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Primers and Probes for Zika Virus Detection

Primers and Probes for Zika Virus Detection


Current molecular assays for flaviviruses use specific primers to amplify viral RNA for their detection. If these primers are to specific they may amplify RNA only form one species, or a range of closely related species. To enable a differential diagnostic, a broad range PCR assays may need to be developed to detect all flaviviruses. Historically, several diagnostic protocols using different primer sets have been developed and tested.

To enable detection of newly emerging flaviviruses a two stage process will be needed, as was recommended by Kuno in 1998.

1:  Initially broad range group-reactive primers are used to narrow the range of targets. 

2:  Species-specific primers are used next to determine which flavivirus is actually present.


However, if a totaly new speciies has emerged RNA sequencing of the genome will be needed for its characterization.

Zika virus (ZIKV) is an arbovirus transmitted by mosquitoes. Arboviruses are viruses that belong to any of several groups of RNA-containing viruses that are transmitted by bloodsucking arthropods, such as ticks, fleas, or mosquitoes. These viruses can cause encephalitis, yellow fever, or dengue fever, or similar feverish symptoms. Zika virus is an emerging mosquito-borne flavivirus first circulating in Asia and Africa but now also in the new world. When humans are infected an influenza-like syndrome is induced that is associated with retro-orbital pain, oedema, lymphadenopathy, or diarrhea. Flaviviruses include several pathogenic agents that can cause severe illness in humans. Some of them are known to expand their geographical range.

T
he Flaviviridae family consists of more than 70 virus species. It includes many arthropodae (insect, spider, crustacean)-borne viruses including the highly pathogenic yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV) and dengue virus (DENV). 

Flaviviruses are grouped into three epidemiologically distinct groups:

(1) The mosquito-borne group,
(2) The tick-borne group,
(3) and the unknown vector viruses.

These viruses are enveloped positive-stranded RNA viruses with a genome of approximately 11 kB. The viral genome encodes a single large polyprotein from which three structural proteins and seven non-structural proteins are produced. Structural proteins are Capsid (C), Envelope (E), and Membrane (M) protein. Non-structural proteins are NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 proteins.

Clinical diagnosis of flavivirus infections can be difficult due to unspecific symptoms. Typically symptoms can vary from mild to severe as well as to viral hemorrhagic fever. Many of these viruses are transmitted through a common vector. Diagnosis of Zika fever requires virus isolation and serology. However, this type of diagnostic is time-consuming or cross-reactive. During the first week of the infection, viral RNA can often be identified in serum. Therefore, RT-PCR is the preferred test for the Zika virus. Because the presence of a virus in the blood stream decreases over time, a negative RT-PCR collected 5-7 days after symptom onset does not exclude a Zika virus infection. Therefore serologic testing should also be performed as a follow-up test.


Design of primers and probes


For the design of primers and probes full-length genomic sequences for flaviviruses can be retrieved from NCBI or similar databases. Various bioinformatic tools are available for sorting and alignment of sequences. The goal is to identify conserved sequence regions to allow for the detection of the virus families in a first pass analysis. Results from sequence alignments of different published virus strain sequences can be used to identify highly conserved regions as well as highly divergent regions identified in flavivirus sequences enabling the design of unique primer and probe sets or pairs to allow for detection of unique virus strains.

Patel et al. (2013) developed a one-step quantitative reverse transcription PCR for the rapid detection of flaviviruses. The research group designed a rapid, sensitive TagMan probe-based quantitative RT-PCR (qRT-PCR) assay for the simultaneous detection of several flaviviruses. For the design of the Pan-Flavi primers, the conserved NS5 gene region was used. The primers and probes tested by Patel et al. are listed in table1:

Table 1: Oligonucleotide sequence of primers and probes used in Pan-Flavi qRT-PCR assay

 

Primer / Probe

Sequence

pmol

Orientationa

Positionb

Tm (°C)

Flavi all S

TACAACATgATggggAARAgAgARAA

10

S

8993–9019

54.7

DEN4 F

TACAACATgATgggRAAACgTgAGAA

10

S

8996–9019

60.18

Flavi all AS 1

gTCCCANCCDgCKgTRTC

10

AS

9236–9253

52.5

 

gTCCCATCCAgCKgTRTCATC

5

AS

9236–9256

57.1

Flavi all AS 2

gTgTCCCAgCCNgCKgTgTCATCWgC

10

AS

9232–9260

69.6

Flavi all probe 1

FAM-AARggHAgYMgNgCCA+TH+T+g+g+T-BBQ

5

S

9044–9065

69–83

Flavi all probe 2

FAM-Tg+gTWYATgTggYTNg+gRgC-BBQ

5

S

9062–9082

62–75

Flavi all probe 3 mix

FAM-Tg+gTWYATgT+ggYTNg+gRgC—BBQc

5

S

9062–9082

66-79

 

FAM-CCgTgCCATATggTATATgTggCTgggAgC-BBQd

0.5

S

9052–9081

74.7

 

FAM-TTTCTggAATTTgAAgCCCTgggTTT-BBQe

0.5

S

9086–9012

68

Flavi all S2

TACAACATgATgggMAAACgYgARAA

10

S

8996–9019

58.2

Flavi all AS4

gTgTCCCAGCCNgCKgTRTCRTC

10

S

9235–9260

64.1

 

Tm: temperature, Bridged-Nucleic Acid (LNA) bases are written as ‘ + _’, e.g., +A.

Degenerate bases: R = (A/G), W = (A/T), K = (T/G), Y = (C/T), N = (A/G/T/C).

a S: sense orientation, AS: antisense orientation.

b YFV strain; accession no: NC 002031.

c Flavi all probe, d Flavi probe YFV, e Flavi probe DEN4.

Final Pan-Flavi assay comprise of Flavi all S, Flavi all S2 & Flavi all AS4 primer and Flavi all probe 3 mix.

 

Patel et al. state in their paper: “At present, RT-PCR in nested or hemi-nested format is used most frequently. It requires sequencing for the identification of viruses and needs approximately one day for experimentation. In addition, this carries a high risk of contamination caused by open handling of PCR products, increasing the potential for false positives. In contrast, the Pan-Flavi assay presented here requires only some 50 minutes for specific and sensitive detection of several flaviviruses in one reaction. The detection sensitivity of YFV, DENV, TBEV, JEV and WNV, using the described Pan-Flavi assay, was close to or as good as the species-specific qRT-PCR assays. These differences may have resulted from deviant reactions conditions, enzyme kits and instruments, which were used as published or in-house established, and it is to mention, that WNV and JEV assays were performed in a two-step PCR which is known to be more sensitive than the one-step procedure.”

Maher-Sturgess et al. (2008) described a degenerate primer set for the amplification of flavivirus RNA to generate an 800 base pair (bp) cDNA product. The amplified region encoded part of the methyltransferase and most of the RNA-dependent-RNA-polymerase (NS5) coding sequence. One-step RT-PCR testing allowed the isolation of the RNA fragment of 60 different flavivirus strains and sequencing of cDNA from each virus isolated. Database searches were used to confirm the identity of the template RNA.

According to Maher-Sturgess et al. (2008) the Flav100F and Flav200R primers have the potential to detect emerging and related flaviviruses without prior serological evidence or additional primer design. This primer pair allow for the rapid detection at the genus level. In addition Maher-Sturgess et al. designed a set of universal primer that allowed the amplification of different flaviviruses.

Table 2: Universial Primers for the detection of Flaviviruses via PCR amplification.

Primer

Sequence

Notes

YF-F

   aat tcc act cat gaa atg tac

Yellow fever virus

Flav100F

5’-AAY TCI ACI CAI GAR ATG TAY-3’

Maher-Sturgess et al. (2008)

Flav200R

5’-CCI ARC CAC ATR WAC CA-3’

 

 

 

 

Forward primer

5’-AAR TAC ACA TAC CAR AAC AAA GTG GT-3’

Faye et al. 2013

Reverse primer

5’-TCC RCT CCC YCT YTG GTC TTG-3’

Faye et al. 2013

16 nt BNA/LNA-probe

FAM-CTY AGA CCA GCT GAA R-BBQ

Faye et al. 2013

 

 

 

E F1269-F

5’-GAG GCT GGG AAA TGG CTG-3’

Grubaugh et al., 2013

E F2225-R

5’-CCT CCA ACT GAT CCA AAG TCC CA-3’

Grubaugh et al., 2013

NS3 F5015-F

5’-GTG GTT GGN CTG TAT GGN AA-3’

Grubaugh et al., 2013

NS3 F5807-R

5’-CCC ATT TCT GAG ATG TCA GT-3’

Grubaugh et al., 2013

NS5 F8276d-F

5’-AAY TCN CAN CAN GAR ATG TAY-3’

Grubaugh et al., 2013

NS5 F9063d-R

5’-CCN ARC CAC ATR WAC CA-3’

Grubaugh et al., 2013

 

 

 

Unifor

5’-tgg ggn aay srn tgy ggn ytn tty gg-3’

Faye et al. 2008

Unirev

5’-CCN CCH RNN GAN CCR AAR TCC CA-3’

Faye et al. 2008

Mounifor2

5’-GGR DRM DTB KWA AYV TGY GCN AWR TT-3’

Faye et al. 2008

Mounirev2

5’-CCN ATN SWR CTH CCH KHY YTR WRC CA-3’

Faye et al. 2008

ZIKVENVF

5’-GCT GGD GCR GAC ACH GGR ACT-3’

Faye et al. 2008

ZIKVENVR

5’-RTC YAC YGC CAT YTG GRC TG-3’

Faye et al. 2008

 

 

 

ZIKV 835

5’-TTG GTC ATG ATA CTG CTG ATT GC-3’

Lanciotti et al. 2007

ZIKV 911c

5’-CCT TCC ACA AAG TCC CTA TTG C-3’

Lanciotti et al. 2007

ZIKV 860-FAM

5’-CGG CAT ACA GCA TCA GGT GCA TAG GAG-3’

Lanciotti et al. 2007

ZIKV 1086

5’-CCGCTG CCC AAC ACA AG-3’

Lanciotti et al. 2007

ZIKV 1162c

5’-CCA CTA ACG TTC TTT TGC AGA CAT-3’

Lanciotti et al. 2007

ZIKV 1107-FAM

5’-AGC CTA CCT TGA CAA GCA GTC AGA CAC TCA A-3’

Lanciotti et al. 2007

 

 

 

3PNC-2R

5′-GCT CAG GGA GAA CAA GAA CCG-3′

Grard et al. 2007

Priming on viral RNA.

 

Reverse oligonucleotide located in the 3′ non-coding region.

NS3 region

 

 

LIV coding sequence

X1

5′-YIR TIG GIY TIT AYG GIW WYG G-3′

4913–4935

X2

5′-RTT IGC ICC CAT YTC ISH DAT RTC IG-3′

5707–5733

TB-5′UTR-S F

5′-AAA AGA CAG CTT AGG AGA ACA AGA-3′

NS5 gene

TB-3′UTR-R

5′-AGA ACA AGA ACC GCC CCC CC-3′

 

 

 

 

FLAVI-1 S

5’-AAT GTA CGC TGA TGA CAC AGC TGG CTG GGA CAC-3’

Ayers et l. 2006

FLAVI-2 A

5’-TCC AGA CCT TCA GCA TGT CTT CTG TTG TCA TCC A-3’

 

 

Segments 9273-9305 and 10,102-10,136 of the West Nile virus NY 2000 (GenBank accession #AF404756).

 

 

 

 

FU1

5’-TAC AAC ATG ATGGGA AAG AGA GAG AA-3’

Kuno 1998

Cfd2

5’-GTG TCC CAG CCG GCG GTG TCA TCA GC-3’

Kuno 1998

MA

5’-CAT GAT GGG RAA RAG RGA RRA G-3’

Kuno 1998

 Legend: N = A+C+G+T, R = A+G, W = A+T, Y= C+T, H = A+C+T;  V = A+C+G, M = A+C, I = inosine.

Reference


M. Ayers, D. Adachi, G. Johnson, M. Andonova, M. Drebot, R. Tellier; A single tube RT-PCR assay for the detection of mosquito-borne flaviviruses. Journal of Virological Methods Volume 135, Issue 2, August 2006, Pages 235–239.

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Chambers, T. J., Hahn, C. S., Galler, R., and Rice, Ch. M.; Flavivirus genome organization, expression and replication. Annu. Rev. Microbiol. 1990. 44:649-6488.


Oumar Faye, Ousmane Faye, Diawo Diallo, Mawlouth Diallo, Manfred Weidmann and Amadou Alpha Sall; Quantitative real-time PCR detection of Zika virus and evaluation with field-caught Mosquitoes. Virology Journal 2013, 10:311, 1-8.


Grard G
, Moureau G, Charrel RN, Lemasson JJ, Gonzalez JP, Gallian P, Gritsun TS, Holmes EC, Gould EA, de Lamballerie X.; Genetic characterization of tick-borne flaviviruses: new insights into evolution, pathogenetic determinants and taxonomy. Virology. 2007 Apr 25;361(1):80-92. Epub 2006 Dec 13.

Grubaugh, N. D., McMenamy, S. S., turell, M. J., and Lee, J. S.; Multigene dedection and identification of mosquito-borne RNA viruses using an oligonucleotide microarray. PLOS Negl Trop Dis 7(8): e2349. Doi: 10.1371/journal.pntd.0002349. 1-16.

Goro Kuno; Universial diagnostic RT-PCR protocol for arboviruses. Journal of Virological Methods 72 (1998) 27-41.


Lanciotti, R. S., Kosoy, O. L., Laven, J. J., Velez, J. O., Lambert, A. J., Johnson, A. J., Stanfield, S. M., Duffy, M. R., 2008. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerging Infectious Diseases Vol. 14, No. 8, August 2008. www.cdc.gov/eid.


Sheryl L Maher-Sturgess, Naomi L Forrester, Paul J Wayper, Ernest A Gould, Roy A Hall, Ross T Barnard and Mark J Gibbs; Universal primers that amplify RNA from all three flavivirus subgroups. Virology Journal20085:16 DOI: 10.1186/1743-422X-5-16.
http://virologyj.biomedcentral.com/articles/10.1186/1743-422X-5-16

Pranav Patel, Olfert Landt, Marco Kaiser, Oumar Faye, Tanja Koppe, Ulrich Lass, Amadou A Sall and Matthias Niedrig; Development of one-step quantitative reverse transcription PCR for the rapid detection of flaviviruses. Virology Journal 2013, 10 :58. http://www.virologyj.com/content/10/1/58 METHODOLOGY Open Access