|Year : 2012 | Volume
| Issue : 4 | Page : 391-396
Development and evaluation of reverse-transcription loop-mediated isothermal amplification for rapid detection of human immunodeficiency virus type 1
Xihong ZHAO1, Xiaoping CHEN2, Youhong ZHANG2, Xiaowei HE3, Wenmei LI4, Lei SHI5, Xingzhou CHEN2, Zhenbo XU3, Nanjing ZHONG3, Guiyuan JI6, Liansheng YANG3, Jihua WANG7
1 Key Laboratory for Green Chemical Process of Ministry of Education, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430073; College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China
2 Key Laboratory for Green Chemical Process of Ministry of Education, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430073, China
3 College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China
4 College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640; Guangzhou Wondfo Biotech Co., Ltd, Guangzhou 510530, China
5 Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (HUST), Wuhan 430030, China
6 Center for Disease Control and Prevention of Guangdong Province, Institute of Public Health, Guangzhou 510030, China
7 Guangzhou Wondfo Biotech Co., Ltd, Guangzhou 510530, China
|Date of Submission||27-Jan-2012|
|Date of Acceptance||30-May-2012|
|Date of Web Publication||24-Nov-2012|
Key Laboratory for Green Chemical Process of Ministry of Education, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430073; College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640
Source of Support: Research Fund of Wuhan Institute of Technology (10113201), China; National Natural Science Foundation of China (81172178), Conflict of Interest: None
Purpose: The objective of this study was to establish a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid detection of human immunodeficiency virus type 1 (HIV-1). Materials and Methods: The HIV-1 integrase gene region was selected because it was a conserved part of the HIV-1 genome. Six primers specific to eight regions of the HIV-1 integrase gene were designed. A total of 171 samples (18 HIV-1 confirmed positive samples and 153 serum specimens were collected in this study) were tested by RT-LAMP and reverse-transcription polymerase chain reaction (RT-PCR). After amplification in an isothermal water bath for 45 min, samples containing HIV-1 generated the expected ladder-like products while other viruses generated no product. Results: The sensitivity and specificity of the RT-LAMP assay were evaluated by comparison with RT-PCR. The assay was significantly more sensitive than normal gel-based RT-PCR. Conclusion: Because it is specific and simple, the RT-LAMP assay can be widely applied in clinical laboratories for rapid detection of HIV-1.
Keywords: Human immunodeficiency virus type 1, reverse-transcription loop-mediated isothermal amplification (RT-LAMP), integrase, Rapid detection
|How to cite this article:|
ZHAO X, CHEN X, ZHANG Y, HE X, LI W, SHI L, CHEN X, XU Z, ZHONG N, JI G, YANG L, WANG J. Development and evaluation of reverse-transcription loop-mediated isothermal amplification for rapid detection of human immunodeficiency virus type 1. Indian J Med Microbiol 2012;30:391-6
|How to cite this URL:|
ZHAO X, CHEN X, ZHANG Y, HE X, LI W, SHI L, CHEN X, XU Z, ZHONG N, JI G, YANG L, WANG J. Development and evaluation of reverse-transcription loop-mediated isothermal amplification for rapid detection of human immunodeficiency virus type 1. Indian J Med Microbiol [serial online] 2012 [cited 2020 May 28];30:391-6. Available from: http://www.ijmm.org/text.asp?2012/30/4/391/103757
| ~ Introduction|| |
Human immunodeficiency virus type 1 (HIV-1), the retrovirus that causes the acquired immunodeficiency syndrome (AIDS), is one of the leading causes of death worldwide. As of December 2009, an estimated 33.3 million people worldwide were living with HIV, while 2.6 (2.3-2.8) million people were newly infected with HIV and 1.8 (1.6-2.1) million people died of HIV-relaxd causes.  There are two types of HIV virus : HIV type 1(HIV-1) is the predominant virus worldwide, while HIV-2 differs from HIV-1 in its lower pathogenicity and higher lever of intrasubtype strain diversity.  Early diagnosis of HIV has been considered as a critical entry point for prevention, treatment and care. 
The residual risk of transfusion-acquired infection is attributed primarily to donations during the antibody-negative, preseroconversion window, the period between infection and detectable seroconversion.  For HIV-1, that period is about 3 to 4 weeks for the contemporary serological tests by HIV-1 antibodies screened. , That residual risk led to investigations into newer technologies for direct detection of the virus to further close the HIV-1 diagnostic window. In recent years, direct detection of the virus has also been achieved to reduce the window period between infection and serological detection by nucleic acid amplification techniques, which enable detection of the viral genome. ,, Previous studies demonstrated the detection of HIV-1 RNA in plasma approximately 1 week earlier than detection of p24 antigen and about nearly 2 weeks earlier than antibodies detection test.  Current testing methods based on nucleic acid amplification are implemented to screen for HIV infected blood donated during window period, however, yielding as high as 1% false-positive rates compared to the antibodies test. , Furthermore, HIV-1 RNA assay requires for trained personnel, operating space, equipment and reagents would hamper its application in resource-limited setting.  Rapid, sensitive, specific, and cost-effective screening of donated blood to prevent transmission of infectious remains challenging.
Notomi et al. reported a novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP) that amplifies DNA with high specificity, efficiency, and speed under isothermal conditions. This method relies on an auto-cycling strand displacement DNA synthesis performed by the Bst DNA polymerase; Bst DNA polymerase has a high displacement activity, to release a single stranded DNA, which then forms a hairpin to initiate the starting loop for cyclic amplification. Amplification proceeds in cyclical order, each strand being displaced during elongation with the addition of new loops with every cycle, , which is different from PCR in that four or six primers perform the amplification of the target gene. The amplification uses isothermal conditions between 59-65°C, and the amplification product is a mixture of many different sizes of stem-loop DNAs with several inverted repeats of the target sequence and cauliflower-like structures with multiple loops.  Compared with PCR, LAMP is advantageous as the LAMP primers do not need thermal denaturation, long circulation times, disordered gel electrophoresis. LAMP could instead of PCR as a novel amplification method in detection microbes and viruses. In the previously research, we had developed and evaluated LAMP assays for detecting different foodborne microbiology. ,,,
The LAMP assay has also been used with RNA templates by combining reverse transcription reactions with LAMP (RT-LAMP), simply through addition of a heat-stable reverse-transcriptase. , This method has been applied for the detection of many kinds of viruses, such as porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), dengue virus (DV), severe acute respiratory syndrome corona virus (SRAS), pseudorabies virus (PRV), swine vesicular disease virus (SVDV), influenza virus (IV), human parvovirus B19, HIV-1 and others. ,,,
In this study, a reverse-transcription LAMP assay (RT-LAMP) was developed to detect toward the HIV-1 integrase gene; the HIV-1 integrase gene was targeted because it was a conserved part of the HIV-1 genome. The sensitivity and specificity of the RT-LAMP assay were evaluated by comparison with reverse-transcription polymerase chain reaction (RT-PCR). With this RT-LAMP assay, non-professional can rapidly screening test HIV-1 in resource-limited settings easily.
| ~ Materials and Methods|| |
WHO HIV-1 International Standard (97/656) 100,000 international units (IU) /vial (National Institute for Biological Standard and Control, UK) was used to establish the detection limit of the RT-LAMP assay. ,,
Bst DNA polymerase and MgSO4 (100 mM) were purchased from NEB Biotechnology, and DNA polymerase was purchased from TaKaRa Biotechnology (Dalian, China). Betaine and DMSO were purchased from SIGMA-Aldrich, Shanghai, China.
Template preparation and extraction of RNA
Total RNA was extracted from plasma using an RNA extraction kit (TaKaRa Biotechnology, Dalian, China). Briefly, total RNA was extracted from plasma samples with TRIzol® reagent in accordance with the manufacturer's instructions. The RNA was immediately stored at-70°C until used for the one step RT-LAMP or RT-PCR reaction.
Based on more than 100 published HIV-1 sequences, the highly conserved sequences in the HIV-1 integrase p31 coding region were selected as the target sequence. Primers were designed for RT-LAMP according to alignment analysis of integrase gene sequences in GenBank. All primers were designed using Primer Explorer V4 software (Fujitsu System Solutions Ltd., Tokyo, Japan). For target gene, a set of inner primers (including forward and backward inner primer), outer primers (including F3 and B3) and loop primers (including LF and LB, to accelerate reaction) was designed for LAMP to target 8 distinct regions. Forward inner primer (FIP) consisted of the complementary sequence of F1, a T-T-T-T linker and F2; backward inner primer (BIP) consisted of B1C, a T-T-T-T linker and the complementary sequence of B2C. The outer primers F3 and B3 located outside of the F2 and B2 regions, while loop primers LF and LB located between F2 and F1 or B1 and B2, respectively. Upload the selected primer information file for the loop primer designing, which was saved in the regular primer designing. The primers for RT-LAMP were shown in [Table 1], among them, F3 and B3 primers were applied in RT-PCR.
|Table 1: Details of RT-PCR and RT-LAMP primers designed for detection of HIV-1 integrase gene sequences|
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Estabishment of RT-LAMP assay and optimization of the RT-LAMP protocol
Based on the previous reports,  RT-LAMP reaction was carried out in a conventional water-bath by mixing 2.0 μM each of FIP and BIP primer, 0.2 μM each of F3 and B3 primer, 0.8 μM each of LF and LB primer, 10 mM each deoxynucleoside triphosphate, 0.65 U of the avian myeloblastosis virus reverse transcriptase (Invitrogen), 8 U of Bst DNA polymerase (New England Biolabs) using the supplied 1× buffer (containing 2 mM of MgSO4, 0.8 M betaine) and 2 μl of extracted RNA. The amplification reaction was performed at 63°C for 45 min and heated at 80°C for 10 min to terminate the reaction. RT-LAMP products were analyzed by 2% agarose gel electrophoresis with the voltage of 100 V for 30 min, and were visualized by staining with ethidium bromide. The result could also be observed directly without staining because of the white precipitate of magnesium pyrophosphate or the green color produced by the intercalating dye in positive reactions. When the amplification was completed, 2 μl of coloring agent (SYBR Green I, Invitrogen, Wisconsin, USA) was added to each test tube and mixed. The test tubes were then visual examined. The color of reaction mixture will turn green in the presence of RT-LAMP amplified products, while it remains orange in reaction mixture with no amplification. The sensitivities of gel electrophoresis analysis and visual observation by addition of SYBR Green I were compared.
RT-PCR was carried out in reaction volume containing 10 mM deoxynucleoside triphosphate, 2 U PrimeScript™Rtase, 5 U of Taq polymerase (TaKaRa) using the supplied 10× buffer, 1.0 μM each of primers F3 and B3, and template of 1.0 μl of extracted RNA from the samples of HIV-1 infection plasma. According to the following cycling parameters : Initial incubation at 50°C for 30 min for cDNA synthesis, followed by initial PCR activation at 94°C for 5 min and then 28 cycles of 94°C for 30 s, 55 C for 30 s and 72 C for 1 min, then final elongation for 5 min at 72 C. RT-PCR was carried out by using the Gene Amplification PCR system TC-XP-A (Bioer technology). RT-PCR products were subjected to electrophoresis on a 2% agarose gel.
Sensitivity of the RT-LAMP assay relative to RT-PCR for the detection of HIV-1 International Standard
The sensitivity of the RT-LAMP assay vs. RT-PCR for the detection of HIV-1 was determined using serial dilutions of WHO HIV-1 International Standard RNA template.
Detection of HIV-1 in clinical samples by RT-LAMP and RT-PCR
A total of 171 samples were tested in this study, among 18 HIV-1 positive samples by Western Blot confirmed and 153 serum specimens from healthy blood donors screened by ELISA kits previously. RNA was extracted from each sample (see Section 2.2) and was used as a template for RT-LAMP and RT-PCR.
| ~ Results|| |
Optimization of the conditions of RT-LAMP assay
In order to determine the optimal conditions of RT-LAMP assays, RNA from HIV-1 infected plasma was used as target template. The specific amplification generated many ladder-like pattern bands on agarose gel due to its characteristic structure. There was no amplification of the negative control.
RT-LAMP assays were under isothermal condition between 59°C and 65°C. No significant difference were observed, however, the LAMP product amplified at 63°C showed slightly larger amount of DNA when compared to other temperatures (data not shown), which was consistent with studies previously. Reaction times of RT-LAMP assays were varied in 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min and 120 min, under 63°C. Without loop primers, amplification products could not be observed until 60 min. While with loop primers, the amplification was initially detected at 30 min, and reached maximal at 45 min. There was no difference in amplification pattern using the additional primers. RT-LAMP assays were performed with omission of one or two of the primers, under 63°C for 60 min. However, no amplification could be observed in the absence of FIP, BIP, F3 or B3 primer. For each of the primers plays an indispensable role in auto-cycling strand placement and forming the loop out structure. 
The results of the RT-LAMP reaction were determined easily and quickly by visual inspection without magnification or staining. If the positive reaction product is present, the SYBR Green I dye inserts into the double-stranded DNA after the reaction, and the product turns green; otherwise, the dye does not insert into the double-stranded DNA and the tube remains blue [Figure 1]. These observations agreed with gel electrophoresis results.
|Figure 1: Detection of HIV-1 by RT-LAMP. Detection of the RTLAMP products was confirmed by visual inspection with 2% agarose gel electrophoresis (a) and SYBR Green І (b), (a) 1, positive reaction ; 2, negative reaction, (b) 1, positive reaction ; 2, negative reaction|
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Optimal amplification of HIV-1 RNA was achieved by incubation of F3, B3, FIP, BIP, LF, LB primers, 8 U of Bst DNA polymerase and 1 U of AMV reverse transcriptase enzyme with the target RNA at 63°C for 45 min.
Sensitivity and specificity of RT-LAMP assays
The WHO HIV-1 International Standard (97/656) 100,000 IU/vial diluted to 6200, 620, 310, 155, 75.5, 38.7 copies/ml was used as template to test the sensitivity of HIV-1 RT-LAMP. The detection limit of RT-LAMP was tested and compared with RT-PCR by using the same templates at identical concentrations and this was done in triplicate at each concentration of templates. Davis  reported that one IU was equivalent to 0.62 genome copies. The reproducible 100% detection limit of the RT-LAMP assay and RT-PCR was 155 copies/ml, 620 copies/ml, respectively, indicating that RT-LAMP was at least 4-fold more sensitive than RT-PCR [Figure 2].
|Figure 2: Sensitivity of RT-LAMP and RT-PCR for the detection of serial dilutions of HIV-1 international standard RNA template. 1, 38.7 copies/ml;2 75.5 copies/ml;3, 155 copies/ml;4, 310 copies/ml;5, 620 copies/ml;6, 6200 copies/ml|
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Evaluation of RT-LAMP assays using clinical samples
To evaluate the clinical sensitivity and specificity of RT-LAMP method, a total of 171 samples (18 HIV-1 confirmed positive samples by western blot and 153 serum specimens from healthy blood donors were collected in our lab) were tested by RT-LAMP and RT-PCR [Table 2]. The overall sensitivities of RT-LAMP were higher than that of RT-PCR.
|Table 2: Detection of HIV-1 in clinical specimens by RTLAMP compared RT-PCR|
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Among 18 HIV-1 positive specimens confirmed by Western Blot previously, all the specimens were also positive by RT-LAMP, but 1 positive specimen was missed by RT-PCR. The detection rate of RT-PCR and RT-LAMP were 18/18 (100%) and 17/18 (94.4%) for the clinical HIV-1 positive specimens in this study, respectively.
Among 153 HIV-1 negative specimens from healthy blood donors screened by ELISA kits, all the specimens were also negative by RT-LAMP, but 2 specimens yielded a false-positive reaction, so the specificities of the RT-LAMP and RT-PCR were 100% and 98.7%, respectively.
| ~ Discussion|| |
The ELISA test is considered as "gold standard method" for rapid and large-scale screening of samples in HIV diagnosis.  But ELISA tests require for trained personnel and equipments. Current assays based on nucleic acid amplification yielded a high false-positive rate. Rapid and direct detection of HIV-1 virus for screening of donated blood remains challenging in resource-limited region. RT-LAMP utilizes four specifically designed primers and isothermal Bst DNA polymerase, which have strand displacement activity. The amplicons are seen as multiple bands on agarose gel electrophoresis, in contrast to the single band seen with RT-PCR. This method is simple and rapid, highly sensitive and specific for target gene.
Successful amplification of RT-LAMP method relies on the specificities of designed primers. In order to develop RT-LAMP method for the specific detection of HIV-1, we successfully designed specific primers by targeting the highly conserved region of HIV-1 gene of integrase. As a result, RT-LAMP method produced many bands of different sizes for HIV-1 integrase.
In this study, the rapid and sensitive diagnostic assay based on RT-LAMP technology was developed to detect HIV-1. When the sensitivity of HIV-1 RT-LAMP was compared to that of RT-PCR in the HIV-1 international standard (97/656) 100,000 IU/virus dilution series, RT-LAMP was approximately 4-fold more sensitive than RT-PCR. The greater sensitivity of HIV-1 RT-LAMP was also reflected in the clinical samples, as some positive samples detected by HIV-1 RT-LAMP were missed by RT-PCR. This is basically consistent with prior results reported by Hosaka et al. Recently, Curtis  and Hosaka  reported that RT-LAMP assay for detection of HIV-1 RNA and DNA. Curtis et al. applied the LAMP technology to detect the HIV-1 in plasma and the whole blood of infected individuals for the first time.  Then, Hosaka et al. developed the RT-LAMP assay to detect HIV-1 group M RNA in plasma.  However, they indicated that RT-LAMP could confirm assay the HIV-1. But, confirmatory tests for HIV antibodies (Western blot or immunofluorescence assay), in contrast to screening tests, are technically difficult to perform, require subjective interpretation. Desirable features in a confirmatory assay include a high degree of sensitivity, specificity, reproducibility, and the potential for automation.  In our study, no false-positive specimen was observed, but some research reports discovered that RT-LAMP might cause false-positive reactions. , Nonetheless, the reliability of this assay should be evaluated by large-scale specimen investigation immediately. Furthermore, to develop a RT-LAMP assay for detection simultaneously of HIV-1 and HIV-2 is very essential in endemic area.
The RT-LAMP method described in this study is a valuable alternative for the detection of HIV-1. Because the RT-LAMP assay is a simple diagnosis tool in which the reaction is carried out in a single tube by mixing the buffers, primers, reverse transcriptase, template, and DNA polymerase and incubating the mixture at 63°C for 1 h in a regular laboratory water bath that provides a constant temperature of 63°C, which is cheaper than the machines of PCR or ELISA. As a novel nucleic acid amplification method, this RT-LAMP was known as a rapid, specific, sensitive, cost-effective and easy-operating alternative for detection of clinical specimen. However, RT-PCR restricted its application to clinical laboratories as demanding operation and expensive kits and equipment. On the other hand, the specificity of the RT-LAMP is extremely high because it uses six primers recognizing eight distinct regions on the target DNA. Furthermore, considerably less time is required to obtain a result using SYBR Green I stain, compared with traditional gel electrophoresis. The assay is suitable to be used under field conditions for the rapid diagnosis of HIV-1 on site, which would allow expedited control and hygiene measures to be implemented to prevent spread of HIV-1 infection.
| ~ Acknowledgments|| |
This work was supported by Science Research Fund of Wuhan Institute of Technology (10113201), China; National Natural Science Foundation of China (81172178).
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[Figure 1], [Figure 2]
[Table 1], [Table 2]
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