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      Development of field-applicable endogenous internally controlled recombinase-aided amplification (EIC-RAA) assays for the detection of human papillomavirus genotypes 6 and 11 using sample releasing agent

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          Abstract

          Objective

          Human papillomavirus (HPV) 6 and 11 are the two most common low-risk HPV subtypes, accounting for more than 90% of condyloma acuminatum. A simple, accurate and rapid screening method to be applied in community-level hospitals is in high demand.

          Methods

          Endogenous internally controlled recombinase-assisted amplification (EIC-RAA) assays for HPV6 and 11 were performed in a single closed-tube at 39 °C within 30 min. The sensitivity and specificity of EIC-RAA were examined using recombinant plasmids and pre-tested HPV DNA. A total of 233 clinical samples were collected, and the DNA was extracted by traditional multi-step extraction, or sample releasing agent, before analysis by EIC-RAA. For comparison, HPV detection via Quantitative real-time PCR (qPCR) was also performed.

          Results

          The sensitivity of EIC-RAA analysis was 10 copies/reaction for HPV6, 100 copies/reaction for HPV11, and 100 copies/reaction for the human β-globin gene. No cross-reaction was observed with other HPV subtypes. Clinical performance of the EIC-RAA assay achieved a 100% of concordance rate with the commercial HPV qPCR kit. Further, the EIC-RAA assay achieved a 100% of concordance rate when using multi-step extracted DNA and sample releasing agent-processed DNA.

          Summary

          The EIC-RAA assay for HPV6 and 11 detection possesses the advantages of accuracy, simplicity and rapidity, and demonstrates great potential to be used in community-level hospitals for field investigation.

          Highlights

          • •

            The EIC-RAA assays for HPV6 and 11 possess high sensitivity and specificity.

          • •

            The endogenous human β-globin can effectively eliminate false-negatives or invalid amplification results, increasing the reliability of the EIC-RAA assay.

          • •

            The EIC-RAA assays for the detection of HPV 6 and 11 using sample releasing agent show the advantages of fast detection speed and simple operation.

          Abstract

          Human papillomavirus 6 and 11; Human β-globin gene; Endogenous internally controlled recombinase-assisted amplification; Sample releasing agent.

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          Most cited references25

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          Both beta-actin and GAPDH are useful reference genes for normalization of quantitative RT-PCR in human FFPE tissue samples of prostate cancer.

          QINGCAI WANG, Peter V Danenberg, Kathleen Danenberg … (2008)
          Beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been frequently considered as constitutive house keeping genes for RT-PCR and used to normalize changes in specific gene expressions. However, these expressions have been shown to be affected by the sample type and experimental conditions. We investigated which housekeeping gene is useful to study gene expression of paraffin embedded human tissue samples of prostate cancer. Fifteen pairs of cancer and corresponding normal tissue were obtained from patients with prostate cancer. We evaluated gene expression of beta-actin, GAPDH, androgen receptor (AR), and heat-shock 70-kd protein 5 (HSPA5) using laser captured microdissection and quantitative RT-PCR. AR and HSPA5 gene expression were normalized to each of these reference genes using the 2(-DeltaDeltaCt) method of relative quantification. The quantity 2(Ct(normal)-Ct(cancer)) divided by ratio of cDNA(cancer)/cDNA (normal) was used for comparing differences between cancer and normal tissue in GAPDH and beta-actin expression. Ct value of beta-actin was significantly correlated with that of GAPDH (r = 0.443, P = 0.014). AR and HSPA5 gene expression levels using beta-actin for normalization were significantly correlated with these gene expression levels using GAPDH (AR; r = 0.689, P = 0.004, HSPA5; r = 0.879, P < 0.001). Both reference genes were expressed more highly in cancer tissue than in normal tissue, with that of GAPDH being significantly different between cancer tissue and normal tissue (P = 0.029). The good correlation between gene expression values obtained when using beta-actin and GAPDH as reference genes suggests that either gene is a valid denominator for gene expression studies in prostate cancer.
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            A rapid and sensitive recombinase aided amplification assay to detect hepatitis B virus without DNA extraction

            Xin-xin Shen, Fang-zhou Qiu, Li-Ping Shen … (2019)
            Background Hepatitis B virus (HBV) infection is the major public health problem worldwide. In clinical practice, serological and molecular assays are the most commonly used diagnostic methods to detect HBV infection in clinical practices. Methods Here we present a rapid and sensitive recombinase aided amplification assay (RAA) to detect HBV at 39.0 °C for 30 min without DNA extraction from serum samples. The analytical sensitivity of RAA assay was 100 copies per reaction and showed no cross reaction with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The universality of RAA assay was validated by testing of 41 archived serum samples with predefined HBV genotypes (B, C and D). Results A total of 130 archived suspected HBV infected serum samples were detected by commercial qPCR with DNA extraction and RAA assay without DNA extraction (heat-treatment). Compared with qPCR assay as a reference, the RAA assay obtained 95.7% sensitivity and 100% specificity and a kappa value of 0.818. Conclusions We developed a rapid, convenient, highly sensitive and specific method to detect HBV without DNA extraction in clinical samples. This RAA method of HBV detection is very suitable for clinical testing.
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              Loop‐mediated isothermal amplification method for detection of human papillomavirus type 6, 11, 16, and 18

              Masanori Hagiwara, Hajime Sasaki, Koma Matsuo … (2007)
              Abstract A new method was developed for detection of human papillomavirus (HPV) by loop‐mediated isothermal amplification (LAMP), which was compared with the polymerase chain reaction (PCR), and real‐time PCR for specificity and sensitivity. All initial validation studies with the control DNA proved to be type‐specific. In order to evaluate the reliability of HPV type‐specific LAMP detecting HPV DNA from clinical samples, tissue specimens were obtained from 27 patients with external genital polypoid lesions. The histologic diagnoses included condyloma acuminatum (n = 21), bowenoid papulosis (n = 2), seborrheic keratosis (n = 2), epidermolytic acanthoma (n = 1), and hairy nymphae (n = 1). HPV‐6 DNA and HPV‐11 DNA were detected in 18 and 3 of 21 condylomata acuminata, respectively, and there was no simultaneous infection. HPV‐16 DNA was detected in one of two bowenoid papuloses. HPV DNA was not detected in the seborrheic keratoses, epidermolytic acanthoma, and hairy nymphae. These results correlated perfectly with those from real‐time PCR analysis. Most positive samples contained high copy numbers of HPV DNA. HPV‐11 DNA was detected in one case that could not be detected by PCR. The average reaction time was about 59 min. There was a linear correlation between the genome quantity and reaction time to reach the threshold. The LAMP method has an additional advantage as a quantitative method, and is superior in terms of sensitivity, specificity, rapidity, and simplicity, and can potentially be a valuable tool for the detection of HPV DNA. J. Med. Virol. 79:605–615, 2007. © 2007 Wiley‐Liss, Inc.
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                Author and article information

                Contributors
                Xinxin Shen
                Xiuge Rong
                Xuejun Ma
                Journal
                Journal ID (nlm-ta): Heliyon
                Journal ID (iso-abbrev): Heliyon
                Title: Heliyon
                Publisher: Elsevier
                ISSN (Electronic): 2405-8440
                Publication date PMC-release: 02 November 2022
                Publication date Collection: November 2022
                Publication date (Electronic): 02 November 2022
                Volume: 8
                Issue: 11
                Electronic Location Identifier: e11323
                Affiliations
                [a ]North China University of Science and Technology, Tangshan, 063210, China
                [b ]Tangshan Gongren Hospital, Tangshan, 063003, Hebei, China
                [c ]NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Changbai Street, Changping District, Beijing 102206, China
                [d ]Qiqihar Medical University, Qiqihar, Heilongjiang, 161000, China
                [e ]Hebei General Hospital, Shijiazhuang, 050051, Hebei, China
                Author notes
                [∗ ]Corresponding author. maxj@ 123456ivdc.chinacdc.cn
                [∗∗ ]Corresponding author. gyrongxiuge@ 123456163.com
                [∗∗∗ ]Corresponding author. x616815@ 123456126.com
                [1]

                Anna He, Cheng Fang and Yue Ming contributed equally to this work.

                Article
                Publisher Item ID: S2405-8440(22)02611-1 Publisher ID: e11323
                DOI: 10.1016/j.heliyon.2022.e11323
                PMC ID: 9647352
                PubMed ID: 36387484
                SO-VID: e69db939-8c8d-450d-b723-7d3ffc4899da
                Copyright © © 2022 The Author(s)
                License:

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                Date received : 1 April 2022
                Date revision received : 25 June 2022
                Date accepted : 24 October 2022
                Categories
                Subject: Research Article

                Keywords: human papillomavirus 6 and 11,human β-globin gene,endogenous internally controlled recombinase-assisted amplification,sample releasing agent
                Data availability:
                Keywords: human papillomavirus 6 and 11, human β-globin gene, endogenous internally controlled recombinase-assisted amplification, sample releasing agent

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