Sensitive Multiplex Real-time RT-qPCR Assay for the Detection of Filoviruses

Abstract

Filoviruses are important etiological agents of emergent diseases with high mortality rates. Traditionally, filovirus fever diseases have primarily been a burden of African countries; however, global interconnectedness has increased the probability of the worldwide spread of filoviruses. Therefore, national healthcare organizations need tools for managing filovirus risk, including diagnostic kits based on real-time reverse transcription PCR (RT-qPCR), as this is the most suitable method for diagnosing filovirus fever diseases. Here we describe a real-time RT-qPCR assay for filovirus detection. This assay is a further development of our previously reported EBOV (Zaire)-Fl kit. Two sets (FiloA-Fl and FiloB-Fl) of real-time RT-qPCR assays for the detection of filoviruses were developed and evaluated using armored RNA phage particles (ARs) as positive controls. The limit of detection of the assay was 5x10² copies/ml of the AR-positive control for the FiloA-Fl set and 5x10³ copies/ml of the AR-positive control for the FiloB-Fl set. Our assay provides a rapid and sensitive tool for detecting filoviruses. The high specificity and sensitivity of the assay make it useful for clinical and epidemiologic investigations in the field of filovirus fever diseases and their etiological agents.

Filoviruses are important etiologic agents of emergent diseases with high mortality rates. Filovirus fever diseases have primarily been a burden of African countries, but global interconnectedness has increased the probability of their worldwide spread. Therefore, national healthcare organizations need tools for managing filovirus risk. This article describes a real-time RT-qPCR assay for filovirus detection.

The family Filoviridae comprises 7 species divided into 3 genera: Ebolavirus, Marburgvirus, and Cuevavirus. Four of these species—Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SUDV), Bundibugyo ebolavirus (BDBV), and Marburg virus (MARV)—are the causative agents of severe fevers among individuals with Ebola fever disease (EFD) and Marburg fever disease (MFD), which have similar clinical symptoms.¹ Reston ebolavirus (RESTV) is not a human pathogen, and Tai Forest ebolavirus (TAFV) has been detected only in 1 case of dengue-like syndrome, with a nonfatal outcome.² Lloviu cuevavirus was recently isolated from bats in Spain,³ though its pathogenicity in humans has not yet been studied.

Humanity first faced filovirus fevers in 1967, when an outbreak of MFD occurred in Germany and in the former Yugoslavia.⁴ Nine years later, in 1976, 2 simultaneous outbreaks of EFD with a total of 602 cases occurred in Sudan and Zaire. These outbreaks were caused by SUDV and EBOV, respectively.^5,6 Subsequently, EFD and MFD outbreaks occurred regularly but did not constitute a great threat to humanity, because they were always limited by the number of cases and usually localized in remote areas of Africa.

However, the situation changed in 2014, when the largest ever recorded epidemic of EFD spread throughout 5 African countries: Guinea, Liberia, Sierra Leone, Nigeria, and Senegal.⁷ A total of 28,646 cases and 11,323 deaths had been recorded by March 27, 2016.⁸ Despite joint efforts by the world community, elimination of the outbreak took nearly 2 years, and sporadic cases of EFD are still being reported in Guinea and Liberia.⁹ Another problem is the large number of convalescents, whose physiological liquids (ie, aqueous humor, sweat, urine, vaginal secretions, conjunctival fluid, feces, and breast milk) contain viable virus for a long time after clinical recovery.^10-12 Therefore, these individuals require close surveillance and control.

For this reason, rapid, sensitive, and specific methods for filovirus detection are needed for the diagnosis of EFD and MFD as well as for the surveillance and identification of viral sources in the environment. Here, we describe a real-time reverse transcription PCR (RT-qPCR) assay for the detection of filoviruses. This assay is a further development of our previously reported EBOV (Zaire)-Fl assay.¹³

Materials and Methods

Identification of Conserved Sites and Design of the Assay

All sequences of SUDV, MARV, BDBV, RESTV, and TAFV available in GenBank (NCBI) were aligned to identify conserved sites suitable for targeting using real-time RT-qPCR. The alignments were performed using the BioEdit 7.2.5 software package (Ibis Biosciences, USA).

Next, a 135-nucleotide fragment of the NP gene (nt positions 1199-1334 in the reference sequence of MARV, GenBank accession number KR063674) (Figure 1), a 130-nucleotide fragment of the VP30 gene (nt positions 9012-9142 in the reference sequence of SUDV, GenBank accession number KT878488) (Figure 2a), a 97-nucleotide fragment of the VP40 gene (nt positions 4733-4830 in the reference sequence of TAFV, GenBank accession number KU182910) (Figure 2b), an 85-nucleotide fragment of the NP gene (nt positions 87-172 in the reference sequence of BDBV, GenBank accession number KU182911) (Figure 2c), and an 86-nucleotide fragment of the L gene (nt positions 12896-12959 in the reference sequence of RESTV, GenBank accession number JX477166) (Figure 2d) were manually selected as targets for amplification. Additionally, a 113-nucleotide fragment of the L gene, described previously, was used for ZEBOV targeting.¹³

Figure 1.

Partial Nucleotide Sequence Alignment of Marburg Virus (L-gen). Partial sequences of Marburg virus (MARV): JX458825-JX458858, Z12132, Z29337, AY358025, KC545387-KC545388, M72714. Sequences of the forward (f) and reverse (r) primers and probe (p) targeting this region are indicated in the frames.

Figure 2.

Partial Nucleotide Sequence Alignments of Filoviruses. A: Sudan ebolavirus (VP30-gen). Partial sequences of Sudan ebolavirus (SUDV): KC242783, KC545383, KC545389-KC545392, KC589025, AY729654, EU338380, FJ968794, JN638998. B: Tai Forest ebolavirus (VP40-gen). Partial sequences of Tai Forest ebolavirus (TAIFV): FJ217162, NC_014372, JA48902. C: Bundibugyo ebolavirus (NP-gen). Partial sequences of Bundibugyo ebolavirus (BDBV): JA489018, KC545393-KC545396, FJ217161, NC_014373, HC918461. D: Reston ebolavirus (L-gen). Partial sequences of Reston ebolavirus (RESTV): AY769362, AB050936, JX477166, FJ621585, FJ621583, JX477165, FJ621584, AF522874, NC_004161. Sequences of the forward (f) and reverse (r) primers and probes (p) targeting these regions are indicated in the frames.

The probability of homodimer, heterodimer, or hairpin formation by the primers and probes was optimized using Oligonucleotide Properties Calculator (http://biotools.nubic.northwestern.edu/OligoCalc.html) and UNA Fold Web Server (http://unafold.rna.albany.edu).

We divided our assay into 2 sets due to limitations of the number of fluorescence channels for the real-time PCR instrument. The first FiloA-Fl set was designed to detect the “most frequent” filoviruses: ZEBOV, SUDV, and MARV. The second FiloB-Fl set was designed to detect BDBV, TAIFV, and nonpathogenic to humans RESTV.

Real-time Reverse Transcription qPCR Assay

A real-time RT-qPCR assay was designed for the detection of filoviruses (Tables 1 and 2). The sequences of all primers and specific probes were tested using BLAST. If necessary, nucleotides were added to the 3'-ends of the probes to form a hairpin. A ZEBOV-specific probe and a TAFV-specific probe were covalently attached to the fluorescent reporter dye carboxyfluorescein (FAM) at the 5' ends and to the black hole quencher (BHQ1) at the 3' ends. A SUDV-specific probe and a BDBV-specific probe were covalently attached to the fluorescent reporter dye rhodamine 6G (R6G) at the 5' ends and to the black hole quencher (BHQ1) at the 3' ends. A MARV-specific probe and a RESTV-specific probe were covalently attached to the fluorescent reporter dye rhodamine X (ROX) at the 5' ends and to the black hole quencher (BHQ2) at the 3' ends. The primers and probes were synthesized by the Bioorganic Synthesis Branch in the Central Research Institute for Epidemiology, Moscow.

Table 1.

Design of the FiloA-Fl Set

Species	Primer/probe	Sequence (5′-3′)	Probe Type	Product Size	Gene Target
EBOV	rt-EBOV-f	ATT TgA ATg ggg TCC AAT TgC C	TaqMan	113 bp	L
	rt-EBOL-r	AAg CAg TRC CTA TAC Tag CCA
	EBOV-prb	FAM-AgT CCC TTA AAA Cgg CTA CAA GAA Tgg gAC-BHQ1
SUDV	rt-SUDV-f	AgA gTC ATC TTA ggA ggg AAA AC	TaqMan	130 bp	VP30
	rt-SUDV-r	TgA TTg TCT ATC CCA CTg TTg C
	SUDV-prb	R6G-AgA CCA AgC TgA ATC AgT TCT CgA ggT C-BHQ1
MARV	rt-MARV-f	ACT gAA ATC ACA CAC AgT CAg AC	TaqMan	135 bp	NP
	rt-MARV-r	ACT gTg ACA CCC gAT TCT gTg
	MARV-prb	ROX-TWg CCg TCC TCA gCC AgA AAC gg-BHQ2
IC-STI87-rec	STI-87f	N/A	TaqMan	117 bp	Artificial target
	STI-87r	N/A
	STI-87-prb	Cy5-(X)₂₅-BHQ2

Table 2.

Design of the FiloB-Fl Set

Species	Primer/probe	Sequence (5′-3′)	Probe Type	Product Size	Gene Target
TAFV	rt-TAFV-f	TAT CTg gCC CgA AAg TgC TgA Tg	TaqMan	97 bp	VP40
	rt-TAFV-r	Tgg TAT ATg ATg CTA gCA TAA Tgg CAg
	TAFV-prb	FAM-CAA ATC CCA ATC Tgg CTT CCT CTg ggA T-BHQ1
BDBV	rt-BDBV-f	ATT gAA ATT gAg ATT CTA ATC TCg AC	TaqMan	85 bp	NP
	rt-BDBV-r	TgC CAA ggA gCA gAT gAC TTC
	BDBV-prb	R6G-TCC CCA ATA CCA ACA CTg AgA ATT ggg-BHQ1
RESTV	rt-RESTV-f	TTC ACT ACC CAT gAT TAA ggA TC	TaqMan	86 bp	L
	rt-RESTV-r	AAg TCA CTA ATC ACC TTY gTA gAg
	RESTV-prb	ROX-TCT ATg ggA ATT CTA TCA TCT TAA TCA CCC ATA-BHQ2
IC-STI87rec	STI-87f	N/A	TaqMan	117 bp	Artificial target
	STI-87r	N/A
	STI-87-prb	Cy5-(X)₂₅-BHQ2

The targeting fragments were amplified and quantified in 2 25-μl reactions containing 10 μl of the sample (RNA template and RNase-free water), 0.2 μM each of forward and reverse primer, 0.2 μM of each specific probe, 2.5 μl dNTPs (1.76 mM, AmpliSens, Russia), 5 μl 1-step RT-PCR mix2 FEP/FRT (AmpliSens, Russia), 0.25 μl TM-revertase (AmpliSens, Russia), and 0.5 μl TaqF polymerase (AmpliSens, Russia).

The thermal cycling parameters were 50°C for 15 min, followed by 95°C for 15 min, and then 45 cycles of 95°C for 10 s and 60°C for 20 s. Fluorescence was observed at 60°C in a Rotor-Gene Q (Qiagen, Germany).

Positive DNA Controls

A PCR fragment containing the target regions and some flanking nucleotides was generated using step-out amplification, as previously described.¹³ The final PCR products were purified using a MinElute Gel Extraction kit (Qiagen, Germany), ligated into the pGEM-T plasmid vector (Promega, USA) and transformed into Escherichia coli (XL1-Blue strain).¹⁴ Recombinant plasmids from individual clones were purified using a Plasmid Miniprep kit (Axygen, USA), and the orientation and absence of mutations in the cloned PCR fragment were evaluated by Sanger sequencing using an ABI-Prism 3500 XL (Applied Biosystems, USA). Plasmids were quantified, diluted in elution buffer (AmpliSens, Russia), mixed in equal amounts, and used as external positive controls (EPC) in the FiloA-Fl and FiloB-Fl sets of the assay.

Plasmid quantification was achieved via droplet digital PCR using the QX100 system (Bio-Rad, USA) with ddPCR Supermix for Probes (Bio-Rad, USA) and species-specific primers and probes.

Armored Positive RNA Controls

Armored RNA phage particles (ARs) were prepared as positive RNA controls for real-time RT-PCR detection of filoviruses, as previously described.¹³ After RNA extraction using the RIBO-prep extraction kit (AmpliSens, Russia), the ARs were quantified by RT droplet digital PCR using the QX100 system (Bio-Rad, USA), One-step ddPCR Supermix for Probes (Bio-Rad, USA), and appropriate filovirus-specific primers and probes. The ARs were mixed in equal amounts and used as external positive armored controls (ARCs) in the FiloA-Fl and FiloB-Fl sets of the assay.

Armored Internal Control

To assess the efficiency of RNA extraction, the STI-87rec (AmpliSens, Russia) exogenous internal control (IC) was added to the reagent mixture. STI-87rec is an artificial RNA sequence (150 nt, GC content 50%) surrounded by an MS2-derived protective protein coat. STI-87rec-specific primers and a Cy5-labeled probe were included in the reaction mixture of the FiloA-F1 and the FiloB-Fl sets.

Analytical Sensitivity

Analytical sensitivity was evaluated using a series of 10-fold dilutions of ARs. Briefly, known concentrations of ARs were diluted 10-fold using RNase-free elution buffer (AmpliSens, Russia), added to serum for a final volume of 100 μl, extracted using the RIBO-prep extraction kit (AmpliSens, Russia, in accordance with the manufacturer's instructions), and then tested in the FiloA-Fl and FiloB-Fl sets to establish standard curves and the limit of detection (LOD). The limit of detection was set as the minimal dilution detected in 3 replicates.¹⁵

Analytical Specificity

The ability of the developed assay to detect ZEBOV, SUDV, and MARV was evaluated using high-titer RNA from ZEBOV, SUDV, and MARV, which were kindly provided by State Research Center of Virology and Biotechnology VECTOR. Potential cross-reactivity was assessed using high-titer solutions of viral RNA and DNA from 19 viral species belonging to 9 viral families, which are a part of the collection of Central Research Institute for Epidemiology. The summary of RNA/DNA used in the study is shown in Table 3.

Table 3.

List of viral species used in this study

Species	Abbreviation	Family	Genus	Type of Nucleic Acid
Zaire ebolavirus	EBOV	Filoviridae	Ebolavirus	RNA
Sudan ebolavirus	SUDV	Filoviridae	Ebolavirus	RNA
Marburg virus	MARV	Filoviridae	Marburgvirus	RNA
Lassa virus	LASV	Arenaviridae	Arenavirus	RNA
Tahyna virus	TAHV	Bunyaviridae	Orthobunyavirus	RNA
Batai virus	BATV	Bunyaviridae	Orthobunyavirus	RNA
Inkoo virus	INKV	Bunyaviridae	Orthobunyavirus	RNA
Crimean-Congo hemorrhagic fever virus	CCHFV	Bunyaviridae	Nairovirus	RNA
Dhori virus	DHOV	Orthomyxoviridae	Thogotovirus	RNA
Flu A/H1N3	FLUAV(H1N3)	Orthomyxoviridae	Influenzavirus A	RNA
Flu A/H3N2	FLUAV(H3N2)	Orthomyxoviridae	Influenzavirus A	RNA
Flu B	FLUBV	Orthomyxoviridae	Influenzavirus B	RNA
Yellow fever virus	YFV	Flaviviridae	Flavivirus	RNA
West Nile virus	WNV	Flaviviridae	Flavivirus	RNA
Zika virus	ZIKV	Flaviviridae	Flavivirus	RNA
Tick-borne encephalitis virus	TBEV	Flaviviridae	Flavivirus	RNA
Sindbis virus	SNDBV	Togaviridae	Alphavirus	RNA
Chikungunya virus	CHIKV	Togaviridae	Alphavirus	RNA
Rubella virus	RUBV	Togaviridae	Rubivirus	RNA
Kemerovo virus, strain 21/10	KEMV-21/10	Reoviridae	Orbivirus	RNA
Tribec virus, strain Tr19	TRBV-Tr19	Reoviridae	Orbivirus	RNA
Human rotavirus A	RVA	Reoviridae	Rotavirus	RNA
Enteric cytopathic human orphan virus 11	ECHO11	Picornaviridae	Enterovirus	RNA
Human immunodeficiency virus 1	HIV-1	Retroviridae	Lentivirus	RNA
Rabies virus	RABV	Rhabdoviridae	Lyssavirus	RNA
Human cytomegalovirus 5	HCMV-5	Herpesviridae	Cytomegalovirus	DNA
Human parvovirus B19	B19	Parvoviridae	Erythroparvovirus	DNA
Middle East respiratory syndrome coronavirus	MERS	Coronaviridae	Betacoronavirus	RNA

Diagnostic Sensitivity and Specificity

Diagnostic sensitivity and specificity were assessed using serum samples from ZEBOV-positive (n = 51) and ZEBOV-negative (n = 164) patients with clinical symptoms of fever, as well as from individuals without any clinical symptoms (n = 50). All human samples were collected, previously tested for ZEBOV, and kindly provided by the Virology Laboratory of Hemorrhagic Fevers Research Project, Gamal Abdel Nasser University, Guinea Conakry. A RealStar^® Ebolavirus RT-PCR Kit 1.0 (Altona Diagnostics GmbH) was used for ZEBOV detection. Viral RNA was extracted from 140 μl of serum using the QIAmp viral RNA kit (Qiagen, Germany) and examined immediately after extraction.

Results

Two sets (FiloA-Fl and FiloB-Fl) of real-time RT-PCR assays for the detection of filoviruses were developed and evaluated. The melting temperatures and GC contents of all primers and the length of the amplicons were adjusted to be similar for efficient multiplex reactions. The analytical specificity of the assay was studied using a representative sampling of viral and human RNA/DNA. No cross-reactions were observed. The LOD of the assay was 5 × 10² copies/ml of the AR-positive control for the FiloA-Fl set and 5 × 10³ copies/ml of the AR-positive control for the FiloB-Fl set. Standard detection was linear in the range from 10⁵ copies/ml (Ct 28) to 5 × 10² copies/ml (Ct 36) for the EBOV AR (R² = 0.99), 10⁵ copies/ml (Ct 27) to 5 × 10² copies/ml (Ct 37) for the SUDV AR (R² = 0.98), 10⁵ copies/ml (Ct 27) to 5 × 10² copies/ml (Ct 37) for the MARV AR (R² = 0.98), 10⁵ copies/ml (Ct 31) to 10³ copies/ml (Ct 39) for the TAIFV AR (R² = 0.99), 10⁵ copies/ml (Ct 29) to 10³ copies/ml (Ct 36) for the BDBV AR (R² = 0.97), and 10⁵ copies/ml (Ct 31) to 10³ copies/ml (Ct 39) for the RESTV AR (R² = 0.98). Within these ranges, efficiencies were assessed at 94% for EBOV, 87% for SUDV, 97% for MARV, 91% for TAIFV, 99% for BDBV, and 86% for RESTV.

A total of 215 clinical samples and 50 samples from healthy humans assessed by the Virology Laboratory of Hemorrhagic Fevers Research Project, Gamal Abdel Nasser University, Guinea Conakry, were also simultaneously tested using RealStar^® Ebolavirus RT-PCR Kit 1.0 (Altona Diagnostics GmbH), with 51 samples testing positive and 214 negative. No discordant samples were observed. Thus, diagnostic sensitivity regarding ZEBOV and diagnostic specificity reached 100%.

Discussion

Filoviruses are important etiologic agents of emergent diseases with high mortality rates. Filovirus fever diseases were traditionally a burden in African countries, but global interconnection has raised the probability of filoviruses spreading worldwide. Therefore, national healthcare organizations need tools for managing filovirus fever disease risks. These tools should include diagnostic kits based on RT-qPCR, because it is the most suitable method compared with enzyme-linked immunosorbent assay (ELISA) or rapid diagnostic tests (RDTs) for filovirus fever disease diagnosis.¹⁶ Many assays and commercial kits for filovirus fever disease diagnosis have been developed in several countries.^15,17-19 Some of these kits have been approved by the World Health Organization,²⁰ but only a few were evaluated during the recent epidemic in West Africa.

The assay described above is mainly intended for use in the mobile laboratory of the Russian special antiepidemic team (SAET) and in the Russian Federation for diagnostic purposes. Unfortunately, we faced some limitations during assay development, namely the lack of RESTV, BDBV, and TAIFV RNAs. Therefore, our assay was evaluated using clinical samples only for ZEBOV and using ARs as well as in silico analysis for the other filoviruses. Nevertheless, the analytical characteristics of the developed assay are comparable with the best of any developed assays for Ebola fever disease and filovirus fever disease diagnostics (see Table 4).²⁰ Based on our experience of using the EBOV (Zaire)-Fl assay during the Ebola outbreak in Guinea in 2014-2016, we expect this assay to work well.

Table 4.

Comparison of IVD Assays that Have Been Approved for EVD Diagnostics

Assay Name	Manufacturer	Kit Format	Viruses Detected	Gene Target	Controls	Through-put: Time to Results	Limit of Detection (LOD)	Approval
RealStar^® Filovirus RT-PCR Kit 1.0 (CE-IVD)	Altona Diagnostics GmbH (Qiagen)	RT-qPCR	ZEBOV, BEBOV, RESTV, SEBOV, TAFV, MARV	L	^†C+, IVTC,^{a ††}IC (Globin)	4-6 (2)^b	3,4 × 10³ copies/ml	WHO
RealStar^® Ebolavirus RT-PCR Kit 1.0 (FDA EUA)	Altona Diagnostics GmbH (Qiagen)	RT-qPCR	ZEBOV, BEBOV, RESTV, SEBOV, TAFV	L	C+, IVTC, IC (Globin)	4-6 (2)	3,4 × 10³ copies/ml	WHO/ USFDA
Liferiver™ Ebola virus (EBOV) real time RT-PCR kit	Shanghai ZJ Bio-Tech Co., Ltd	RT-qPCR	ZEBOV, BEBOV, RESTV, SEBOV, TAFV without differentiation	NP	C+, IC (Globin)	4-6 (2)	4,23 × 10⁴ copies/ml	WHO
Xpert^® Ebola Assay	Cepheid	Lab-on-chip RT-qPCR	ZEBOV	NP,GP	C+, IVTC, IC (Globin)	1,5	(1,34-4,23) × 10⁴ copies/ml	WHO/ USFDA/EUFDA
FilmArray Biothreat-E test	BioFire, Biomerieux	Lab-on-chip RT-qPCR	ZEBOV	L	—	1.25	6 × 10⁴ PFU/ml = 2 × 10⁵ copies/ml	USFDA
FilmArray NGDS BT-E Assay	BioFire Defense, LLC for US Dept. of Defense	Lab-on-chip RT-qPCR	ZEBOV	NP	—	1.25	10⁴ PFU/ml = 3,4 × 10⁴ copies/ml	USFDA
LightMix^® Ebola Zaire rRT-PCR Test	Roche	RT-qPCR	ZEBOV	L	C+, IVTC, IC (Globin)	4-6 (1,5)	4.78 PFU/ml = 2 × 10⁴ copies/ml	USFDA
EZ1 Real-time RT PCR Assay	US Dept. of Defense (in house)	RT-qPCR	ZEBOV	GP	C+, IVTC, IC (Globin)	4-6 (1,5)	5 PFU/ml = 2 × 10⁴ copies/ml	USFDA
CDC Ebola Virus NP Real-time RT-PCR Assay	US CDC (in house)	RT-qPCR	ZEBOV	NP	C+, IVTC, IC (Globin)	4-6 (1,5)	30 TCID₅₀ /reaction	USFDA
CDC Ebola Virus VP40 Real-time RT-PCR Assay	US CDC (in house)	RT-qPCR	ZEBOV, SUDV,	VP40	C+, IVTC, IC (Globin)	4-6 (1,5)	30 TCID₅₀ /reaction	USFDA
Vector-PCR_RV-Ebola-RG	«Vector» (in house), Russia	qPCR	ZEBOV, SUDV, without differentiation	L	C+	4-6 (1,5)	2 × 10³ copies/ml	Roszdravnadzor of the Russian Federation
AmpliSens® EBOV Zaire-FL	CRIE, Russia	RT-qPCR	ZEBOV	L	C+, ARPC,^c IC (STI)	4-6 (1,5)	5 × 10² copies/ml	Roszdravnadzor of the Russian Federation
OM-Screen-ZEBOV/SUDV/MARV-RV	Sintol Ltd., Russia	RT-qPCR	ZEBOV, SUDV, MARV	—	C+, IVTC, IC (Globin)	4-6 (1,5)	10³ copies/ml	Roszdravnadzor of the Russian Federation

IVTC = in vitro–transcribed positive control.

Time to results, excluding extraction stage, is shown in brackets.

ARPC = armored positive control.

†

C+ = positive PCR control.

††

IC = internal control.

Conclusions

This article reports the development of an RT-PCR assay for filovirus detection. The assay contains all of the necessary components to perform the analysis, including an AR-positive control, positive control for PCR, and an armored internal control. The advantage of this assay is that it allows the verification of all steps of the analysis, including extraction, reverse transcription, and PCR.

Our results revealed that the LOD of the assay is in the range of 5 × 10² to 5 × 10³ copies/ml of ARs. Thus, our real-time RT-PCR assay provides a fast and sensitive tool for filovirus detection. The high specificity and sensitivity of the assay makes it useful for clinical and epidemiologic investigations in the field of filovirus fever diseases.

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