Detection assay for HPV16 and HPV18 by loop‑mediated isothermal amplification with lateral flow dipstick tests

  • Authors:
    • Ratchanida Kumvongpin
    • Patcharee Jearanaikoon
    • Chotchana Wilailuckana
    • Nattaya Sae‑Ung
    • Prinya Prasongdee
    • Sakda Daduang
    • Metee Wongsena
    • Patcharee Boonsiri
    • Wansika Kiatpathomchai
    • Sukumarn Sanersak Swangvaree
    • Alisa Sandee
    • Jureerut Daduang
  • View Affiliations

  • Published online on: March 23, 2017     https://doi.org/10.3892/mmr.2017.6370
  • Pages: 3203-3209
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Abstract

Cervical cancer is the third highest cause of death in developing countries and most commonly results from high‑risk human papillomavirus (HR‑HPV) infection. Among HR‑HPV genotypes, HPV16 and HPV18 are the most prevalent in cervical cancers. Therefore, the present study aimed to develop a detection assay for HPV16 and HPV18 infection using loop‑mediated isothermal amplification (LAMP) with lateral flow dipstick (LFD) tests. This assay is a simplified, user‑friendly method for the visual detection of HPV genotypes. DNA was extracted from clinical tissue samples, and HPV genotyping was performed using nested polymerase chain reaction (PCR). The clinical samples were demonstrated to include 44 HPV16‑positive, 18 HPV18‑positive and 80 HPV‑negative samples. All DNA samples were also used as templates for a LAMP reaction (30 min at 65˚C), and subsequently, a fluorescein isothiocyanate‑labelled probe was hybridized with the reaction product. Finally, the LFD test was performed. The sensitivity of the LAMP‑LFD test was higher than LAMP‑turbidity, exhibiting up to 100‑fold higher sensitivity for HPV16 and 10‑fold higher sensitivity for HPV18. All HPV16 and HPV18‑positive samples generated positive results in both assays; however, 22 samples detected as HPV‑negative by LAMP‑turbidity exhibited positive results by LAMP‑LFD test (22 of 80 samples). Therefore, these samples were further examined using quantitative (q)PCR. The results demonstrated that 20 out of the 22 samples designated positive by LAMP‑LFD, but negative by LAMP turbidity, gave a positive result with qPCR, while the remaining 2 samples were negative by qPCR. The present results suggested that LAMP‑LFD provided higher sensitivity than LAMP‑turbidity and nested PCR. Thus, the LAMP‑LFD test developed in the present study might be useful for the detection of HPV16 and HPV18 in local hospitals.

Introduction

Cervical cancer is the fourth highest cause of death from cancer in women worldwide and the third highest cause of death in Thailand and other developing countries (1). High-risk human papillomavirus (HR-HPV) leads to nearly all cases of cervical cancer, and among HR-HPV genotypes, HPV16 and HPV18 are the most prevalent. HPV16 exhibits the highest frequency as the cause of cervical cancers in women in Thailand and worldwide (24). Many types of commercial HPV tests are available. Most commercial tests are designed to detect the presence of DNA from high-risk HPV in patient samples. For example, signal amplification methods, including Hybrid Capture 2 and Cervista are used for the detection of HPV DNA using an RNA-DNA hybridization probe and chemiluminescence or fluorescence respectively for signal amplification and detection. However, these methods do not detect non-amplified HPV DNA or identify specific HPV genotypes (5). Target amplification using polymerase chain reaction (PCR) is the most outstanding target amplification method, using oligonucleotide primers and thermocycling to amplify DNA. PCR methods offer high sensitivity and specificity in the detection of HPV genotypes (6). However, special devices are required to perform these methods, which are often time consuming and costly.

Loop-mediated isothermal amplification (LAMP), an alternative method for nucleotide amplification under isothermal conditions, has been described previously (7). The LAMP method for HPV genotyping has been successfully developed using turbidity (8). However, observing turbidity using the naked eye is not practical and might be difficult, particularly for the identification of low copy DNA (9).

Lateral flow dipstick (LFD) tests are routinely used for the detection of biological infectious agents and chemical contaminants, including bacteria, viruses, toxins, veterinary drugs and pesticides (10). The interpretation of LFD tests is easy without external instrumentation (11). In the present study, LAMP and LFD methods were combined in order to develop a simple assay for the detection of HPV16 and HPV18 with high sensitivity and high specificity and the LAMP-LFD novel assay was evaluated against the nested PCR assay (a gold standard) using clinical samples of known HPV genotype.

Materials and methods

Samples and DNA extraction

Clinical samples (142 cervical tissues, including 44 HPV16-positive, 18 HPV18-positive and 80 HPV-negative samples) were collected from Ubon Ratchathani Cancer Hospital (Ubon Ratchathani, Thailand) during the year 2015. The human research ethics committee of Ubon Ratchathani Cancer Hospital approved the protocol (EC04/2015). DNA was extracted from all clinical samples using the ExiPrep Dx Viral DNA Kit (Bioneer Corporation, Daejeon, Korea) according to the manufacturer's protocol, and the initial detection of DNA samples for high-risk HPV genotyping was performed using nested PCR according to Sotlar et al (12). All DNA samples were stored at −20°C until further use.

LAMP primers and LAMP conditions

The LAMP primer sets were obtained from a previous study (8). These primer sets (listed in Table I) comprised a 5′biotin-labelled forward inner primer (FIP; termed here as BIP), outer primers (F3, B3) and loop primers (LF, LB), synthesized at Pacific Science Co, Ltd. (Bangkok, Thailand). The optimal conditions for the LAMP method were determined after assessing varying reaction temperatures and times. The 25 µl reaction mixture contained 1.6 µM of each inner primer, 0.2 µM of each outer primer, 0.8 µM of each loop primer, 1.4 mM dNTPs (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 0.8 mM betaine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 6 mM MgSO4, 8 units of Bst 2.0 DNA polymerase (New England BioLabs, Inc., Ipswich, MA, USA), 1x Bst buffer (New England BioLabs, Inc.) and 3 µl of HPV16 and HPV18 plasmid DNA at 104 copies (provided by Professor Ethel-Michele de Villiers, The German Cancer Research Centre, Heidelberg, Germany) (13). The reaction was conducted at different temperatures (60, 63 and 65°C) for 10, 20, 30, 40, 50 and 60 min.

Table I.

Primer sets for LAMP and probe sequences for the LAMP-LFD detection of HPV16 and HPV18.

Table I.

Primer sets for LAMP and probe sequences for the LAMP-LFD detection of HPV16 and HPV18.

PrimerPrimer sequence (5′-3′)Genome position
HPV16
  F3 TCGGTTGTGCGTACAAAG756–773
  B3 AGCCTCTACATAAAACCATCC933–913
  FIP Biotin-TGGGGCACACAATTCCTAGT-CACACACGTAGACATTCGT 838-819/TTTT/774–792
  BIP TCAGAAACCATAATCTACCATGGC-ATTACATCCCGTACCCTCTT 846-869/TTTT/912–893
  LF CCCATTAACAGGTCTTCCAAAGT815–793
  LB CCTGCAGGTACCAATGGGG874–892
  Probe FITC-TACCAATGGGGAAGAGGGTA882–901
HPV18
  F3 TCAGAGGAAGAAAACGATGA689–708
  B3 GTTGCTTACTGCTGGGAT912–895
  FIP Biotin-GCTTCACACTTACAACACATACACAATCAACATTTACCAGCCCG 798-774/TTTT/726–744
  BIP TTGAGCTAGTAGTAGAAAGCTCAGCACGGACACACAAAGGACA 804-828/TTTT/887–870
  LF ACGTTGTGGTTCGGCTCGT763–745
  LB GCATTCCAGCAGCTGTTTCTGAAC842–865
  Probe FITC-TGAACACCCTGTCCTTTGTG861–880

[i] LAMP, loop-mediated isothermal amplification; LFD, lateral flow dipstick; HPV, human papillomavirus virus; F3-B3, outer primers; LF-LB, loop primers; FIP, forward inner primer; BIP, 5′ biotin-labelled forward inner primer.

LAMP-LFD assay conditions

The HPV16 and HPV18 LAMP products were hybridized with the appropriate DNA probe (Pacific Science Co, Ltd.) labelled with fluorescein isothiocyanate (FITC) at the 5′-end, designed according to the HPV16 and HPV18 sequences between the LB and B3 primer targets (Table I). The LAMP-LFD conditions were optimized after assessing various hybridization times and DNA probe concentrations. The reaction mixtures containing LAMP product and DNA probe (200 and 20 pmol, respectively) were incubated at 65°C for 5, 10 or 15 min. Subsequently, 8 µl of hybridized product was added to 150 µl of assay buffer in a new tube (14). An LFD strip (Milenia HybriDetect; Milenia Biotec GmbH, Giessen, Germany) was dipped into the reaction mixture for 5 min. A red-purple line was observed at the control line for all strips, which confirmed that the test was correctly operated.

Sensitivity of LAMP-turbidity and LAMP-LFD assays

To detect sensitivity limits, the HPV16 and HPV18-containing plasmid DNA, varying from 105 to 100 copies, was used as a template for the LAMP reaction. The plasmids pBR322-HPV16 and pBR322-HPV18 were kindly provided by Professor Ethel-Michele de Villiers (13). The LAMP products were detected using LAMP-turbidity (15) compared with LAMP-LFD.

Specificity of the DNA-probe LFD assay

The specificity of the DNA probe was examined using 104 copies of HPV16 and HPV18 plasmid DNA for the LAMP reaction, and subsequently, the LAMP products were detected using the LFD assay. The negative control was performed using the same test, but water was added instead of DNA.

Evaluation of clinical samples

All 142 clinical samples were examined using LAMP-turbidity (15) and LAMP-LFD assays. The sensitivity and specificity of these assays were calculated using standard formulae based on the results of nested PCR.

Use of quantitative PCR (qPCR) to examine discrepant results

All samples with inconsistent results were further analysed using qPCR. The set of primers and probes for E2/E6 of HPV16 and HPV18 were designed according to previous studies (16,17). qPCR was conducted using an Exicycler 96 system (Bioneer Corporation), as previously described (16,17), and 10 µl of the clinical samples were used as the DNA templates. The standard curves were obtained from amplification of a dilution series using 108, 107, 106, 105, 104, 103, 102 and 101 copies of HPV16 and HPV18 plasmid DNA.

Results

Design of the LAMP-LFD HPV test

The LAMP-LFD assay was designed to detect biotin-labelled LAMP products hybridized to a FITC-labelled specific DNA probe (design explained in the schematic of Fig. 1). Subsequently, the FITC-labelled specific DNA probe was recognized by a gold-labelled anti-FITC antibody. This triple-labelled complex was then trapped at a test line using avidin, generating a red-purple band (positive result). By contrast, non-LAMP products hybridized with the FITC-labelled specific probe and bound the gold-labelled anti-FITC antibody, but did not bind avidin, due to lack of biotin; therefore, this complex moved past the test line and was trapped at the control line (18).

LAMP optimal conditions

The LAMP conditions for HPV16 and HPV18 detection were optimized by assaying variable temperatures and reaction duration times, and the turbidity of the amplified product was observed using the naked eye. The results demonstrated that all three temperatures tested (60, 63 and 65°C) generated a slightly different turbidity; however, the highest turbidity was observed at 65°C (data not shown). In addition, the turbidity was initiated at 20 min, but it was difficult to observe under the naked eye. Therefore, the best set of conditions, 65°C for 30 min, was selected for the present study.

LAMP-LFD assay conditions

The specific FITC-labelled DNA probes were hybridized with the HPV16 and HPV18 LAMP products. It was determined that the best conditions for LAMP product hybridization were as follows: 20 pmol of DNA probe as hybridization input and incubation at 65°C for 15 min (data not shown). Subsequently, 8 µl of hybridization product was added to 150 µl of assay buffer in a new tube. Next, the LFD strip was dipped into this reaction mixture for 5 min, and positive result was denoted by the presence of 2 red-purple lines on LFD strips using the naked eye.

Sensitivity of LAMP-turbidity and LAMP-LFD assays

The limits of LAMP-turbidity detection for HPV16 and HPV18 were 103 and 101 plasmid copies, respectively (Figs. 2A and 3A, respectively). The LAMP-LFD assay showed a limit of detection of 101 and 100 copies of HPV16 and HPV18, respectively (Figs. 2B and 3B, respectively). Therefore, the detection limits of LAMP-LFD were ~100 and 10-fold more sensitive for the detection of HPV16 and HPV18, respectively, compared with the LAMP-turbidity method.

Specificity of the DNA-probe LFD assay

The specificity of the DNA probe was examined using 104 copies of HPV16 and HPV18 as templates for HPV18 and HPV16 LAMP, respectively. The results revealed no cross-reactivity between HPV16 and HPV18 using the LAMP-LFD assay (Fig. 4).

Evaluation of LAMP-LFD assay

All 142 clinical samples, including HPV16-positive (n=44), HPV18-positive (n=18) and HPV-negative (n=80) samples, were examined by the LAMP turbidity assay compared with the novel LAMP-LFD assay. The results from the LAMP-turbidity assay revealed that 44 and 18 samples were positive for HPV16 and HPV18, respectively, while the results from the LAMP-LFD assay revealed that 57 samples and 27 samples were positive for HPV16 and HPV18, respectively (Table II). Therefore, a higher number of samples gave positive readings with the novel LAMP-LFD assay developed in the present study than with the nested PCR, gold standard method.

Table II.

Comparison of LAMP-turbidity and LAMP-LFD assays for HPV16 and HPV18 detection.

Table II.

Comparison of LAMP-turbidity and LAMP-LFD assays for HPV16 and HPV18 detection.

HPV16HPV18


ResultsNested PCRLAMP-turbidityLAMP-LFDNested PCRLAMP-turbidityLAMP-LFD
Positive444457181827
Negativea404027404031
Total848484585858

a Results repeated by quantitative PCR for HPV16 in 40-27=13 clinical samples and for HPV18 in 40-31=9 clinical samples. LAMP, loop-mediated isothermal amplification; LFD, lateral flow dipstick; HPV, human papillomavirus.

qPCR of discrepant results

The samples with inconsistent results between LAMP-LFD assay and the gold standard nested PCR method were further analysed using qPCR. The limits of qPCR detection for HPV16 and 18 were 103 copies (data not shown). When the 22 samples that exhibited inconsistent results between the LAMP-LFD assay and the gold standard method were repeated using qPCR, the results demonstrated that 13 of 13 samples and 7 of 9 samples were positive by qPCR for HPV16 and HPV18, respectively, while 2 samples generated negative results by qPCR (Tables III and IV).

Table III.

Analysis of clinical sample discrepant results by quantitative PCR.

Table III.

Analysis of clinical sample discrepant results by quantitative PCR.

HPV16HPV18


ResultsLAMP-LFDReal time PCRLAMP-LFDReal time PCR
Positive131397
Negative  0  002
Total131399

[i] PCR, polymerase chain reaction; HPV, human papillomavirus; LAMP, loop-mediated isothermal amplification; LFD, lateral flow dipstick.

Table IV.

qPCR confirmation of the LAMP-LFD discrepant results obtained using the gold standard method.

Table IV.

qPCR confirmation of the LAMP-LFD discrepant results obtained using the gold standard method.

Clinical no.LAMP-LFD resultsqPCR resultsViral load (copy/ng DNA)
  1HPV 16HPV 169.49
  2HPV 16HPV 164.45
  3HPV 16HPV 1611.85
  4HPV 16HPV 163.09
  5HPV 16HPV 162.71
  6HPV 16HPV 1611.4
  7HPV 16HPV 1618.07
  8HPV 16HPV 1610.92
  9HPV 16HPV 166.59
10HPV 16HPV 1650.43
11HPV 16HPV 1631.09
12HPV 16HPV 1618.82
13HPV 16HPV 1612.48
14HPV18NegativeNot detected
15HPV18HPV1825.98
16HPV18NegativeNot detected
17HPV18HPV184.18
18HPV18HPV1851.15
19HPV18HPV185.43
20HPV18HPV189.13
21HPV18HPV1863.13
22HPV18HPV18117.42

[i] qPCR, quantitative polymerase chain reaction; LAMP, loop-mediated isothermal amplification; LFD, lateral flow dipstick; HPV, human papillomavirus.

Discussion

LAMP is an alternative method for nucleotide amplification (7). This fast, simple and inexpensive amplification method can be performed within 1 h under isothermal conditions, and visualization of LAMP amplification products can be detected using the naked eye through several methods, such as turbidity, fluorescence and colour change (19). However, specific and nonspecific products cannot be separated using these detection methods. Therefore, to avoid false-positive results, LAMP products can be hybridized to specific probes (20) and subsequently detected using LFD. LFD is sufficient for the detection of hybridized LAMP products (21,22), and the results are easy to read without the use of carcinogens, such as ethidium bromide. LAMP detection methods have been successfully developed for HPV16 and HPV18 using visual turbidity and gel electrophoresis, and these assays can be completed within 70 and 115 min, respectively (15). In the present study, the same LAMP primer sets were used as previously published, but LAMP-LFD combined detection provided higher sensitivity and was completed within a shorter time period compared with a previous report (8).

The detection limits of LAMP-turbidity for HPV16 and HPV18 were 103 and 101 copies, respectively, and the assay was completed in 30 min. The detection limits of LAMP-LFD for HPV16 and HPV18 were 101 and 100 copies, respectively, and the assay was completed in 45 min. Therefore, the sensitivity of LAMP-LFD was higher than LAMP-turbidity, and the signal for LAMP-LFD was easy to read using the naked eye.

LAMP-turbidity and LAMP-LFD were further evaluated using 142 clinical samples (Table II), and the results revealed that the sensitivity and specificity of LAMP-turbidity for HPV16 and HPV18 detection were 100%. The sensitivity of LAMP-LFD for HPV16 and HPV18 was 100%, while the specificity was 67.5 and 77.5%, respectively. The decrease in LAMP-LFD specificity may be due to the fact that 22 of 80 HPV-negative samples generated positive results with the LAMP-LFD method. Therefore, those 22 samples were further analysed using qPCR (Tables III and IV). The results demonstrated that all 13 samples that generated positive HPV16 results with LAMP-LFD were also demonstrated HPV16-positive by qPCR. However, out of the 9 samples that were positive for HPV18 by LAMP-LFD, only 7 were also HPV18-positive by qPCR. Further analysis of the remaining 2 samples that generated negative results by qPCR revealed that the detection limit of the qPCR, although 10 µl of clinical sample was used as DNA template, increased ~3.3-fold, potentially reflecting a low concentration of HPV viral DNA. Therefore, detection of HPV16 and HPV18 using LAMP-LFD demonstrated higher sensitivity compared with nested PCR and did not require two reaction steps for PCR cycling (13). However, to avoid cross-contamination during the detection of the DNA products, the use of uracil DNA glycosylase has been recommended (23). In conclusion, LAMP-LFD is a rapid and simple method for the highly sensitive and specific detection of HPV16 and HPV18. Thus, LAMP-LFD might be useful as an HPV16 and HPV18 diagnostic tool in local hospitals or field studies.

Acknowledgements

This work was financially supported by grants from the Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences and HPV & EBV and Carcinogenesis Research Group, Khon Kaen University, Thailand (grant no. KKU-2556).

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Volume 15 Issue 5

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Spandidos Publications style
Kumvongpin R, Jearanaikoon P, Wilailuckana C, Sae‑Ung N, Prasongdee P, Daduang S, Wongsena M, Boonsiri P, Kiatpathomchai W, Swangvaree SS, Swangvaree SS, et al: Detection assay for HPV16 and HPV18 by loop‑mediated isothermal amplification with lateral flow dipstick tests. Mol Med Rep 15: 3203-3209, 2017
APA
Kumvongpin, R., Jearanaikoon, P., Wilailuckana, C., Sae‑Ung, N., Prasongdee, P., Daduang, S. ... Daduang, J. (2017). Detection assay for HPV16 and HPV18 by loop‑mediated isothermal amplification with lateral flow dipstick tests. Molecular Medicine Reports, 15, 3203-3209. https://doi.org/10.3892/mmr.2017.6370
MLA
Kumvongpin, R., Jearanaikoon, P., Wilailuckana, C., Sae‑Ung, N., Prasongdee, P., Daduang, S., Wongsena, M., Boonsiri, P., Kiatpathomchai, W., Swangvaree, S. S., Sandee, A., Daduang, J."Detection assay for HPV16 and HPV18 by loop‑mediated isothermal amplification with lateral flow dipstick tests". Molecular Medicine Reports 15.5 (2017): 3203-3209.
Chicago
Kumvongpin, R., Jearanaikoon, P., Wilailuckana, C., Sae‑Ung, N., Prasongdee, P., Daduang, S., Wongsena, M., Boonsiri, P., Kiatpathomchai, W., Swangvaree, S. S., Sandee, A., Daduang, J."Detection assay for HPV16 and HPV18 by loop‑mediated isothermal amplification with lateral flow dipstick tests". Molecular Medicine Reports 15, no. 5 (2017): 3203-3209. https://doi.org/10.3892/mmr.2017.6370