of HBV viral genes is needed in the construction of an anti-HBV virus drug screening and evaluation system, which in turn allows targeted screening of drugs for the treatment of the HBV virus.

Chikungunya Virus Diagnosis: A Review of Current Antigen Detection Methods.

Chikungunya is a mosquito-borne viral disease caused by the chikungunya virus (CHIKV). CHIKV is expanding at an alarming rate, potentially spreading and establishing endemicity in new areas where competent vectors are present. The dramatic spread of CHIKV in recent years highlights the urgent need to take precautionary measures and investigate options for control. It is crucial in developing nations where diagnostic tools are limited, and symptoms are similar to other prevalent diseases such as malaria and dengue. The most reliable method for diagnosing chikungunya virus is viral gene detection by RT-PCR. Alternative methods like detecting human antibody and viral antigen can also be used, especially in areas where resources are limited. In this review, we summarize the limited data on antigen detection immunoassays. We further explain the essential structural elements of the virus to help comprehend the scientific concepts underlying the testing methods, as well as future methods and diagnostic approaches under investigation.

Development of a novel real-time RT-PCR method using peptide nucleic acid (PNA) probes for detecting and genotyping of viral haemorrhagic septicaemia virus (VHSV)

Viral haemorrhagic septicaemia (VHS) is one of the most serious viral diseases in salmonid and olive flounder farms. The causative agent of VHS is the VHS virus (VHSV), which has been classified into four genotypes (I–IV), based on sequence analysis of the genes encoding for nucleoprotein, glycoprotein, and non-structural viral protein. Among the various diagnostic methods, real-time reverse transcription PCR method based on TaqMan-probe (RT-qPCR) is a stable, rapid, specific, and highly sensitive method for viral gene detection. However, the currently accepted diagnostic method based on RT-qPCR can only detect viral presence and load, and does not provide information about viral genotype. Peptide nucleic acids (PNAs) are artificially synthesized DNA analogues with an uncharged peptide backbone. PNA probes can effectively detect a target gene by amplification and a specific melting temperature signal. It was reported that PNA probes can effectively distinguish between mismatched sequences based on their different melting temperatures in amplified PCR products. The present study reports a novel real-time RT-PCR method for simultaneous detection and genotyping of VHSV using PNA probes. The newly-developed method showed a sensitivity similar to that of the infectious titre by fish cell cultures inoculated with the virus, except for genotype IVa, where viral inoculation in cell culture showed a 10-fold higher sensitivity than the novel method. The melting point analysis to distinguish the four genotypes was performed on 80 VHSV isolates representing all known genotypes, showing that this novel real-time RT-PCR can distinguish between all VHSV genotypes without the need of further sequencing.

Prognostic Prediction for Therapeutic Effects of Mutian on 324 Client-Owned Cats with Feline Infectious Peritonitis Based on Clinical Laboratory Indicators and Physical Signs.

Feline infectious peritonitis (FIP) is a fatal disease classified as either effusive, non-effusive ('dry'), or a mixture ('mixed') of the forms of FIP, with mixed showing signs of both effusive and dry. To determine whether the therapeutic effect of Mutian on dry and mixed FIP can be predicted using clinical indicators before starting treatment, we entered 161 cats with mixed FIP and 163 cats with dry FIP into this study. Physical assessments, the reverse transcriptase-PCR detection of viral genes, and clinical laboratory tests (hematocrit, albumin/globulin ratio, serum amyloid A, α1-acid glycoprotein, and total bilirubin) were performed before Mutian was administered. These indicators were compared between the FIP groups that survived after receiving Mutian for 84 days and those that died before the completion of treatment. Significant differences in body temperature, appetite, and activity scores were confirmed between the surviving and non-surviving groups. The therapeutic effect was insufficient when total bilirubin levels increased in cats with the mixed form. In both of the FIP types, therapeutic effects were difficult to obtain when neurological clinical signs were observed. The therapeutic effects of Mutian on the cats with dry and mixed FIP can be predicted based on pre-treatment body temperature, appetite scores, and activity scores, as well as the presence of neurological signs.

Research Note: Evaluation of the cross protective efficacy of a subtype B aMPV vaccine against virulent subtype A aMPV by different administration routes

Avian metapneumovirus (aMPV) causes respiratory and reproductive diseases in birds, including chickens. In the chicken industry, live vaccines against aMPV subtypes A and B, which are the major aMPV subtypes, are widely used to control disease caused by aMPV. In this study, we evaluated the cross protective efficacy of a live aMPV subtype B vaccine administered via 3 different routes (nasal, spray, and oral) against virulent aMPV subtype A in chickens. At 3 wk after vaccination of 1-wk-old specific-pathogen-free chickens, we measured the serological responses. On the same day, we challenged the birds with aMPV subtype A. Protection was evaluated by viral gene detection and histopathological examination at 3 and 5 days postchallenge. Although there were differences in the serological responses according to administration route, all vaccinated birds showed complete protection at 5 days postchallenge. Regardless of administration route, genome of challenge virus was not detected in vaccinated group, and there were significant differences between vaccinated birds and control group. Overall, our results demonstrated that a subtype B aMPV vaccine can provide cross protection against virulent subtype A aMPV in chickens.

Comparison of the SARS-CoV-2 Rapid Antigen Test with Real-Time RT-PCR Assay for Laboratory Diagnosis of COVID-19 in Bangladesh

Background: The authenticity and importance of RT-PCR testing cannot be overstated, for resource-limited settings like Bangladesh, especially in places where RT -PCR facilities are unavailable, rapid antigen testing could be an important supportive tool in SARS-CoV-2 diagnosis.
 Objective: The aim of this study was to compare rapid SARS-CoV-2 antigen detection test with RT-PCR for viral gene detection assay.
 Methodology: This cross-sectional study was performed at National Institute of Laboratory Medicine and Referral Center (NILMRC), Dhaka between March and April 2021. The nasopharyngeal swab samples were obtained from COVID-19 suspected cases collected from NILMRC virology lab. RT-PCR testing was conducted by Novel Coronavirus Nucleic Acid Diagnostic Kit. Rapid antigen testing was conducted using Standard Q COVID-19 Ag test.
 Results: The median age was 35.2 years. Among the confirmed cases, 63.0% were male patients. A total of 68 samples came out as positive and 226 were negative using both methods. Additionally, four more positive cases were detected by the Rapid Antigen Testing method. The sensitivity and specificity of the rapid antigen test was found to be 94.0% and 99.0% respectively.
 Conclusions: Rapid antigen test and rt-PCR showed almost similar sensitivity and specificity.
 Bangladesh Journal of Infectious Diseases, April 2022;9(Suppl_1): S24-S27

Enhanced Specificity in Loop-Mediated Isothermal Amplification with Poly(ethylene glycol)-Engrafted Graphene Oxide for Detection of Viral Genes.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method that allows the simple, quick, and low-cost detection of various viral genes. LAMP assays are susceptible to generating non-specific amplicons, as high concentrations of DNA primers can give rise to primer dimerization and mismatched hybridizations, resulting in false-positive signals. Herein, we reported that poly(ethylene glycol)-engrafted nanosized graphene oxide (PEG-nGO) can significantly enhance the specificity of LAMP, owing to its ability to adsorb single-stranded DNA (ssDNA). By adsorbing surplus ssDNA primers, PEG-nGO minimizes the non-specific annealing of ssDNAs, including erroneous priming and primer dimerization, leading to the enhanced specificity of LAMP. The detection of complementary DNAs transcribed from the hepatitis C virus (HCV) RNA was performed by the PEG-nGO-based LAMP. We observed that the inclusion of PEG-nGO significantly enhances the specificity and sensitivity of the LAMP assay through the augmented difference in fluorescence signals between the target and non-target samples. The PEG-nGO-based LAMP assay greatly facilitates the detection of HCV-positive clinical samples, with superior precision to the conventional quantitative real-time PCR (RT-qPCR). Among the 20 clinical samples tested, all 10 HCV-positive samples are detected as positive in the PEG-nGO-based LAMP, while only 7 samples are detected as HCV-positive in the RT-qPCR. In addition, the PEG-nGO-based LAMP method significantly improves the detection precision for the false-positive decision by 1.75-fold as compared to the LAMP without PEG-nGO. Thus, PEG-nGO can significantly improve the performance of LAMP assays by facilitating the specific amplification of target DNA with a decrease in background signal.

A review on corona virus disease 2019 (COVID-19): current progress, clinical features and bioanalytical diagnostic methods.

A new epidemic of acute respiratory viral pneumonia was discovered in central China at the end of 2019. The disease was given the name coronavirus disease 2019 (COVID-19), and the virus that caused this disease was known as severe acute respiratory syndrome coronavirus (SARS-CoV-2). So far, diagnostic methods have been focused on (a) human antibody detection, (b) viral antigen detection and (c) viral gene detection, the latter using RT-PCR being the most accurate approach. In this paper, we present a summary of the COVID-19 pandemic, clinical features and epidemiology and pathogenesis. Also, we focus on the recent advances in bioanalytical diagnostic methods based on various techniques for SARS-CoV-2 sensing that have recently been published (2020–2021). Furthermore, we present the mechanisms, advantages and disadvantages of the most common biosensors for COVID-19 detection, which include optical, electrochemical and piezoelectric biosensors as well as wearable and smart nanobiosensors, immunosensors, aptasensors and genosensors.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00604-022-05167-y.

RNA-extraction-free diagnostic method to detect SARS-CoV-2: an assessment from two states, India

AbstractWith increasing demand for large numbers of testing during the coronavirus disease 2019 pandemic, alternative protocols were developed with shortened turn-around time. We evaluated the performance of such a protocol wherein 1138 consecutive clinic attendees were enrolled; 584 and 554 respectively from two independent study sites in the cities of Pune and Kolkata. Paired nasopharyngeal and oropharyngeal swabs were tested by using both reference and index methods in a blinded fashion. Prior to conducting real-time polymerase chain reaction, swabs collected in viral transport medium (VTM) were processed for RNA extraction (reference method) and swabs collected in a dry tube without VTM were incubated in Tris–EDTA–proteinase K buffer for 30 min and heat-inactivated at 98 °C for 6 min (index method). Overall sensitivity and specificity of the index method were 78.9% (95% confidence interval (CI) 71–86) and 99% (95% CI 98–99.6), respectively. Agreement between the index and reference method was 96.8% (k= 0.83,s.e.= 0.03). The reference method exhibited an enhanced detection of viral genes (E, N and RNA-dependent RNA polymerase) with lower Ct values compared to the index method. The index method can be used for detecting severe acute respiratory syndrome corona virus-2 infection with an appropriately chosen primer–probe set and heat treatment approach in pressing time; low sensitivity constrains its potential wider use.

SARS-CoV-2: Comparative analysis of different RNA extraction methods

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection.

COVID-19 diagnosis —A review of current methods

A fast and accurate self-testing tool for COVID-19 diagnosis has become a prerequisite to comprehend the exact number of cases worldwide and to take medical and governmental actions accordingly. SARS-CoV-2 (formerly, 2019-nCoV) infection was first reported in Wuhan (China) in December 2019, and then it has rapidly spread around the world, causing ~14 million active cases with ~582,000 deaths as of July 2020. The diagnosis tools available so far have been based on a) viral gene detection, b) human antibody detection, and c) viral antigen detection, among which the viral gene detection by RT-PCR has been found as the most reliable technique. In this report, the current SARS-CoV-2 detection kits, exclusively the ones that were issued an “Emergency Use Authorization” from the U.S. Food and Drug Administration, were discussed. The key structural components of the virus were presented to provide the audience with an understanding of the scientific principles behind the testing tools. The methods that are still in the early research state were also reviewed in a subsection based on the reports available so far.

Epidemiological Survey of Severe Fever with Thrombocytopenia Syndrome Virus in Ticks in Nagasaki, Japan

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease endemic in East Asia. Transmitted to other organisms by infected ticks, the SFTS virus (SFTSV) and is endemic to Nagasaki in western Japan. However, epidemiological information regarding SFTSV in Nagasaki ticks has not been available to date. In this study, we began by examining the sensitivities of SFTSV gene detection by real-time RT-PCR and virus isolation in cultured cells and mice. These methods could detect SFTSV in the samples containing more than 4 × 100 ffu. Next, we attempted to isolate SFTSV and to detect viral gene in 2,222 nymph and adult ticks collected from May to August 2013 among seven regions of Nagasaki. However, neither virus isolation nor viral gene detection were confirmed in the tick pools. SFTSV positivity rates are considered to be very low in ticks, and viral loads are also very limited. Further investigations increasing the number of ticks and including larval samples as well as improved detection methods, may be required to find SFTSV-positive ticks in this region.

Development and evaluation of SYBR Green-I based quantitative PCR assays for herpes simplex virus type 1 whole transcriptome analysis

There is an emerging need for viral gene specific quantitative PCR (qPCR) assays that validate and complement whole transcriptome level technologies, including microarray and next generation sequencing. Therefore, a compilation of qPCR assays that represented the breadth of the entire Herpes simplex virus type 1 (HSV-1) genome were developed and evaluated. SYBR Green-I-based quantitation of each of the 74 HSV-1 lytic genes enabled accurate and reproducible detection of viral genes using a minimal number of reaction conditions. The amplification specificity of these assays for HSV-1 target genes was confirmed by amplicon size and purity determination on agarose gels, melt temperature dissociation curve analysis, and direct DNA sequencing of amplified products. Analysis of representative target genes demonstrated that these assays accurately and reproducibly quantified target gene expression across a wide and linear range of detection. In addition, minimal intra- and inter-assay variability was observed with significant well-to-well and plate-to-plate/assay-to-assay precision. To evaluate the utility of the developed qPCR assay system, kinetic profiles of viral gene expression were determined for an array of representative genes from all HSV-1 transcriptional gene classes. Collectively, these data demonstrate that the compiled optimized qPCR assays is a scalable and cost-effective method to assess HSV-1 gene expression with broad application potential, including investigation of pathogenesis and antiviral therapies. In addition, they can be employed to validate and complement evolving technologies for genome-wide transcriptome analysis.

Investigation of an Idiopathic Lesion in Redclaw Crayfish Cherax Quadricarinatus Using Suppression Subtractive Hybridization

Lesions indicative of putative parvovirus infection were detected in a population of freshwater redclaw crayfish ( Cherax quadricarinatus ) displaying hypertrophied nuclei, while these changes were undetected in another population. These changes were characterised by hypertrophic nuclei with marginated chromatin but without Cowdry type A (CA) intranuclear inclusion bodies. Suppression subtractive hybridization (SSH) was performed to generate a cDNA forward library between the two populations of crayfish. A total of 323 sequences were analysed from SSH libraries. These sequences were grouped into 76 consensus sequences and clustered into 8 categories based on sequence homology from the NCBI GenBank database. Even though histopathological observations consistently revealed lesions presumptive of putative parvovirus in C. quadricarinatus as previously reported, the molecular method failed to confirm a viral aetiology. Despite the absence of viral gene detection and therefore a conclusion of an idiopathic aetiology, the health status of these two populations of redclaw crayfish was different, resulting in differentially expressed immune-related genes in the two populations, with some genes being up-regulated in the hypertrophied nuclei population. Furthermore, the absence of parvovirus from the SSH libraries indicates that perhaps the viral messenger RNA was in too low a concentration to be detected or does not have any poly(A) tail which the SSH methodology needs to function correctly.

SPF 닭에서 레오바이러스와 아데노바이러스의 단일 혹은 혼합감염에 따른 병원성 비교

Avian reovirus (ARV) and fowl adenovirus (FAdV) were evaluated for pathogenicity in specific pathogen free (SPF) chickens. ARV was isolated from the broilers with history of malabsorption syndrome (MAS). FAdV was isolated from the layer breeders with inclusion body hepatitis and hydropericardium syndrome. Total 6 inoculated groups including 1 un-inoculated group were organized and inoculated with the ARV and/or FAdV by oral route. The minimal pathological lesions and lower viral gene detection rates were present in the ARV inoculated groups compared to those of FAdV or ARV/FAdV inoculated groups. Common gross lesions in the ARV inoculated group were distended intestine with foamy contents and in the FAdV group there were foamy cecal contents and hydropericardium among the evaluation methods such as gross and histological lesion, viral gene detection, body weight and serum chemistry, histopathological lesion score was reliable especially in the liver lesions such as hepatic necrosis and lymphocytic infiltration. However, we did not success to evaluate the synergetic effect of mixed infection of ARV and FAdV in this study. Therefore, we need further study to reproduce malabsorption syndrome of ARV infection using different viral agent such as rotavirus and using different dose of virus.

Fabrication and Use of Electroless Plated Polymer Surface-Enhanced Raman Spectroscopy Substrates for Viral Gene Detection

Surface-enhanced Raman spectroscopy (SERS) substrates are prepared by electroless Ag metallization and vapor phase Au deposition on nanostructured poly(chloro-p-xylylene) (nanoPPX) templates. These substrates exhibit quasi-periodic nanomorphology inherited from the underlying nanoPPX template, resulting in highly reproducible SERS signal (<10% spot-to-spot and substrate-to-substrate signal variation for Au/nanoPPX comprising contributions from substrate imperfections). These substrates are therefore chosen for developing respiratory syncytial virus (RSV) gene detection that requires high sensitivity, stability, and reproducibility of the Raman signal. Metallized nanoPPX films show enhancement factors of ∼104 to 106 that strongly depend on the metallization route and can be optimized by controlling the metallization parameters. RSV gene detection is achieved by using a molecular probe (MP) consisting of a fluorescent moiety and a thiol linker for attachment to the SERS substrate. To detect multiple targets, MPs are designed in two colors (Hex and Cy5 dyes) utilizing a broad range of fluorophores. Our approach provides reproducible dual-mode detection (i.e., fluorescent and SERS) where the assay results generated by fluorescence and SERS are self-confirmatory and eliminate false positives.

Viral Gene Detection in Oral Neoplasms Using the Polymerase Chain Reaction

Eighty-four squamous cell proliferative lesions of the oral cavity and six examples of normal oral squamous mucosa were investigated for the presence of human papillomavirus (HPV) by the polymerase chain reaction (PCR) using universal primers and type specific probes. Human papillomavirus was identified in 1 of 6 histologically normal biopsy specimens, 2 of 4 hyperplasias, 2 of 5 inflammatory lesions, 9 of 36 cases of dysplasia or carcinoma in situ, and 7 of 39 carcinomas. All of the specimens found positive for HPV using the PCR were confirmed using slot blot hybridizations. All HPV-positive cases contained HPV 16. In addition, 2 were co-infected with HPV 11 and 1 was co-infected with HPV 18. Types 6 and 33 were not found. The same genotypes that infect the uterine cervix also were found in the oral cavity. This is the first time universal primers that allow for the detection of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 45, 51-59, and at least 25 other novel types have been used on oral tumors, and represents the largest number of oral samples analyzed by the PCR at this time. Due to lack of clear correlation between HPV infection and histologic findings, however, the role of this virus in oral carcinogenesis remains uncertain.