Rapid detection of Human T-cell leukemia virus-1 in blood donors by a real time loop-mediated isothermal amplification assay

doi:10.21203/rs.3.rs-3144220/v1

Download PDF

Article

Rapid detection of Human T-cell leukemia virus-1 in blood donors by a real time loop-mediated isothermal amplification assay

https://doi.org/10.21203/rs.3.rs-3144220/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

It is essential to detect Human T-cell Leukemia Virus-1 (HTLV-1) accurately and quickly in order to develop prevention strategies to reduce its transmission rates. However, the development of HTLV-1 testing in blood centers (stations) laboratories faces two challenges, including expensive reagents and labor-intensive processes .Thus, for the first time, a rapid real time loop-mediated isothermal amplification (RT-LAMP) was operated to detect HTLV-1 in blood donors.

Methods: As a first step, we combined the hybridization of fluorescent dye with the powerful amplification of LAMP to detect the presence of HTLV-1 plasmids containing PX gene. After that, we validated the method by performing gene test verification on blood samples and attempted to achieve one-step DNA extraction. The method was finally applied to the testing of blood donors for the presence of the HTLV-1 virus.

Results: It was successful to construct an RT-LAMP to detect HTLV-1, and the level of sensitivity was as low as 34 copies of the DNA from an HTLV-1-infected blood sample, which is sufficient for a primary blood screening. It was possible to achieve high specificity in detecting HTLV-1 virus in blood donor. With simply processed blood samples, it has shown good performance in helping establish negative donor pools in areas with high rates of HTLV-1.

Conclusion: With the above method, crudely treated blood samples can be detected in one step, are fluorescent, specific and sensitive, showing great potential for use in hematology and POCT.

Health sciences/Diseases/Haematological diseases

Health sciences/Diseases/Immunological disorders

Health sciences/Diseases/Infectious diseases

Biological sciences/Molecular biology

Biological sciences/Biological techniques/Genomic analysis

HTLV-1

LAMP

Molecular diagnosis

Blood transfusion

It is estimated that at least 20 million individuals globally [1] are infected with the first human retrovirus discovered in the early 1980s[2], human T-cell leukemia virus type 1 (HTLV-1). ATL (adult T cell leukemia) and myelopathy/tropical spastic paraplegia (HTLV-1 associated inebriation) are two of the most common clinical manifestations caused directly by HTLV-1 infection. In immunocompetent individuals, each has an overall risk between 1% and 4%, and an extended latency period of roughly 30 to 50 years[3][4].

Transfusion safety depends on the collecting blood from donors who are not at high risk for transfusion-transmitted illnesses[5]. Among them, HTLV-1 is transmitted primarily through mother-to-child transmission, sexual transmission, contaminated blood products, and needle sharing, which results in the transfusion of cellular blood components. Transfusion of cellular blood components is therefore very important as a possible transmission method[6]. The WHO guidelines for transfusion-transmitted infection screening and the Good Practice Guidelines on Blood Transfusion (EDQM 20th Edition) both recommend the necessity for HTLV blood donor screening, whether common or 'first time only', which be determined based on regional epidemiology[7][8]. In several countries, including the US, Japan, and Sweden, HTLV-1 screening has been conducted between blood donors using various screening techniques[9–11].

HTLV-1 is typically found in resource-limited regions with inadequate infrastructure[12, 13]. Therefore, an accurate, rapid and convenient nucleic acid detection assay for HTLV-1 is urgently needed. As of now, most HTLV-1 measurements in blood centers (stations) are performed through enzyme-linked immunosorbent assays (ELISA) or quantitative polymerase chain reactions (PCR) [14–16], which is inappropriate for point-of-care use. ELIA, however, is time-consuming and expensive, while PCR calls for accurate thermal cycling, which is time-consuming and requires specialized equipment. In order to overcome this problem, loop-mediated isothermal amplification (LAMP) of blood samples was proposed, which allows for shorter time to results and and reliable nucleic acid testing with less technological cost. [17]. However, this assay had the subsequent two disadvantages:(1) because of the significant danger of cross-contamination, this measurement needed tube opening at the end point, which was a unsuitable alternative in LAMP reaction.(2) there was a certain subjectivity in the judgment, which may vary with the operator's different operating experience, so their method may not meet modern people's pursuit of precision medicine.

Here, we described a novel method for RT-LAMP-based HTLV-1 viral identification in blood donors, in which the fluorescent dye was given a new function for HTLV-1 detection. At the same time, we aim to achieve visual detection without unmasking, making the novel approach an easy-to-use donor screening tool..

2.1 | Plasmid construction and blood donors

To obtain the standard HTLV-1 template, the plasmid containing the PX [GenBank:L36905.1] specific sequence was transfected into Escherichia coli DH5a plasmid pUC57. The standard internal reference template containing the ACTB [GenBank:GCA_000001405.29] sequence is also constructed using the same method as described above. The the recombined plasmids were constructed by Sangon Biotech Co., Ltd (Shanghai, China).

The standard HTLV-1 template was used to determine the optimal HTLV-1 RT-Primers and concentrations of dTNPs and Tte UvrD helicase. The standard internal reference template was used to evaluate the performance of the DNA/RNA direct extraction kit .

Blood donors from May 2021 to May 2022 who met the relevant provisions and standards of the Health Examination Requirements for Blood Donors in China and Blood Donation Law of the People's Republic of China were selected from Zhongshan Central Blood Station, Guangdong Province, China. All aspects of this study were performed in accordance with relevant guidelines and approved by the institutional review boards of Zhongshan Central Blood Station (reference number: 2021-01). Participants received written informed consent of the study's purpose and of their right to keep information confidential.

2.2 | Preparation of blood sample

Blood DNA samples from confirmed infected and uninfected with HTLV-1 blood donors between May 2021 to 2022 were used to assess the efficacy and optimization of HTLV-1 RT-LAMP tests.

The DNA of blood samples was extracted by DNA/RNA direct extraction kit (HaiGene Biotech Co.,Ltd, Haerbin, China):(1) 100 µL serum was taken and added into 1.5mLEP tube; (2) Add 500 µL solution DNA/RNA DirOut-A and swirl for 30s; (3) 100µL DNA/RNA DirOut-B solution was added after the whirlpool was completed. After the whirlpool was mixed evenly, the supernatant was taken into the sterile centrifuge tube, which was the template DNA to be tested.

2.3 | Design of RT-LAMP Primers

The primers were generated depending on the sequences of PX genes of HTLV-1 [GenBank:L36905.1] and ACTB [GenBank:GCA_000001405.29] strains. The PrimerExplorer V4 program (http://primerexplorer.jp; Eiken Chemical 125 Co., Ltd., Tokyo, Japan) was used to create primers. The sequences of the HTLV-1 and ACTB RT-LAMP primers are listed in Table S1.

2.4 | RT-LAMP assay protocol

The RT-LAMP reaction was carried out in 25 µL mixture containing 0.2 µM each of F3 and B3 primers, 1.6 µM each of FIP and BIP primers, 0.8 µM each of loop F and loop B primers, 1×ThermoPol® reaction buffer II(New England Biolabs, Ipswich, MA, USA), 1.2 mM betaine (Sigma-Aldrich, Saint Louis, MO, USA), 6 mM MgSO₄(New England Biolabs, Ipswich, MA, USA), 0.05 mM YO-PRO™-1 Iodide(Invitrogen, São Paulo, Brazil), 8 U Bst 3.0 DNA polymerase(New England Biolabs, Ipswich, MA, USA), 5µL DNA template and RNase-free water. Tte UvrD helicase was tested at concentrations ranging from 1×10^− 6 µg to 4×10^− 6 µg(New England Biolabs, Ipswich, MA, USA). dNTPs was assessed at concentrations ranging from 1 mM to 1.6 mM(Invitrogen, Carlsbad, CA, USA). The mixture was subjected to incubation at a temperature of 65°C for a duration of 45 minutes, followed by heating at 80°C for a period of 2 minutes to bring about the termination of the reaction.

In order to monitor for false positives caused by reagent contamination, a background were performed at each run. Meanwhile to monitor for false negatives due to poor DNA extraction, positive control containing ACTB RT-LAMP primers was also included in each run. Simultaneously, the fluorescence intensity of the florescent dye was monitored using a Isothermal amplification fluorescence detector H1600(Hangzhou Bio-gener Biotechnology Co., LTD. )

2.5 | Evaluation of the RT-LAMP assay

To verify the LoD of HTLV-1 RT-LAMP assay, multiple dilutions (3.4×10⁴ 3.4×10³ 3.4×10² 3.4×10¹ 3.4×10⁰ copies per tube) of the DNA from HTLV-1-infected blood were tested on 3 independent cycles.

2.6 | Application of the method using blood samples

HTLV-1 RT-LAMP method was evaluated by the clinical confirmed specimens with and without HTLV-1 infection.

3.1 | Selection of RT-Primers using standard HTLV-1 plasmid

To investigate the feasibility of the method, three pairs of RT-LAMP primers according to the PX genes of HTLV-1 were designed. We carried out RT-LAMP reaction with and without plasmid(background), respectively, to confirm the effect of primers to RT-LAMP rate. The RT-LAMP results (Fig. 1) showed that H3 (Fig. 1D)primers had the best amplification efficiency, but primers H2 primers (Fig. 1C) had the best specificity. The delay in time between the positive test and the background(without plasmid) was declared as the ΔTq.

As we can see from the column picture (Fig. 1A), there was a longest time gap (more than 25min), which demonstrated the good specificity of H2 primers in detecting the HTLV-1 target.

3.2 | Optimization of HTLV-1 RT-LAMP tests

The HTLV-1 RT-LAMP tests were adapted for optimum doses of dNTPs and Tte UvrD, which decreases secondary structure formation in guanine-cytosine (GC)-rich sectors.

3.2.1 | Optimization of dNTPs concentration

The dNTPs at concentrations of 1 (Fig. 2B), 1.2 (Fig. 2C), 1.4 (Fig. 2D) and 1.6(Fig. 2E)mM were set respectively, which showed that the background (without plasmid) time firstly increased and then decreased with the increase of dNTPs concentration. As we can see from the column picture (Fig. 2A), there was a longest time gap (29.67 min) in 1.4 mM, which demonstrated the good specificity of betaine at concentrations of 1.4 mM in detecting the HTLV-1 target.

3.2.2 | Optimization of Tte UvrD helicase concentration

Tte UvrD helicase at concentrations of 1×10^− 6(Fig. 3B), 2×10^− 6(Fig. 3C), 3×10^− 6 (Fig. 3D)and 4×10^− 6(Fig. 3E) µg were set respectively, which showed that the background (without plasmid) time increased with the increase of Tte UvrD helicase concentration. As we can see from the column picture (Fig. 3A), when the concentration of the enzyme was increased to 4×10^− 6 µg((Fig. 3E), the the background did not appear and the amplification efficiency was not significantly reduced, which demonstrated the good specificity of Tte UvrD helicase at concentrations of 4×10^− 6 µg in detecting the HTLV-1 target.

3.3 | Performance of the RT-LAMP assay

By amplifying ten-fold dilutions of from 3.4×10⁴ 3.4×10³ 3.4×10² 3.4×10¹ copies in triplicate, the linear of standard curve for HTLV-1 RT-LAMP was determined. Figure 4B depicted the linear connection between the time-to-positive readings and the log₁₀ of the DNA copies, with a coefficient of determination (R²) of 0.9417, as demonstrated in Fig. 4A.

3.4 | Evaluation of the assay using blood samples

To investigate the applicability of this method in real-life applications, the method was additionally used to HTLV-1 test by the DNA of infected and uninfected blood donors.

The fluorescence curves of internal reference genes presented in Fig. 5 demonstrated the blood samples were simply treated with by the DNA/RNA direct extraction kit that is commercially available and could be detected by RT-LAMP. Meanwhile, Fluorescence curves of samples are present Fig. 4A and absent in Fig. 4B showed that the primers designed in this paper could distinguish between infected and uninfected HTLV-1 blood sample.

ELISA, WB, and PCR are the most frequently used methods for HTLV-1 detection[19]. Due to the use of recombinant proteins that are specific for HTLV-I, previous studies have indicated that WB[20] is more sensitive than ELISA[21]. However, WB requires expensive equipment, is technically challenging, and requires expertise in interpretation. Compared with previous LAMP method used in HTLV detection[17], our RT-LAMP assay provides a simple, faster and more efficient method for HTLV-1 virus detection. In our study, a real-time fluorescence method was used to analyze the results instead of agarose gel electrophoresis, which avoids cross-contamination and aerosol generation. At the same time, the traditional endpoint method has been replaced by fluorescence amplification curve, which is more convenient for the examiner's interpretation rather than subjective naked eye observation.

Primer design and selection is the most important step in the RT-LAMP test, which is slightly more sophisticated than PCR[23]. High sensitivity and sequence conservation avoid false negative results, while high saccuracy and limited primer-dimers decrease false positive results. Therefore, three pairs of RT-LAMP primers were generated depending on PX gene of HTLV-1. There was a longest time gap (more than 25 min), which demonstrated the good specificity of H2 primers in detecting the HTLV-1 target. The specificity of the HTLV-1 RT-LAMP assay was also affected by concentrations of dNTPs and Tte UvrD helicase. By setting different concentrations of components, we extended the time gap time of the screened H2 primers from the initial 25min to no ΔTq values, which was sufficient to avoid false positive results.

Additionally, this assay differed from the LAMP reactions that had previously been reported as they lacked a quantitative ability and only showed the existence of HTLV-1. In this research, a specific standard curve of HTLV-1 was obtained from 3.4×10⁴ to 3.4×10¹ copies, and the measurement coefficient R² = 0.9417. However, the concentration of the human genome during ordinary blood extraction was normally 10⁴-10⁵copies per microliter, which is sufficient for common clinical diagnostic purposes[24]. Thus, the sensitivity of our strategy was enough to detect clinical blood sample, which was also validated in the experiment above.

Here, we described a quick and affordable method for detecting HTLV-1 at a set temperature. The proposed HTLV-1 RT-LAMP test makes use of fluorescent dye and the isothermal amplification, which leads to a significant differences in amplification specificity.

Our approach depends mainly on fluorescent dye hybridization to reduce the impacts of laboratory cross-contamination brought on by electrophoretic analysis, in contrast to recently described lamp-based viral assays. Besides, the fluorescence amplification curve replaces the traditional end-point fluorescence method, which is more convenient for the interpretation of inspectors rather than subjective eye-based observation.

In summary, the utilization of this approach yields several advantages, including: (1) the exclusive reliance on RT-LAMP for the entirety of the test, which enables the detection of blood samples in an isothermal fashion; (2) the integration of a fluorescent dye into the assay, facilitating single-step visual detection that can be discerned by the unaided eye and mitigating the potential for laboratory cross contamination. (3) The method exhibits sensitivity and specificity levels that are comparable to those of the conventional detection platform, thereby providing a justification for its utilization in clinical tests. (4) The fluorescent dye has the ability to detect HTLV viruses, thereby indicating that this approach may hold significant potential for the detection of other viruses that are transfusion-induced, such as HIV, HCV, and HBV. Given these advantages, the proposed approach can be implemented with a basic temperature controller, making it highly suitable for healthcare decentralization in countries that have limited resources. And it is anticipated that this method will provide a reliable platform for screening for infections transmitted by transfusion.

Author Contributions

Ziqiang Wang designed RT-LAMP primers, Huiyan Lin and Yonglun Wu performed out RT-LAMP tests, Fei Pu and Qiao Li gathered analyzed data, Feng GeandHengyi Ou generated images, Tingyan Liao and Rui Peng summarized research results data. Ziqiang Wang drafted the manuscript. Zhicheng Caisupervised and controlled the progress of the research project. All authors have read and agreed to the published version of the manuscript.

Acknowledgment

I would like to thank Ainong Sun for the technical support of this project and Rui hong He for the administrative support of this project.

Funding

The research was funded by The third batch of social welfare and basic research projects of ZhongShan in 2021 (Major and Key Projects of Medical and Health) Topic Six: Youth Training Program（grant 2021B3021）.

Conflict of Interest

All authors have no conflict.

Data availability

All data generated or analysed during this study are included in the current study.

Willems L, Hasegawa H, Accolla R, Bangham C, Bazarbachi A, Bertazzoni U, et al. Reducing the global burden of HTLV-1 infection: an agenda for research and action. Antivir Res. 2017;137:41–8.
Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc Natl Acad Sci U S A. 1980;77(12):7415–7419. doi: 10.1073/pnas.77.12.7415.
Manns A, Hisada M, La Grenade L. Human T-lymphotropic virus type I infection. Lancet. 1999;353:1951–8.
Martin F, Fedina A, Youshya S, Taylor GP. A 15-year prospective longitudinal study of disease progression in patients with HTLV-1 associated myelopathy in the UK. J Neurol Neurosurg Psychiatry. 2010;81:1336–40.
Williams P, O'Flaherty N, Field S, Waters A. Human T-lymphotropic virus in Irish blood donors: Impact on future testing strategy. Transfusion. 2022;62(9):1799–1807. doi:10.1111/trf.17017
Pique C, Jones KS. Pathways of cell-cell transmission of HTLV-1. Front Microbiol. 2012;3:378.
Transfusion ECoB. Good practice guidelines for blood establishment required to comply with directive 2005/62/EC. European Commission: Standards and specifications for implementing the quality system in blood establishments; 2018..
Screening donated blood for transfusion-transmissible infections: Dent Rec. 2009:36.
Satake M, Yamaguchi K, Tadokoro K. Current prevalence of HTLV-1 in Japan as determined by screening of blood donors. J Med Virol. 2012; 84(2):327–35.
Pillonel J, Le Marrec N, Girault A, David D. Laperche S: [epidemiological surveillance of blood donors and residual risk of blood-borne infections in France, 2001 to 2003]. Transfusion clinique et biologique: journal de la Societe francaise de transfusion sanguine. 2005;12(3):239–46.
Malm K, Ekermo B, Hillgren K, Britton S, Fredlund H, Andersson S. Prevalence of human T-lymphotropic virus type 1 and 2 infection in Sweden. Scand J Infect Dis. 2012;44(11):852–9.
Einsiedel, L.; Spelman, T.; Goeman, E.; Cassar, O.; Arundell, M.; Gessain, A. Clinical associations of Human T-Lymphotropic Virus type 1 infection in an indigenous Australian population. PLoS Negl. Trop. Dis. 2014, 8, e2643.
Van Tienen, C.; van der Loeff, M.F.S.; Peterson, I.; Cotten, M.; Holmgren, B.; Andersson, S.; Vincent, T.; Sarge-Njie, R.; Rowland-Jones, S.; Jaye, A.; et al. HTLV-1 in rural Guinea-Bissau: Prevalence, incidence and a continued association with HIV between 1990 and 2007. Retrovirology 2010, 7, 50
Zhao J, Zhao F, Han W, et al. HTLV screening of blood donors using chemiluminescence immunoassay in three major provincial blood centers of China. BMC Infect Dis. 2020;20(1):581. Published 2020 Aug 6. doi:10.1186/s12879-020-05282-2
Estes, M.C.; Sevall, J. Multiplex PCR using real time DNA amplification for the rapid detection and quantitation of HTLV I or II. Mol. Cell. Probes 2003, 17, 59–68.
Dehée, A.; Césaire, R.; Désiré, N.; Lézin, A.; Bourdonné, O.; Béra, O.; Plumelle, Y.; Smadja, D.; Nicolas, J.-C. Quantitation of HTLV-I proviral load by a TaqMan real-time PCR assay. J. Virol. Methods 2002, 102, 37–51.
Campos, K.R.; Santos, F.L.N.; da Silva Brito, V.; Gonçalves, N.L.S.; Araujo, T.H.A.; Galvão-Castro, B.; Caterino-de-Araujo, A. Line Immunoassay for Confirmation and Discrimination of Human T-Cell Lymphotropic Virus Infections in InconclusiveWestern Blot Serum Samples from Brazil. J. Clin. Microbiol. 2019, 58.
Gomes Y, Caterino-de-Araujo A, Campos K, et al. Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid and Accurate Confirmatory Diagnosis of HTLV-1/2 Infection. Viruses. 2020;12(9):981. Published 2020 Sep 4. doi:10.3390/v12090981.
Fani M, Rezayi M, Meshkat Z, et al. Current approaches for detection of human T-lymphotropic virus Type 1: A systematic review. J Cell Physiol. 2019;234(8):12433–12441. doi:10.1002/jcp.28087.
Naderi M, Paryan M, Azadmanesh K, Rafatpanah H, Rezvan H, Mirab Samiee S. Design and development of a quantitative real time PCR assay for monitoring of HTLV-1 provirus in whole blood. J Clin Virol. 2012;53(4):302–307. doi:10.1016/j.jcv.2011.12.033.
Jalaeikhoo H, Soleymani M, Rajaeinejad M, Keyhani M. Prevalence of Human T-lymphotropic virus type 1 (HTLV-1) Infection in Patients with Hematologic Disorders and Non-Hematologic Malignancies in a Tertiary Referral Hospital. Arch Iran Med. 2017;20(4):224–228.
Dehée A, Césaire R, Désiré N, et al. Quantitation of HTLV-I proviral load by a TaqMan real-time PCR assay. J Virol Methods. 2002;102(1–2):37–51. doi:10.1016/s0166-0934(01)00445-1.
Aoi, Y., Hosogai, M., & Tsuneda, S.. (2006). Real-time quantitative lamp (loop-mediated isothermal amplification of dna) as a simple method for monitoring ammonia-oxidizing bacteria. Journal of Biotechnology, 125(4), 484–491.
Lu Y, Ma X, Wang J, et al. Visualized detection of single-base difference in multiplexed loop-mediated isothermal amplification amplicons by invasive reaction coupled with oligonucleotide probe-modified gold nanoparticles. Biosens Bioelectron. 2017;90:388–393. doi:10.1016/j.bios.2016.12.015

No competing interests reported.

SupplementaryMaterial.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

Rapid detection of Human T-cell leukemia virus-1 in blood donors by a real time loop-mediated isothermal amplification assay

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 | Plasmid construction and blood donors

2.2 | Preparation of blood sample

2.3 | Design of RT-LAMP Primers

2.4 | RT-LAMP assay protocol

2.5 | Evaluation of the RT-LAMP assay

2.6 | Application of the method using blood samples

3. Results

3.1 | Selection of RT-Primers using standard HTLV-1 plasmid

3.2 | Optimization of HTLV-1 RT-LAMP tests

3.2.1 | Optimization of dNTPs concentration

3.2.2 | Optimization of Tte UvrD helicase concentration

3.3 | Performance of the RT-LAMP assay

3.4 | Evaluation of the assay using blood samples

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1