Research Article A One-Step Real-Time RT-PCR Assay for the...

Research ArticleA One-Step Real-Time RT-PCR Assay for the Detection andQuantitation of Sugarcane Streak Mosaic Virus

Wei-Lin Fu12 Sheng-Ren Sun1 Hua-Ying Fu1 Ru-Kai Chen1 Jin-Wei Su2 and San-Ji Gao13

1Key Laboratory of Sugarcane Biology and Genetic Breeding Ministry of Agriculture Fujian Agriculture and Forestry UniversityFuzhou Fujian 350002 China2College of Life Sciences Fujian Agriculture and Forestry University Fuzhou Fujian 350002 China3Guangxi Collaborative Innovation Center of Sugarcane Industry Guangxi University Nanning Guangxi 530005 China

Correspondence should be addressed to Jin-Wei Su sujinwei8aliyuncom and San-Ji Gao gaosanjiyahoocom

Received 6 October 2014 Revised 4 December 2014 Accepted 4 December 2014

Academic Editor Akikazu Sakudo

Copyright copy 2015 Wei-Lin Fu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Sugarcane mosaic disease is caused by the Sugarcane streak mosaic virus (SCSMV genus Poacevirus family Potyviridae) which iscommon in some Asian countries Here we established a protocol of a one-step real-time quantitative reverse transcription PCR(real-time qRT-PCR) using the TaqMan probe for the detection of SCSMV in sugarcane Primers and probes were designed withinthe conserved region of the SCSMV coat protein (CP) gene sequences Standard single-stranded RNA (ssRNA) generated by PCR-based gene transcripts of recombinant pGEM-CP plasmid in vitro and total RNA extracted from SCSMV-infected sugarcane wereused as templates of qRT-PCRWe further performed a sensitivity assay to show that the detection limit of the assay was 100 copiesof ssRNA and 2 pg of total RNA with good reproducibility The values obtained were approximately 100-fold more sensitive thanthose of the conventional RT-PCR A higher incidence (686) of SCSMV infection was detected by qRT-PCR than that (486)with conventional RT-PCR in samples showing mosaic symptoms SCSMV-free samples were verified by infection with Sugarcanemosaic virus (SCMV) or Sorghum mosaic virus (SrMV) or a combination of both The developed qRT-PCR assay may become analternative molecular tool for an economical rapid and efficient detection and quantification of SCSMV

1 Introduction

Sugarcane mosaic virus (SCMV) Sorghum mosaic virus(SrMV) and Sugarcane streak mosaic virus (SCSMV) are thethree viruses associated with the mosaic disease of sugarcane[1] SCMV and SrMV belong to the genus Potyvirus familyPotyviridae while SCSMV has been recently assigned to thegenus Poacevirus of the same family [2] SCSMV that causedmosaic disease has been identified in many Asian countriesincluding Pakistan Bangladesh Sri Lanka Thailand IndiaVietnam Japan Indonesia and China [1 3ndash9] SCSMV isthe major causal pathogens of mosaic disease on sugarcanein India [10] and 10ndash15 yield loss has been attributed tothe disease [4] Recently SCSMV has been noted to becomewidespread in themainChinese sugarcane producing regions[9]

SCSMV viruses are rod-shaped with an average dimen-sion of 890 times 15 nm and are similar to the members of the

familyPotyviridae [4]Theviral genome consists of a positive-sense single-stranded RNA (ssRNA) of approximately 10kilobase (kb) in length excluding a poly-A tail at the 31015840terminus [6 8] SCSMV is transmitted vegetatively duringvirus-infected cane cutting under natural conditions [11 12]In addition to sugarcane sorghum was found to be a naturalhost for SCSMV [13] Moreover other Poaceae species suchas maize millet Sudangrass Johnson grass crabgrass andcrowfoot grass can be infected bymechanical sap inoculationand cutting knife by SCSMV [6 12 14 15] Triticum mosaicvirus (TrMV genus Poacevirus family Potyviridae) which isclosely related to SCSMV is transmitted by an eriophyid mite[16] whereas the aphidsAphis craccivoraMyzus persicae andRhopalosiphum maidis serve as vectors for the potyvirusesSCMVand SrMV [14]However a vector for the transmissionfor SCSMV has not yet been identified [10 11]

SCSMV SCMV and SrMV cause mosaic symptomssuch as interveinal chlorotic specks streaks or stripes on

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 569131 9 pageshttpdxdoiorg1011552015569131

2 BioMed Research International

sugarcane leaves and it is difficult to distinguish the causalvirus on the basis of these symptoms alone Moreover thesymptom expression can vary depending on age of infectionenvironment and host cultivar [1] Thus it is important todistinguish the three viruses accurately and reliably Manydiagnostic methods are available for the detection of SCSMVin sugarcane plants including microscopic examination ofviral morphology [4] double antibody sandwich-enzymelinked immunosorbent assay (DAS-ELISA) and direct anti-gen coating- (DAC-)ELISA tests [17] reverse transcriptionpolymerase chain reaction (RT-PCR) [10] immunocapture-RT-PCR (IC-RT-PCR) and duplex-immunocapture-RT-PCR(D-IC-RT-PCR) [18 19] and one-step quadruplex RT-PCR[20] However conventional PCR diagnostic approaches arenot suitable for large scale diagnoses because they are time-consuming and have low sensitivity Although ELISA wasfound to be useful for routine large scale testing of virusit requires high quality antibodies and is limited by lowsensitivity

Hence the development of quick and less expensivediagnostic approaches is imperative to allow for the accurateand sensitive distinction among the SCSMV SCMV andSrMV More recently the stem-loop quantitative RT-PCR(qRT-PCR) method was used to detect the miRNA encodedby the SCSMV genome [21] The aim of the present studywas to develop a real-time RT-PCR assay for the detection ofSCSMV in sugarcane Thus our study reports a sensitive andreliable one-step TaqMan-based qRT-PCR assay for SCSMVfor the first time

2 Material and Method

21 Plant Material A total of 35 of the visible top dewlapleaf samples exhibitingmosaic symptoms among commercialsugarcane cultivars (Saccharum spp hybrids) were collectedin 2013 inGuangdong Guangxi YunnanHainan Fujian andGuizhou provinces China All collected leaf samples werestored at minus80∘C until RNA extraction

22 RNA Extraction and Purification Total RNA wasextracted from 100mg of sugarcane fresh leaf tissue usingTRIZOL Reagent (InvitrogenLife Technologies CarlsbadCA USA) according to the manufacturerrsquos instructionsRNase-free H

2O (50120583L) was added and then solutions were

stored at minus20∘C RNA was analyzed using a 1 agarosegel electrophoresis to ascertain the quality of RNA andthe absence of DNA contamination The concentration ofeach RNA sample was measured with micro-volume UV-Vis spectrophotometer (NanoVue Plus GE Healthcare Pitts-burgh PA USA) Only the RNA samples with A260A280ratio (an indication of protein contamination) of 19ndash21 andA260A230 ratio (an indication of reagent contamination)greater than 20 were used and 100 ng120583L RNA was subjectedto qRT-PCR and conventional RT-PCR assays

23 Primers and Probes Design In order to develop a real-time RT-PCR assay primers and probes were designedaccording to the sequences of SCSMV CP gene published inGenBank library using the Primer Express software version

20 (ABI Applied Biosystems Foster City CA USA) Forconventional RT-PCR detection of SCSMV a set of primersSCSMV-CPF2 and SCSMV-CPR2 were designed to corre-spond with the 572 base pair (bp) fragment of SCSMV CPgene All primers and probes were targeted in the conservedregion within the fragmentThe TaqMan probes were labeledwith 6-carboxy-fluorescein (FAM excitation wavelength494 nm emission wavelength 521 nm) reporter dye at 51015840-endand 6-carboxytetramethylrhodamine (TAMRA) fluorescentquencher at the 31015840-end The details of all primers and probesare listed in Table 1

24 RNA Transcripts of SCSMV CP Synthesis In VitroSCSMV CP fragment (572 bp) was amplified with SCSMV-CPF2 and SCSMV-CPR2 from YN-YZ211 isolates (GenBankacc no KJ187047) and inserted into the pGEM-T Easy(Promega Madison WI USA) and then it transformed theplasmid into competent cell of Escherichia coli strain DH5120572The right inserted PCR product was verified by sequencingPositive-sense single-strand RNA (ssRNA) was transcribedusing the RiboMAX Large Scale RNA Production Systems-T7 Kit (Promega) using 2120583L linearized recombinant plasmidDNA (1 120583g) as the template and then the plasmid DNAwas digested with RNase-free DNase I at 37∘C for 20minThe RNA was purified with RNAclean kit (BioTeke BeijingChina) and then was quantified using NanoVue Plus

25 One-Step Real-Time qRT-PCR Assay One-step real-timeqRT-PCR assay was performedwith the final volume of 20120583Lusing the one-step PrimeScript RT-PCRKit (TaKaRaBiotechDalian China) following the manufacturerrsquos instructionsEach reaction was carried out using 1 120583L of ssRNA transcriptsfrom pGEM-CP plasmid or 2 120583L of total RNA extractedfrom collected samples as template on the ABI 7500 Real-Time PCR Detection System (Applied Biosystems USA)During the amplification process the fluorescence intensityof the reporter dye (FAM) and a quencher dye (TAMRA)were recorded These data allowed the calculation of thenormalized reporter signal which is linked to the amount ofproduct amplifiedThe threshold cycle (Ct values number ofamplification cycles for the fluorescence to reach the thresh-old) refers to the number of amplification cycles requiredfor a significant increase in the reporterrsquos fluorescence Thedata were analyzed with ABI 7500 Real-Time PCR DetectionSystem program

Upstream and downstream primers were subjected toa 3 times 3 optimization matrix using a concentration of 100200 and 400 nmol120583L for each concentration of primerunder 200 nmol120583L probe concentration Subsequently theconcentration of the TaqMan probe was optimized ssRNAtranscripts synthesized in vitro were used as template Themost suitable program and parameter were reverse tran-scription of the RNA at 42∘C for 5min The optimumdenaturation time was 10 sec at 95∘C Annealing-extensiontime and temperature were 34 sec at 60∘C

A standard curve was generated using tenfold serialdilutions ranging from 109 to 102 copies of ssRNA transcriptsor 100 ng to 1 pg of total RNA extracted from sugarcaneleaf with SCSMV-infected YN-YZ211 isolate Ct values were

BioMed Research International 3

Table 1 List of the primers and probes used in this study

Virus Primer name Sequence (51015840-31015840) Fragment size (bp)

SCSMV

SCSMV-CPF2 TCATMTCTTCATCRGCCGC 572SCSMV-CPR2 ATCTTCYCTACGCAGGTCCG

SCSMV-QPCR-F1 CGGGAAACCCATAATACCAC 115SCSMV-QPCR-R1 GTCGATTTCTGCTGGTGAGASCSMV-QPCR-P1 FAM-TGCTGCATTGATTTCGTGATGGTG-TAMRASCSMV-QPCR-P2 FAM-TGCTGCATTGATTTTGTGATGGTG-TAMRA

SCMV SCMV-F4 GTTTTYCACCAAGCTGGAACAGTC 900SCMV-R3 AGCTGTGTGTCTCTCTGTATTCTC

SrMV SrMV-F ACAGCAGAWGCAACRGCACAAGC 850SrMV-R CTCWCCGACATTCCCATCCAAGCC

SCMV-F4SCMV-R3 and SrMV-FSrMV-R published in Alegria et al [22] and Xie et al [20] respectivelyM = AC Y = CT W = AT R = AG and W = AT in primer sequencesFAM 6-carboxyfluorescein TAMRA 6-carboxy tetramethyl rhodamine

measured in three duplicates and plotted against the knowncopy numbers of the standard samplesThese standard curveswere determined to estimate the reaction efficiency andthe quantification of viral target in the unknown samplesThe reaction efficiency under our experimental conditionswas addressed as and calculated with the formula 119864 =(10minus1slopeminus 1) times 100

26 Conventional RT-PCR In parallel the same set of tenfoldserial dilutions of ssRNA and total RNA were carried outby conventional RT-PCR assay using SCSMV-CPF2 andSCSMV-CPR2 primers for sensitivity tests SCSMV wasdetected in the field samples using the abovementionedprimers In addition to this primer pair two other sets ofprimers SCMV-F4 and SCMV-R3 published by Alegria et al(2003) [22] and SrMV-F and SrMV-R reported by Xie etal (2009) [20] were used for screening the SCMV andSrMV respectively PCR amplification was performed in atotal volume of 25 120583L containing 2 120583L RNA with 1 120583L Prime-Script one-step Enzyme Mix (TaKaRa shanghai China)400 nmolL of each of the primers and 200120583molL of dNTPsmixture The RT-PCR program was reverse transcription ofRNA at 50∘C for 30min and PCR performed at 94∘C 2min35 cycles at 94∘C 30 sec 55∘C for SCSMV or 52∘C for SrMVor 65∘C for SCMV 30 sec and 72∘C 1min followed by a final72∘C extension step for 5min The RT-PCR amplified prod-ucts were detected on a 15 agarose gel and then cloned intopMD19-T vectors (TaKaRa Dalian China) for sequencing

27 Sequence Analysis SCSMV SrMV and SCMV sequencesobtained with individual primers were analyzed by BLASTsearch in NCBI (httpblastncbinlmnihgov) Alignmentof the identified CP sequence (572 nt) from SCSMV YN-YZ211 isolate and other 24 SCSMV sequences published inGenBank were performed using MUSCLE algorithm [23]implemented in MEGA software program version 5 [24]Phylogenetic tree was generated by the neighbor-joiningmethod with 1000 bootstrap replications using MEGA 5

3 Results

31 Sequence Alignment and Primer Specificity The phylo-genetic tree of the SCSMV CP or P1 sequences was usedto report the three SCSMV genotypes from Asian countries[9 10] In the present study three SCSMV genotypes (I IIand III) were also clustered by the phylogenetic tree based onthe CP nucleotide (572 nt) sequences from YN-YZ211 isolateand 24 representative isolates published in the GenBankdatabase (Figure 1(a)) Two SCSMV genotypes (SCSMV-Iand -III) from the Chinese isolates were observed and YN-YZ211 isolatewas grouped as the SCSMV-III genotype All theIndian isolates were clustered into the SCSMV-II genotypeThe real-time PCR primers and probes were designed basedon a highly conserved region of the SCSMV CP sequencesThe primers and probes specificities were evaluated basedon multiple sequence alignment (Figure 1(b)) Sense primerof SCSMV-QPCR-F1 was completely identified with thesequences of three SCSMV genotypes while the antisenseprimer of SCSMV-QPCR-R1 had one variable nucleotide(ACT) positioned at the 51015840 end of the primer One variablenucleotide (CT) was located in the middle of the proberegion Therefore the two specific probes were designed fordeveloping a TaqMan-based real-time qRT-PCR in this study

32 Standard Curve of the Real-Time qRT-PCR AssaysThe optimum concentration for both upstream and down-stream primers of SCSMV real-time RT-PCR was found tobe 200 nmol120583L The concentration of the TaqMan probe(SCSMV-QPCR-P1 or -P2) was optimized as 200 nmol120583LssRNA was synthesized through RNA transcripts of pGEM-CP in vitro to obtain the standard curve for the real-time qRT-PCR assays The standard curves were established by usingtenfold serial dilutions of the standard ssRNA ranging from102 to 109 copies to determine the end-point limit of detectionand for the one-step real-time qRT-PCR assays Furthermorethe total RNA extracted from the sugarcane leaf was infectedwith the SCSMV YN-YZ211 isolate and diluted to a tenfold


Co94012-3IndiaAM749393 PAK2PakistanU75456 PAKPakistanNC_014037

M55ChinaJQ954717 M126ChinaKC588929

IND671IndiaJN941985 CB671-1IndiaDQ421788 IdnIndonesiaAB563503 kaIndiaAY193783 KA1IndiaGQ386844

taIndiaAY189681 tnIndiaAY193784 TN1IndiaGQ386845

AP1IndiaGQ386843 Andhra PradeshIndiaY17738

TPTIndiaGQ246187 CB86-1IndiaEF079667

Sorghum2IndiaEU883391 M111ChinaJQ954711

S-3IndiaEU650178 THA-NP3ThailandJN163911 YN-YZ211ChinaKJ187047

IDIndonesia-ChinaJF488066 JP1Japan-ChinaJF488064

JP2Japan-ChinaJF488065

9679

97100

88

78

100

93

99100

99

95

78

001

I

II

III

(a)

I

II

III

CGGGAAACCCATAATACCACACAATGCAGTGAAGGCAGGATTGACAACACCAGAGTTTTATCCTTGCTGCATTGATTTCGTGATGGTGAACATCCTCTCACCAGCAGAAATCGACCGGGAAACCCATAATACCACACAATGCAGTGAAGGCAGGATTGACAACACCAGAGTTTTATCCTTGCTGCATTGATTTCGTGATGGTGAACATCCTCTCACCAGCAGAAATCGACCGGGAAACCCATAATACCACACAATGCAGTGAAGGCAGGATTGACAACACCAGAGTTTTATCCTTGCTGCATTGATTTCGTGATGGTGAACATCCTCTCACCAGCAGAAATCGACCGGGAAACCCATAATACCACACAATGCAGTGAAGGCAGGATTGACAACACCAGAGTTTTATCCTTGCTGCATTGATTTCGTGATGGTGAACATCCTCTCACCAGCAGAAATCGACCGGGAAACCCATAATACCACACAATGCAGTGAAGGCAGGATTGACAACACCAGAGTTTTATCCTTGCTGCATTGATTTCGTGATGGTGAACATCCTCTCACCAGCAGAAATCGACCGGGAAACCCATAATACCACACAATGCAGTGAAGGCAGGATTGACCACACCAGAGTTTTATCCTTGCTGCATTGATTTCGTGATGGTGAACATCCCCTCACCAGCAGAAATCGACCGGGAAACCCATAATACCACACAATGCATTGAAGGCAGGATTGACAACACCAGAGTTTTATCCTTGCTGCATTGATTTCGTGATGGTGAACATCCTCTCACCAGCAGAAATCGACCGGGAAACCCATAATACCACACAATGCAGTGAAGGCAGGATTGACCACACCAGAGTTTTATCCTTGCTGCATTGATTTCGTGATGGTGAATATCCTCTCACCAGCAGAAATCGACCGGGAAACCCATAATACCACACAATGCAGTAAAGGCAGGATTGACCACACCAGAGTTTTATCCTTGCTGCATTGATTTTGTGATGGTGAACATCCTCTCACCAGCAGAAATTGACCGGGAAACCCATAATACCACACAATGCAGTAAAGGCAGGATTGACCACACCAGAGTTTTATCCTTGCTGCATTGATTTTGTGATGGTGAACATCCTCTCACCAGCAGAAATTGACCGGGAAACCCATAATACCACACAATGCAGTAAAGGCAGGATTGACCACACCAGAGTTTTATCCTTGCTGCATTGATTTTGTGATGGTGAACATCCTCTCACCAGCAGAAATTGACCGGGAAACCCATAATACCACACAATGCAGTAAAGGCAGGATTGACCACACCAGAGTTTTATCCTTGCTGCATTGATTTTGTGATGGTGAACATCCTTTCACCAGCAGAAATTGACCGGGAAACCCATAATACCACACAATGCAGTAAAGGCAGGATTGACCACACCAGAGTTTTATCCTTGCTGCATTGATTTTGTGATGGTGAACATCCTTTCACCAGCAGAAATTGACCGGGAAACCCATAATACCACACAATGCAGTAAAGGCAGGATTGACCACACCAGAGTTTTATCCTTGCTGCATTGATTTTGTGATGGTGAACATCCTTTCACCAGCAGAAATTGACCGGGAAACCCATAATACCACACAATGCAGTAGAGGCAGGATTGACCACACCAGAGTTTTATCCTTGCTGCATTGATTTTGTGATGGTGAACATCCTCTCACCAGCAGAAATTGACCGGGAAACCCATAATACCACACAATGCAGTAAAGGCAGGATTGACCACACCAGAGTTTTATCCCTGCTGCATTGATTTTGTGATGGTGAACATCCTCTCACCAGCAGAAATTGACCGGGAAACCCATAATACCACATAATGCAGTAAAGGCAGGATTAACAACTCCAGAGTTTTATCCTTGCTGCATTGATTTTGTGATGGTGAATATCCTCTCACCAGCAGAAATAGACCGGGAAACCCATAATACCACATAATGCAGTAAAGGCAGGATTAACAACTCCAGAGTTTTATCCTTGCTGCATTGATTTTGTGATGGTGAATATCCTCTCACCAGCAGAAATAGACCGGGAAACCCATAATACCACATAATGCAGTAAAGGCAGGATTAACAACTCCAGAGTTTTATCCTTGCTGCATTGATTTTGTGATGGTGAATATCCTCTCACCAGCAGAAATAGACCGGGAAACCCATAATACCACATAATGCAGTAAAGGCAGGATTAACAACTCCAGAGTTTTATCCTTGCTGCATTGATTTTGTGATGGTGAATATCCTCTCACCAGCAGAAATAGACGGGGAAACCCATAATACCACATAATGCAGTAAAGGCAGGATTAACAACTCCAGAGTTTTATCCTTGCTGCATTGATTTTGTGATGGTGAACATCCTCTCACCAGCAGAAATAGACCGGGAAACCCATAATACCACATAATGCAGTAAAGGCAGGATTAACAACTCCAGAGTTTTATCCTTGCTGCATTGATTTTGTGATGGTGAATATCCTCTCACCAGCAGAAATAGACCGGGAAACCCATAATACCACATAATGCAGTAAAGGCAGGATTAACAACTCCAGAGTTTTATCCTTGCTGCATTGATTTTGTGATGGTGAATATCCTCTCACCAGCAGAAATAGACCGGGAAACCCATAATACCACATAATGCAGTAAAGGCAGGATTAACAACTCCAGAGTTTTATCCTTGCTGCATTGATTTTGTGATGGTGAATATCCTCTCACCAGCAGAAATAGACCGGGAAACCCATAATACCACATAATGCAGTAAAGGCAGGATTAACAACTCCAGAGTTTTATCCTTGCTGCATTGATTTTGTGATGGTGAATATCCTCTCACCAGCAGAAATAGACTGCTGCATTGATTTCGTGATGGTGTGCTGCATTGATTTTGTGATGGTGTCTCACCAGCAGAAATCGACCGGGAAACCCATAATACCAC

Co94012-3AM749393PAK2U75456PAKNC 014037M55JQ954717M126KC588929CB671-1DQ421788IND671JN941985IdnAB563503kaAY193783KA1GQ386844taAY189681tnAY193784TN1GQ386845AP1GQ386843Andhra PradeshY17738TPTGQ246187CB86-1EF079667Sorghum2EU883391M111JQ954711S-3EU650178THA-NP3JN163911YN-YZ211KJ187047IDJF488066JP1JF488064JP2JF488065SCSMV-QPCR-P1SCSMV-QPCR-P2SCSMV-QPCR-R1SCSMV-QPCR-F1Consensus

11511511511511511511511511511511511511511511511511511511511511511511511511524242020

(b)

Figure 1 (a) Phylogenetic tree derived from the CP nucleotide sequences (572 nt) of Sugarcane streak mosaic virus (SCSMV) The tree wasgenerated usingMEGA 5 by the neighbor-joiningmethodwith 1000 bootstrap replicatesThe bootstrap confidence values are indicated whengt60 The NCBI accession number of the sequences used in the comparison is shown along with isolatesrsquo name and the isolates from thisstudy were marked with a black triangle (b) Primers and probes sequences used in qRT-PCR assay aligned with the same set of CP sequencesby DNAMAN version 8

serial solution (1 pg120583L to 100 ng120583L) for establishing thestandard curves Ct values (three replicates) were measuredand plotted against the known copy numbers of the standardsamplesThe standard curves were generated by two differentprobes SCSMV-QPCR-P1 and SCSMV-QPCR-P2

Standard curves together with the reaction efficienciesand slopes are shown in Figure 2 For ssRNA as templatethe standard curve covered a linear range of eight ordersof magnitude (Figures 2(a) and 2(b)) The slopes from thestandard curve of SCSMV-QPCR-P1 and SCSMV-QPCR-P2

probes were minus3220 and minus3581 efficiency (119864 ) = 104 and902 and 1198772 = 0999 and 0998 respectively For total RNAas template the standard curves covered a linear range of sixorders of magnitude (Figures 2(c) and 2(d)) We observedthat the standard curve for SCSMV-QPCR-P1 and SCSMV-QPCR-P2 probes had similar slopes of minus3365 and minus3364and 1198772 = 0994 and 0997 respectively Both standard curvesshowed the same efficiency of 100 This demonstrated thatthe TaqMan real-time RT-PCR protocol was feasible for thequantification of the SCSMV pathogen


05

10152025303540

1 2 3 4 5 6 7 8 9 10

Cycle

thre

shol

d (C

t)

Log starting quantity copy number

y = minus3220x + 4143Efficiency = 104

R2 = 0999

(a)

05

10152025303540

1 2 3 4 5 6 7 8 9 10Log starting quantity copy number

Cycle

thre

shol

d (C

t) Efficiency = 902

y = minus3581x + 4093

R2 = 0998

(b)

0

5

10

15

20

25

30

35

0 1 2 3 4 5Picograms of template total RNA (log scale)

Cycle

thre

shol

d (C

t)

y = minus3365x + 3253

R2 = 0994

Efficiency = 100

(c)

0

5

10

15

20

25

30

35


Cycle

thre

shol

d (C

t)

y = minus3364x + 3140

R2 = 0997

Efficiency = 100

(d)

Figure 2 Standard curves for TaqMan probes-based real-time qRT-PCR using SCSMV CP transcripts (positive-sense single-stranded RNAssRNA) ((a) and (b)) and total RNA ((c) and (d)) of plant infected SCSMV YN-YZ211 isolate as templates A tenfold dilution serial standardtemplate containing ssRNA ranged from 1 times 109 to 1 times 102 copiesreaction Total RNA templates ranged from 2 pg to 200 ngreaction Ctvalues were measured in triplicate and plotted against the known copy numbers (log scale) or picograms of template total RNA (log scale)Two different probes SCSMV-QPCR-P1 ((a) (c)) and SCSMV-QPCR-P2 ((b) (d)) were developed

33 Assessment of the Real-Time qRT-PCR Assays In orderto compare the end-point sensitivity between TaqMan qRT-PCR and conventional RT-PCR assays the same set oftemplates of six tenfold serial dilutions (107ndash102 copies) ofstandard ssRNA were used in conventional RT-PCR withtwo pairs of primers (SCSMV-CPF2 and -CPR2 and SCSMV-QPCR-F1 and -R1) The expected amplicons of 572 bp and115 bp were obtained from the reaction which were con-firmed by agarose gel electrophoresis The detection limitswere 1 times 104 copies for each set of primers (Figures 3(a) and3(b)) However we observed a titer as low as 1 times 102 copiesof standard ssRNA in a one-step TaqMan real-time RT-PCRassay (Figures 2(a) and 2(b))Thus our results suggested thatthe real-time qRT-PCR was 100-fold more sensitive than theconventional RT-PCR

The same set of templates that is the six tenfold dilutions(from 100 ng120583L to 1 pg120583L) of total RNA were also usedin the conventional RT-PCR Figures 3(c) and 3(d) presentthe typical data from a serial dilution of total RNA extractedfrom the SCSMV-infected leaf with a detection limit of 200 pgusing either of the primer pairs that is SCSMV-CPF2 and-CPR2 and SCSMV-QPCR-F1 and -R1 However the mini-mum detection limit of real-time RT-PCR assay was as lowas 2 pg (Figures 2(b) and 2(d)) We deduced that more than

100-fold sensitivity in the one-step TaqMan-based real-timeqRT-PCR assay compared with the conventional RT-PCR

34 Application of Real-Time RT-PCR Assays A total of 35sugarcane samples with mosaic symptoms were collectedfrom the sugarcane fields in China for determination ofSCSMV infection using both one-step real-time qRT-PCRand conventional RT-PCR assays Among the 35 samplesqRT-PCR identified 686 (2435) positives while only 486(1735) of samples were detected as positive by the conven-tional RT-PCR assay (Table 2) The virus titer of SCSMV-infected field samples ranged from 102 to 106 copies120583L Torule out false negatives in SCSMV infection in additionto using SCSMV-uninfected sugarcane sample as negativecontrol SCSMV-infected sample and water were also usedas positive and blank controls Furthermore all of the RNAsamples were checked for SCMV and SrMV detection usingconventional RT-PCR assay We found that 11 SCSMV-freesamples which were verified by qRT-PCR and conventionalRT-PCR were infected with either SCMV or SrMV or bothOn the other hand to exclude nonspecific amplification allqRT-PCR or conventional RT-PCR products were verified bysequencing and their nucleotide sequences were comparedusing the BLAST tool (httpblastncbinlmnihgov) Thesefindings indicated that qRT-PCR yields more accurate results


750500

250

M1 107 106 105 104 103 102 NC H2O(b

p)

(a)

200

100

M2 107 106 105 104 103 102 NC H2O

(bp)

(b)

H2O

750500

250

(bp)

M1 200ng 20ng 2ng 200pg 20pg 2pg NC

(c)

200

100(bp)

H2OM2 200ng 20ng 2ng 200pg 20pg 2pg NC

(d)

Figure 3 Gel-based sensitivity tested using the two set primers of qRT-PCR (SCSMV-QPCR-F1 and -R1) ((a) and (c)) and conventional RT-PCR (SCSMV-CPF2 and -CPR2) ((b) and (d)) The same set of templates (107ndash102 copiesreaction) of SCSMV CP transcripts (positive-sensesingle-stranded RNA ssRNA) was used in (a) and (b) and the same set of diluted solutions (2 pg to 200 ngreaction) of total RNA extractionof sugarcane leaf infected SCSMV YN-YZ211 isolate were carried out in (c) and (d) M1 DL 2000 DNA marker M2 20 bp DNA laddermarker NC Virus-free total RNA (100 ng)

and helps detect more positive samples in a low virus titer ascompared with the conventional gel-based RT-PCR

4 Discussion

Mosaic disease of sugarcane caused by SCSMV has beenreported in Asian countries including India and China TheSCSMV has become widespread in the sugarcane-growingfields inChina [9]Three SCSMVgenotypes have been identi-fied in China and India based on the phylogenetic trees of theP1 or CP sequences [9 10] All 17 SCSMV isolates identifiedin the present study by conventional RT-PCR shared highnucleotide sequence identity (977ndash998) and were groupedunder the SCSMV-III genotype (Table 2) A representativeisolate YN-YZ211 was used as a positive control The diseasecaused by SCSMV is difficult to distinguish from thosecaused by SCMVand SrMVbecause of similar symptoms andoccasional coinfection with the two viruses RT-PCR reversetranscription loop-mediated isothermal amplification (RT-LAMP) and serological methods are most commonly usedfor detecting sugarcane pathogens however these methodsdo not effectively distinguish among the causal pathogens Inthe present study a one-step qRT-PCR assay using TaqManwas established for the quick detection and quantificationof SCSMV in sugarcane plants This assay yielded highlysensitive results by using a fluorescence signal

The specificity of the one-step qRT-PCR assay hasbeen evaluated based on the multiple sequence alignmentAlthough we found few nucleotides in 51015840 end of primers var-ied among the published SCSMV genotypes this mismatch

would not significantly affect the PCR efficiency Besides onenucleotide varied in probe sequence region between SCSMVgenotypes therefore two different probes (SCSMV-QPCR-P1and -P2) were designed to assess the differences of PCR effi-ciency with the same RNA template A permissible differenceof PCR efficiency (104 for SCSMV-QPCR-P1 and 902for SCSMV-QPCR-P2) was presented for ssRNA template(Figures 2(a) and 2(b)) on the other hand no difference ofPCR efficiency was found between the two probes for totalRNA template (Figures 2(c) and 2(d)) SCSMV-QPCR-P2wasused as a probe for determination of SCSMV infection in thefield samples (as it is showed in Table 2) as its sequence wasidentified to positive control of ssRNA template of YN-YZ211isolateThis developedmethod in this study can be effectivelyapplied in SCSMV diagnoses Moreover none of the othersugarcane causal viruses SCMV SrMV and Sugarcane yellowleaf virus (SCYLV) were detected using the primers andprobes of this qRT-PCR assay (data not shown)

In real-time quantification of PCR assay the Ct value isa parameter reflecting the quantity of templates presented inthe reaction [25] As always the concentration of the templatewas linear in correlation to the Ct values The efficiency(slope) and the coefficient of determination (1198772) of the linearequation were determined to optimize the concentrationsand conditions of the real-time RT-PCR assay The standardserial dilution curve was obtained with a rational qRT-PCR efficiency for SCSMV (902ndash104) in this study usingeither ssRNA transcripted in vitro or plant total RNA astemplate Besides the 1198772 values of SCSMVwere very close to1 (0994ndash0999) Two specific probes designed in this study


Table 2 Detection of SCSMV from field sugarcane samples by real-time qRT-PCRwith SCSMV-QPCR-P2 probe and conventional RT-PCR

Varieties Sampling location qRT-PCR Conventional RT-PCRCt value Copies120583L Result SCSMV SrMV SCMV

FN38 Fuzhou Fujian 3053 plusmn 028 82 times 102 + mdash + [KC984957] mdashFN15 Fuzhou Fujian na na minus mdash + [KC984956] mdashROC22 Fuzhou Fujian 3037 plusmn 016 89 times 102 + mdash + [KC984964] + [KC899320]FN39 Zhangzhou Fujian na na minus mdash + [KC984958] mdashROC22 Guangzhou Guangdong na na minus mdash + [KC984980] mdashF160 Suixi Guangdong 2051 plusmn 033 51 times 105 + + [KJ944725] + [KJ944744] mdashGT31 Suixi Guangdong 3351 plusmn 034 12 times 102 + + [KJ944729] + [KJ944745] mdashLC05-136 Suixi Guangdong na na minus mdash + [KJ944746] mdashROC22 Suixi Guangdong 3268 plusmn 042 20 times 102 + + [KJ944727] + [KJ944741] mdashYZ05-49 Suixi Guangdong 1934 plusmn 014 11 times 106 + + [KJ944728] + [KJ944742] mdashYZ07-2384 Zhanjiang Guangdong 1672 plusmn 023 58 times 106 + + [KJ944726] + [KJ944736] mdashGT11 Wangmo Guizhou 3255 plusmn 030 22 times 102 + + [KC985070] + [KC985029] mdashGZ18 Wangmo Guizhou 3139 plusmn 05 46 times 102 + + [KC985071] mdash mdashYZ03-45 Wangmo Guizhou 3221 plusmn 015 27 times 102 + + [KC985072] + [KC985030] mdashYG35 Kaiyuan Yunnan 1842 plusmn 036 19 times 106 + + [KC985085] + [KC985056] mdashYN01-58 Kaiyuan Yunnan 3126 plusmn 025 50 times 102 + + [KC985086] + [KC985061] mdashYZ05-211 Kaiyuan Yunnan 2119 plusmn 029 33 times 105 + + [KC985090] + [KC985064] mdashYT93-159 Lincang Yunnan 3259 plusmn 031 21 times 102 + mdash + [KC985060] mdashFN15 Danzhou Hainan 3235 plusmn 022 25 times 102 + mdash + [KC985031] mdashYT55 Danzhou Hainan 3152 plusmn 027 42 times 102 + mdash + [KC985038] mdashROC22 Haikou Hainan 3207 plusmn 007 30 times 102 + + [KC985074] mdash mdashYZ05-49 Lingao Hainan 1644 plusmn 027 69 times 106 + + [KC985075] + [KC985041] mdashGT02-281 Beihai Guangxi 3213 plusmn 019 29 times 102 + + [KC985069] + [KC984999] mdashFN1110 Bingyang Guangxi na na minus mdash + [KJ944748] mdashYT93-159 Bingyang Guangxi na na minus mdash + [KJ944750] + [KJ944720]FN39 Laibin Guangxi 3227 plusmn 032 26 times 102 + + [KJ944735] + [KJ944782] mdashROC16 Laibin Guangxi 3217 plusmn 021 28 times 102 + mdash + [KJ944757] mdashROC22 Laibin Guangxi 2960 plusmn 012 15 times 103 + + [KJ944731] + [KJ944764] mdashYG46 Laibin Guangxi 3292 plusmn 035 17 times 102 + + [KJ944734] + [KJ944774] mdashYR06-189 Laibin Guangxi 3289 plusmn 046 18 times 102 + mdash + [KJ944758] mdashGT05-3084 Longan Guangxi na na minus mdash + [KJ944820] mdashGT07-994 Liuzhou Guangxi na na minus mdash + [KJ944814] mdashROC22 Liuzhou Guangxi na na minus mdash + [KJ944815] mdashFN15 Shanglin Guangxi na na minus mdash + [KJ944752] mdashROC22 Shanglin Guangxi na na minus mdash + [KJ944754] mdashPositive result (+) if Ct le 35 negative result (minus) if Ct gt 35GenBank acc no of these sequences from conventional RT-PCR product were in bracketsna data not available

can be used in the TaqMan-based real-time RT-PCR forSCSMVTherefore this qRT-PCR assay could be used for thequantitative detection of SCSMV in routine virus diagnoses

High sensitivity is one of the salient features of qRT-PCR In this assay a minimum titer of SCSMV could bedetermined for as low as 100 copies of ssRNA or 2 pg totalRNA qRT-PCRwas at least 100-foldmore sensitive comparedwith conventional RT-PCR techniques Furthermore highersensitivity of qRT-PCR was observed in SCSMV detection

of sugarcane field samples Similar sensitivity results werefound in SCYLV detection with TaqMan probe-based qRT-PCR assay [26 27] The nonspecific amplification in SCSMVqRT-PCR assay was excluded because all qRT-PCR productswere further verified by sequencing Similarly false negativesfor the qRT-PCR assaywere ruled out by including RNA fromSCSMV-infected and SCSMV-uninfected samples as positiveand negative controls respectively Water was used as a blankcontrol Moreover the samples with mosaic symptom could


not be determined by SCSMV qRT-PCR or regular RT-PCRbut were further detected by infection with SCMV or SrMVor both The virus titers of SCSMV infecting field samplesranged from 102 to 106 copies120583L It is worthy noted thatthe negative SCSMV samples might be false negatives in thecase of the virus titre in leaf samples less than the minimumdetection limit of qRT-PCR or conventional RT-PCR assays

SCSMV is mainly transmitted through virus-infectedcane cuttings among sugarcane-planting countries andregions Therefore it is necessary to develop a specific rapidand sensitive detection method for screening germplasms inquarantine and determine the distribution of the SCSMVin commercial fields A reliable diagnosis is vital for thedevelopment of efficient strategies for viral disease controlTherefore our study reports a highly specific sensitive andreliable qRT-PCR assay using TaqMan probe for the detectionand quantification of SCSMV in sugarcane

5 Conclusion

In this study we firstly established a protocol of TaqManprobe-based real-time qRT-PCR for the detection and quan-tification of SCSMV causing sugarcane mosaic disease ThisqRT-PCR assay is an optional molecular tool to be usedin a rapid sensitive and efficient diagnosis of SCSMV insugarcane quarantine and disease control

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Fu Wei-Lin and Sun Sheng-Ren contributed equally to thiswork

Acknowledgments

This work was funded in part by an Earmark Fund fromthe China Agriculture Research System (CARS-20-2-4) and agrant from theNational Natural Science Foundation of China(31170345)

References

[1] R Viswanathan M Balamuralikrishnan and R KaruppaiahldquoSugarcane mosaic in India a cause of combined infection ofsugarcane mosaic virus and sugarcane streak mosaic virusrdquoSugar Cane International vol 25 no 2 pp 6ndash14 2007

[2] M J Adams and E B Carstens ldquoRatification vote on taxonomicproposals to the International Committee on Taxonomy ofViruses (2012)rdquoArchives of Virology vol 157 no 7 pp 1411ndash14222012

[3] J S Hall B Adams T J Parsons R French L C Lane and SG Jensen ldquoMolecular Cloning Sequencing and PhylogeneticRelationships of a New Potyvirus Sugarcane Streak Mosaic

Virus and a Reevaluation of the Classification of the Potyviri-daerdquo Molecular Phylogenetics and Evolution vol 10 no 3 pp323ndash332 1998

[4] M Hema J Joseph K Gopinath P Sreenivasulu and H SSavithri ldquoMolecular characterization and interviral relation-ships of a flexuous filamentous virus causing mosaic diseaseof sugarcane (Saccharum officinarum L) in Indiardquo Archives ofVirology vol 144 no 3 pp 479ndash490 1999

[5] M Chatenet C Mazarin J C Girard et al ldquoDetection ofSugarcane streak mosaic virus in sugarcane from several Asiancountriesrdquo Sugar Cane International vol 23 no 4 pp 12ndash152005

[6] D-L Xu G-H Zhou Y-J Xie R Mock and R Li ldquoCompletenucleotide sequence and taxonomy of Sugarcane streak mosaicvirus member of a novel genus in the family PotyviridaerdquoVirusGenes vol 40 no 3 pp 432ndash439 2010

[7] T A Damayanti and L K Putra ldquoFirst occurrence of Sugarcanestreak mosaic virus infecting sugarcane in Indonesiardquo Journal ofGeneral Plant Pathology vol 77 no 1 pp 72ndash74 2011

[8] W Li Z He S Li et al ldquoMolecular characterization of a newstrain of sugarcane streak mosaic virus (SCSMV)rdquo Archives ofVirology vol 156 no 11 pp 2101ndash2104 2011

[9] Z HeW Li R Yasaka et al ldquoMolecular variability of sugarcanestreak mosaic virus in China based on an analysis of the P1 andCP protein coding regionsrdquo Archives of Virology vol 159 no 5pp 1149ndash1154 2014

[10] R Viswanathan M Balamuralikrishnan and R KaruppaiahldquoCharacterization and genetic diversity of Sugarcane streakmosaic virus causing mosaic in sugarcanerdquoVirus Genes vol 36no 3 pp 553ndash564 2008

[11] M Hema P Sreenivasulu and H S Savithri ldquoTaxonomic posi-tion of sugarcane streak mosaic virus in the family PotyviridaerdquoArchives of Virology vol 147 no 10 pp 1997ndash2007 2002

[12] L K Putra A Kristini E M Achadian and T A DamayantildquoSugarcane streak mosaic virus in Indonesia distribution char-acterisation yield losses and management approachesrdquo SugarTech vol 16 no 4 pp 392ndash399 2014

[13] K P Srinivas C V Subba Reddy B Ramesh P L Kumarand P Sreenivasulu ldquoIdentification of a virus naturally infectingsorghum in India as Sugarcane streak mosaic virusrdquo EuropeanJournal of Plant Pathology vol 127 no 1 pp 13ndash19 2010

[14] M Hema H S Savithri and P Sreenivasulu ldquoSugarcane streakmosaic virus occurrence purification characterization anddetectionrdquo in Sugarcane Pathology Vol 2 Virus and Phytoplas-ma Disease G P Rao R E Ford M Tosic and D S TeakleEds pp 37ndash70 Science Publishers Enfield NH USA 2001

[15] D Singh and G P Rao ldquoSudan grass (Sorghum sudanenseStapf) a new sugarcane streak mosaic virus mechanical hostrdquoGuangxi Agricultural Sciences vol 41 no 5 pp 436ndash438 2010

[16] D L Seifers T J Martin T L Harvey J P Fellers and J PMichaud ldquoIdentification of the wheat curl mite as the vector ofTriticum mosaic virusrdquo Plant Disease vol 93 no 1 pp 25ndash292009

[17] M Hema H S Savithri and P Sreenivasulu ldquoAntibody andnucleic acid probe-based techniques for detection of sugarcanestreak mosaic virus causing mosaic disease of sugarcane inIndiardquo Current Science vol 81 no 8 pp 1105ndash1108 2001

[18] M Hema N Kirthi P Sreenivasulu and H S SavithrildquoDevelopment of recombinant coat protein antibody based IC-RT-PCR for detection and discrimination of sugarcane streakmosaic virus isolates from Southern IndiardquoArchives of Virologyvol 148 no 6 pp 1185ndash1193 2003


[19] C V S Reddy P Sreenivasulu and G Sekhar ldquoDuplex-im-munocapture-RT-PCR for detection and discrimination of twodistinct potyviruses naturally infecting sugarcane (Saccharumspp hybrid)rdquo Indian Journal of Experimental Biology vol 49no 1 pp 68ndash73 2011

[20] Y Xie M Wang D Xu R Li and G Zhou ldquoSimultaneousdetection and identification of four sugarcane viruses by one-step RT-PCRrdquo Journal of Virological Methods vol 162 no 1-2pp 64ndash68 2009

[21] C Viswanathan J Anburaj and G Prabu ldquoIdentification andvalidation of Sugarcane streak mosaic virus-encoded microR-NAs and their targets in sugarcanerdquo Plant Cell Reports vol 33no 2 pp 265ndash276 2014

[22] O M Alegria M Royer M Bousalem et al ldquoGenetic diversityin the coat protein coding region of eighty-six sugarcanemosaicvirus isolates from eight countries particularly fromCameroonand Congordquo Archives of Virology vol 148 no 2 pp 357ndash3722003

[23] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[24] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[25] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993

[26] J Korimbocus D Coates I Barker and N BoonhamldquoImproved detection of Sugarcane yellow leaf virus using a real-time fluorescent (TaqMan)RT-PCR assayrdquo Journal of VirologicalMethods vol 103 no 2 pp 109ndash120 2002

[27] S J Gao Y H Lin Y B Pan et al ldquoMolecular characterizationand phylogenetic analysis of Sugarcane yellow leaf virus isolatesfrom Chinardquo Virus Genes vol 45 no 2 pp 340ndash349 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


sugarcane leaves and it is difficult to distinguish the causalvirus on the basis of these symptoms alone Moreover thesymptom expression can vary depending on age of infectionenvironment and host cultivar [1] Thus it is important todistinguish the three viruses accurately and reliably Manydiagnostic methods are available for the detection of SCSMVin sugarcane plants including microscopic examination ofviral morphology [4] double antibody sandwich-enzymelinked immunosorbent assay (DAS-ELISA) and direct anti-gen coating- (DAC-)ELISA tests [17] reverse transcriptionpolymerase chain reaction (RT-PCR) [10] immunocapture-RT-PCR (IC-RT-PCR) and duplex-immunocapture-RT-PCR(D-IC-RT-PCR) [18 19] and one-step quadruplex RT-PCR[20] However conventional PCR diagnostic approaches arenot suitable for large scale diagnoses because they are time-consuming and have low sensitivity Although ELISA wasfound to be useful for routine large scale testing of virusit requires high quality antibodies and is limited by lowsensitivity

Hence the development of quick and less expensivediagnostic approaches is imperative to allow for the accurateand sensitive distinction among the SCSMV SCMV andSrMV More recently the stem-loop quantitative RT-PCR(qRT-PCR) method was used to detect the miRNA encodedby the SCSMV genome [21] The aim of the present studywas to develop a real-time RT-PCR assay for the detection ofSCSMV in sugarcane Thus our study reports a sensitive andreliable one-step TaqMan-based qRT-PCR assay for SCSMVfor the first time

2 Material and Method

21 Plant Material A total of 35 of the visible top dewlapleaf samples exhibitingmosaic symptoms among commercialsugarcane cultivars (Saccharum spp hybrids) were collectedin 2013 inGuangdong Guangxi YunnanHainan Fujian andGuizhou provinces China All collected leaf samples werestored at minus80∘C until RNA extraction

22 RNA Extraction and Purification Total RNA wasextracted from 100mg of sugarcane fresh leaf tissue usingTRIZOL Reagent (InvitrogenLife Technologies CarlsbadCA USA) according to the manufacturerrsquos instructionsRNase-free H

2O (50120583L) was added and then solutions were

stored at minus20∘C RNA was analyzed using a 1 agarosegel electrophoresis to ascertain the quality of RNA andthe absence of DNA contamination The concentration ofeach RNA sample was measured with micro-volume UV-Vis spectrophotometer (NanoVue Plus GE Healthcare Pitts-burgh PA USA) Only the RNA samples with A260A280ratio (an indication of protein contamination) of 19ndash21 andA260A230 ratio (an indication of reagent contamination)greater than 20 were used and 100 ng120583L RNA was subjectedto qRT-PCR and conventional RT-PCR assays

23 Primers and Probes Design In order to develop a real-time RT-PCR assay primers and probes were designedaccording to the sequences of SCSMV CP gene published inGenBank library using the Primer Express software version

20 (ABI Applied Biosystems Foster City CA USA) Forconventional RT-PCR detection of SCSMV a set of primersSCSMV-CPF2 and SCSMV-CPR2 were designed to corre-spond with the 572 base pair (bp) fragment of SCSMV CPgene All primers and probes were targeted in the conservedregion within the fragmentThe TaqMan probes were labeledwith 6-carboxy-fluorescein (FAM excitation wavelength494 nm emission wavelength 521 nm) reporter dye at 51015840-endand 6-carboxytetramethylrhodamine (TAMRA) fluorescentquencher at the 31015840-end The details of all primers and probesare listed in Table 1

24 RNA Transcripts of SCSMV CP Synthesis In VitroSCSMV CP fragment (572 bp) was amplified with SCSMV-CPF2 and SCSMV-CPR2 from YN-YZ211 isolates (GenBankacc no KJ187047) and inserted into the pGEM-T Easy(Promega Madison WI USA) and then it transformed theplasmid into competent cell of Escherichia coli strain DH5120572The right inserted PCR product was verified by sequencingPositive-sense single-strand RNA (ssRNA) was transcribedusing the RiboMAX Large Scale RNA Production Systems-T7 Kit (Promega) using 2120583L linearized recombinant plasmidDNA (1 120583g) as the template and then the plasmid DNAwas digested with RNase-free DNase I at 37∘C for 20minThe RNA was purified with RNAclean kit (BioTeke BeijingChina) and then was quantified using NanoVue Plus

25 One-Step Real-Time qRT-PCR Assay One-step real-timeqRT-PCR assay was performedwith the final volume of 20120583Lusing the one-step PrimeScript RT-PCRKit (TaKaRaBiotechDalian China) following the manufacturerrsquos instructionsEach reaction was carried out using 1 120583L of ssRNA transcriptsfrom pGEM-CP plasmid or 2 120583L of total RNA extractedfrom collected samples as template on the ABI 7500 Real-Time PCR Detection System (Applied Biosystems USA)During the amplification process the fluorescence intensityof the reporter dye (FAM) and a quencher dye (TAMRA)were recorded These data allowed the calculation of thenormalized reporter signal which is linked to the amount ofproduct amplifiedThe threshold cycle (Ct values number ofamplification cycles for the fluorescence to reach the thresh-old) refers to the number of amplification cycles requiredfor a significant increase in the reporterrsquos fluorescence Thedata were analyzed with ABI 7500 Real-Time PCR DetectionSystem program

Upstream and downstream primers were subjected toa 3 times 3 optimization matrix using a concentration of 100200 and 400 nmol120583L for each concentration of primerunder 200 nmol120583L probe concentration Subsequently theconcentration of the TaqMan probe was optimized ssRNAtranscripts synthesized in vitro were used as template Themost suitable program and parameter were reverse tran-scription of the RNA at 42∘C for 5min The optimumdenaturation time was 10 sec at 95∘C Annealing-extensiontime and temperature were 34 sec at 60∘C

A standard curve was generated using tenfold serialdilutions ranging from 109 to 102 copies of ssRNA transcriptsor 100 ng to 1 pg of total RNA extracted from sugarcaneleaf with SCSMV-infected YN-YZ211 isolate Ct values were




SCSMV









3 Results














9679

97100

88

78

100

93

99100

99

95

78

001

I

II

III

(a)

I

II

III



11511511511511511511511511511511511511511511511511511511511511511511511511524242020

(b)






05

10152025303540

1 2 3 4 5 6 7 8 9 10

Cycle

thre

shol

d (C

t)



R2 = 0999

(a)

05

10152025303540


Cycle

thre

shol

d (C

t) Efficiency = 902

y = minus3581x + 4093

R2 = 0998

(b)

0

5

10

15

20

25

30

35


Cycle

thre

shol

d (C

t)

y = minus3365x + 3253

R2 = 0994

Efficiency = 100

(c)

0

5

10

15

20

25

30

35


Cycle

thre

shol

d (C

t)

y = minus3364x + 3140

R2 = 0997

Efficiency = 100

(d)







750500

250

M1 107 106 105 104 103 102 NC H2O(b

p)

(a)

200

100

M2 107 106 105 104 103 102 NC H2O

(bp)

(b)

H2O

750500

250

(bp)


(c)

200

100(bp)


(d)



4 Discussion















5 Conclusion






Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




SCSMV









3 Results














9679

97100

88

78

100

93

99100

99

95

78

001

I

II

III

(a)

I

II

III



11511511511511511511511511511511511511511511511511511511511511511511511511524242020

(b)






05

10152025303540

1 2 3 4 5 6 7 8 9 10

Cycle

thre

shol

d (C

t)



R2 = 0999

(a)

05

10152025303540


Cycle

thre

shol

d (C

t) Efficiency = 902

y = minus3581x + 4093

R2 = 0998

(b)

0

5

10

15

20

25

30

35


Cycle

thre

shol

d (C

t)

y = minus3365x + 3253

R2 = 0994

Efficiency = 100

(c)

0

5

10

15

20

25

30

35


Cycle

thre

shol

d (C

t)

y = minus3364x + 3140

R2 = 0997

Efficiency = 100

(d)







750500

250

M1 107 106 105 104 103 102 NC H2O(b

p)

(a)

200

100

M2 107 106 105 104 103 102 NC H2O

(bp)

(b)

H2O

750500

250

(bp)


(c)

200

100(bp)


(d)



4 Discussion















5 Conclusion






Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












9679

97100

88

78

100

93

99100

99

95

78

001

I

II

III

(a)

I

II

III



11511511511511511511511511511511511511511511511511511511511511511511511511524242020

(b)






05

10152025303540

1 2 3 4 5 6 7 8 9 10

Cycle

thre

shol

d (C

t)



R2 = 0999

(a)

05

10152025303540


Cycle

thre

shol

d (C

t) Efficiency = 902

y = minus3581x + 4093

R2 = 0998

(b)

0

5

10

15

20

25

30

35


Cycle

thre

shol

d (C

t)

y = minus3365x + 3253

R2 = 0994

Efficiency = 100

(c)

0

5

10

15

20

25

30

35


Cycle

thre

shol

d (C

t)

y = minus3364x + 3140

R2 = 0997

Efficiency = 100

(d)







750500

250

M1 107 106 105 104 103 102 NC H2O(b

p)

(a)

200

100

M2 107 106 105 104 103 102 NC H2O

(bp)

(b)

H2O

750500

250

(bp)


(c)

200

100(bp)


(d)



4 Discussion















5 Conclusion






Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


05

10152025303540

1 2 3 4 5 6 7 8 9 10

Cycle

thre

shol

d (C

t)



R2 = 0999

(a)

05

10152025303540


Cycle

thre

shol

d (C

t) Efficiency = 902

y = minus3581x + 4093

R2 = 0998

(b)

0

5

10

15

20

25

30

35


Cycle

thre

shol

d (C

t)

y = minus3365x + 3253

R2 = 0994

Efficiency = 100

(c)

0

5

10

15

20

25

30

35


Cycle

thre

shol

d (C

t)

y = minus3364x + 3140

R2 = 0997

Efficiency = 100

(d)







750500

250

M1 107 106 105 104 103 102 NC H2O(b

p)

(a)

200

100

M2 107 106 105 104 103 102 NC H2O

(bp)

(b)

H2O

750500

250

(bp)


(c)

200

100(bp)


(d)



4 Discussion















5 Conclusion






Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


750500

250

M1 107 106 105 104 103 102 NC H2O(b

p)

(a)

200

100

M2 107 106 105 104 103 102 NC H2O

(bp)

(b)

H2O

750500

250

(bp)


(c)

200

100(bp)


(d)



4 Discussion















5 Conclusion






Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











5 Conclusion






Acknowledgments


References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article A One-Step Real-Time RT-PCR Assay for the...

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