BACKGROUND: Up to 80% of the US adult population has been exposed to herpes simplex virus (HSV) type 1, primarily during childhood. Also, approximately 20% of the US population has contracted genital herpes from HSV-2 infections. Clinical symptoms can present as fever, headache, malaise, myalgia, and cold sores/lesions that cause pain, itching, dysuria, and vaginal or urethral discharge. A recurrence of infection is common. HSV culturing is characterized by low sensitivity with variable success rates due to shipping conditions. OBJECTIVE: To design and validate a real-time PCR assay capable of simultaneously detecting each HSV subtype. STUDY DESIGN: ATCC-purchased HSV-1 and HSV-2 positive samples and HSV-1 and HSV-2 infected clinical specimens were assayed simultaneously with shared amplification primers and subtype-specific probes against the HSV glycoprotein B gene on a Rotor-Gene 3000 platform. Separately, two PCR reactions were performed in which one primer contained a 5' biotin modification. Single-stranded DNA from the amplicon was purified and Pyrosequenced. RESULTS: The quantitative range of the assay extended from 10(8) through 10(0) copies of each virus (r(2) > 0.991) and specificity was determined by non-amplification of 37 different human pathogens, including other herpesviruses such as VZV, CMV, and EBV. Sensitivity and specificity values of 100% were calculated by concordance analysis between the real-time PCR and the DNA Pyrosequencing results (HSV-1: n = 119, HSV-2: n = 120). Application of this assay to 4581 cervical swab specimens collected from women visiting physicians primarily in six states provided detection rates of 3.1% for HSV-1 and 7.6% for HSV-2. The average age of women infected with HSV-1 was 29.5 versus 35.6 for HSV-2. CONCLUSIONS: This procedure was demonstrated as both highly sensitive and specific for the detection of HSV-1 and HSV-2 in a single reaction. Also, the integration of Pyrosequencing analysis permitted an innovative and rapid verification for each subtype.
BACKGROUND: Up to 80% of the US adult population has been exposed to herpes simplex virus (HSV) type 1, primarily during childhood. Also, approximately 20% of the US population has contracted genital herpes from HSV-2 infections. Clinical symptoms can present as fever, headache, malaise, myalgia, and cold sores/lesions that cause pain, itching, dysuria, and vaginal or urethral discharge. A recurrence of infection is common. HSV culturing is characterized by low sensitivity with variable success rates due to shipping conditions. OBJECTIVE: To design and validate a real-time PCR assay capable of simultaneously detecting each HSV subtype. STUDY DESIGN: ATCC-purchased HSV-1 and HSV-2 positive samples and HSV-1 and HSV-2 infected clinical specimens were assayed simultaneously with shared amplification primers and subtype-specific probes against the HSV glycoprotein B gene on a Rotor-Gene 3000 platform. Separately, two PCR reactions were performed in which one primer contained a 5' biotin modification. Single-stranded DNA from the amplicon was purified and Pyrosequenced. RESULTS: The quantitative range of the assay extended from 10(8) through 10(0) copies of each virus (r(2) > 0.991) and specificity was determined by non-amplification of 37 different human pathogens, including other herpesviruses such as VZV, CMV, and EBV. Sensitivity and specificity values of 100% were calculated by concordance analysis between the real-time PCR and the DNA Pyrosequencing results (HSV-1: n = 119, HSV-2: n = 120). Application of this assay to 4581 cervical swab specimens collected from women visiting physicians primarily in six states provided detection rates of 3.1% for HSV-1 and 7.6% for HSV-2. The average age of women infected with HSV-1 was 29.5 versus 35.6 for HSV-2. CONCLUSIONS: This procedure was demonstrated as both highly sensitive and specific for the detection of HSV-1 and HSV-2 in a single reaction. Also, the integration of Pyrosequencing analysis permitted an innovative and rapid verification for each subtype.
Authors: Joanna S Ellis; Joanne W Smith; Sharleen Braham; Matthew Lock; Katrina Barlow; Maria C Zambon Journal: J Clin Microbiol Date: 2007-03-14 Impact factor: 5.948
Authors: Blanca L Valle; Sebastian Rodriguez-Torres; Elisabetta Kuhn; Teresa Díaz-Montes; Edgardo Parrilla-Castellar; Fahcina P Lawson; Oluwasina Folawiyo; Carmen Ili-Gangas; Priscilla Brebi-Mieville; James R Eshleman; James Herman; Ie-Ming Shih; David Sidransky; Rafael Guerrero-Preston Journal: Cancer Prev Res (Phila) Date: 2020-06-24
Authors: Shadi Shokralla; Xin Zhou; Daniel H Janzen; Winnie Hallwachs; Jean-François Landry; Luke M Jacobus; Mehrdad Hajibabaei Journal: PLoS One Date: 2011-07-27 Impact factor: 3.240
Authors: Svetlana V Shcherbik; Nicholas C Pearce; Marnie L Levine; Alexander I Klimov; Julie M Villanueva; Tatiana L Bousse Journal: PLoS One Date: 2014-03-19 Impact factor: 3.240