Rapid and Real-time Assays for Detection and Quantification of Chikungunya Virus

MM Parida; SR Santhosh; PK Dash; PV Lakshmana Rao

Disclosures

Future Virology. 2008;3(2):179-192. 

In This Article

RT-loop-mediated Isothermal Amplification

Collectively, the real-time RT-PCR amplification methods have several intrinsic disadvantages requiring either a high precision instrument for amplification or an elaborate, complicated method for detection of amplified products. As a result, these rapid molecular tests might not be the method of choice in basic clinical settings of developing countries or in field situations owing to the requirement for sophisticated instrumentation and expensive reagents. It is, therefore, essential to develop simple and economical molecular tests for the above scenarios. In this regard, the loop-mediated isothermal amplification (LAMP) method developed by Eiken Chemical Co. Ltd (Japan) has the potential to replace PCR as a result of its simplicity, rapidity, specificity and cost-effectiveness.

LAMP is a novel method of gene amplification that amplifies nucleic acid with high specificity, efficiency and rapidity under isothermal conditions by employing a set of six specially designed primers that span eight distinct target sequences.[30,31] The LAMP assay is a simple diagnostic tool that is carried out in a single tube by mixing of the buffer, primers, reverse transcriptase and DNA polymerase, and incubating the mixture at 63°C for 30 min. Amplification can be done with RNA templates following the same procedure as with DNA templates, simply through the addition of reverse transcriptase. The detection of gene amplification can be accomplished by agarose gel electrophoresis as well as by real-time monitoring in an inexpensive turbidimeter. In addition, the gene amplification can also be visualized either as turbidity in the form of white precipitate or by employing a fluorescent intercalating dye (SYBR Green I) and using a UV lamp.[32] Being an isothermal amplification, LAMP does not require a thermal cycler and thus can be performed simply with a heating block and/or water bath.

Compared with RT-PCR and real-time PCR, LAMP has the advantages of reaction simplicity and detection sensitivity. The higher sensitivity and specificity of the LAMP reaction is attributed to continuous amplification under isothermal conditions employing six primers recognizing eight distinct regions of the target. Additionally, the higher amplification efficiency of the LAMP reaction yields large amounts of by-product (pyrophosphate ion) leading to a white precipitate of magnesium pyrophosphate in the reaction mixture. Since the increase in turbidity of the reaction mixture - according to the production of precipitate - correlates with the amount of DNA synthesized, real-time monitoring of the LAMP reaction can be achieved by real-time measurement of turbidity. The LAMP assay has emerged as a powerful gene amplification tool for the rapid identification of microbial infections and is being increasingly used by various investigators for the rapid detection and typing of emerging viruses, to include WNV, severe acute respiratory syndrome (SARS), dengue, JEV and so on.[33,34,35,36]

Parida et al. reported the standardization and validation of a one-step single tube accelerated quantitative RT-LAMP (LAMP for RNA amplification) assay targeting the E1 gene for rapid and real-time detection of CHIKV.[37] A set of six primers comprising two outer, two inner and two loop primers that recognize eight distinct regions on the target sequence was designed, employing the LAMP primer designing support software program (Net laboratory, Japan).[38] The details of primer design are shown in Figure 5.[37] Optimal assay conditions with zero background were established for the detection of low levels of CHIKV in acute-phase patient serum samples. The RT-LAMP was found to be tenfold more sensitive than RT-PCR with a detection limit of 20 copy numbers (compared with 200 copy numbers for RT-PCR indicated by the presence of a 205 bp amplicon [Figure 6]).[37] The specificity of the RT-LAMP primers for the E1 gene of CHIKV was established by ruling out the cross reactivity with members of the Flavivirus group that display similar clinical symptoms, such as dengue 1-4, JEV, WNV (e.g., 101) and St. Louis Encephalitis virus (Parton strain), in addition to healthy human serum samples. The quantification of the viral load in the positive samples was extrapolated on the basis of their time of positivity employing the standard curve (Figure 7A).[37] Most of the patient serum samples were found to have very high virus concentration within the range of 2 × 103-2 × 106 copy numbers (Figure 7B).[37]

Primer designing for CHIKV RT-LAMP assay. Construction of two inner primers (FIP & BIP) having both sense and antisense sequences that help in loop formation is depicted. F1C and B2C are the complementary sequences of F1 and B2, respectively. B3: Backward outer primer; BIP: Backward internal primer; BLP: Backward loop primer; CHIKV: Chikungunya virus; F3: Foward outer primer; FIP: Foward internal primer; FLP: Foward loop primer; LAMP: Loop-mediated isothermal amplification; RT: Reverse transcription.

Comparative sensitivity of RT-LAMP with RT-PCR for detection of the CHIKV E1 gene. (A) Sensitivity of RT-LAMP assay as monitored by real-time measurement of turbidity. Shown from left to right are the curves of decreasing concentration of virus from 2 × 108 to 2 × 10-1 copy number of the template in serial tenfold dilution. The detection limit for the assay was 20 copy numbers. (B) Sensitivity of RT-PCR for the detection CHIKV E1 gene as observed by 205-bp amplicon on agarose gel analysis with a detection limit of 200 copy numbers. Lane M: 100 bp DNA ladder (Sigma, USA); Lane 1-11: Different concentration of virus ranging from 2 × 108 to 2 × 10-1 in serial tenfold dilution pattern. CHIKV: Chikungunya virus; LAMP: Loop-mediated isothermal amplification; RT: Reverse transcription.

Quantification of virus load in acute phase patient serum samples by CHIKV RT-LAMP assay. (A) Standard curve for CHIKV-specific RT-LAMP assay generated from the amplification plots between tenfold serially diluted plasmid construct and time of positivity. (B) Quantitative determination of virus concentration in clinical samples employing standard curve. CHIKV: Chikungunya virus; LAMP: Loop-mediated isothermal amplification; RT: Reverse transcription.

The feasibility of CHIKV RT-LAMP for clinical diagnosis was validated with a total of 89 samples comprising 69 acute-phase serum samples and 20 serum-negative samples collected from southern India during the 2006 epidemic. The RT-LAMP assay demonstrated exceptionally higher sensitivity compared with conventional RT-PCR by detecting 21 additional positive cases (p <0. 0001).[37] The field applicability of RT-LAMP assay was also validated by employing an SYBR Green I-mediated naked-eye visualization test. Following incubation at 63°C for 30 min (water bath), the monitoring of RT-LAMP amplification was accomplished by naked eye with the addition of 1 µl of SYBR Green I (1:1000) dye to the amplified products (Figure 8).[37] The comparative evaluation of this field-based SYBR Green I-based RT-LAMP assay with more than 200 clinical samples revealed a very good concordance (93%) with that of RT-PCR. This SYBR Green I-mediated naked-eye visualization RT-LAMP test can be easily used in any peripheral healthcare center by using a simple heating block or routine laboratory water bath, which can provide a constant temperature of 60-65°C. These findings demonstrated that RT-LAMP assay is a valuable tool for rapid, real-time detection as well as quantification of CHIKV in acute phase serum samples without the requirement for any sophisticated equipment, and has potential applications for clinical diagnosis and surveillance of CHIKV in developing countries.

SYBR® Green I fluorescent dye-mediated monitoring of CHIKV RT-LAMP amplification. (A) Naked eye inspection under normal light. The original orange color of the SYBR Green I changed to yellow in the case of positive amplification whereas in a negative control having no amplification, the original orange color is retained. (B) The visual observation of green fluorescence of DNA binding SYBR Green I under UV light. CHIKV: Chikungunya virus; LAMP: Loop-mediated isothermal amplification; RT: Reverse transcription.

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