Establishment of a Multiplex RT-PCR Assay for Detection of Four Sweet Potato RNA Viruses

doi:10.7668/hbnxb.20192971

Abstract

Abstract:

A multiplex RT-PCR detection system was established and studied to target four sweet potato RNA viruses,Sweet potato feathery mottle virus (SPFMV),Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus G (SPVG)and Sweet potato virus C (SPVC),which caused serious damage in sweet potato production. Firstly,the specific primers for four viruses were designed by Premier 5.0 software and synthesized according to the conserved sequences of coat protein(CP)gene of these viruses from GenBank. The cDNA synthesized was used as a template,and 4 couples of selected primers were used simultaneously to amplify the target fragments by PCR. A best multiplex RT-PCR reaction system was tested by optimizing the annealing temperature,extension time,dNTPs amount,template amount,primer concentration and so on and an optimal quadruple RT-PCR system that could simultaneously detect these four sweet potato RNA viruses was established. The results showed that an accurate and sensitive multiplex RT-PCR method for detecting the four sweet potato viruses mentioned above with respective target band had been successfully established by challenge tests in which there would be no crossover among the virus primers. The multiplex RT-PCR method established in this study could detect four kinds of sweet potato virus at the same time. It was not only accurate and sensitive,but also reduced the cost of detection and improved the efficiency of virus detection.

Key words: Sweet potato, Sweet potato feather mottle virus (SPFMV), Sweet potato chlorosis stunt virus (SPCSV), Sweet potato virus G (SPVG), Sweet potato virus C (SPVC), Multiplex RT-PCR

FENG Liting, ZHANG Jianfeng, CHI Shengqi. Establishment of a Multiplex RT-PCR Assay for Detection of Four Sweet Potato RNA Viruses[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(4): 206-211. doi: 10.7668/hbnxb.20192971.

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http://www.hbnxb.net/EN/Y2022/V37/I4/206

Figures/Tables 14

Tab.1 Specific primers of sweet potato virus used

病毒种类 Type of virus	引物编号 Primer number	引物序列(5'-3') Primer sequence(5'-3')	目的片段/bp Destination segment
SPFMV	SPFMV-F	CCCTCCACCACCCACAAT	746
	SPFMV-R	CGGCTGCCTTCATCTGTAA
SPCSV	SPCSV-F	AACCCAAAGAATAAACTACAGTCC	622
	SPCSV-R	AAGTCGCAGCCAGCCCATC
SPVG	SPVG-F	GCAGACAGGACCATTACCAC	487
	SPVG-R	AACCATTAACCCGTTGAGC
SPVC	SPVC-F	GGTTACTGAGCAGGAGGATT	270
	SPVC-R	GGTTCATAAGTTGCGAGGTG

Fig.1 Detection of the virus by RT-PCR using a pair of primer M.DL2000 DNA Marker;1.SPFMV;2.SPCSV;3.SPVG;4.SPVC; 5.Negative control.

Fig.2 Detection of the viruses by RT-PCR using 2 pairs of primers M.DL2000 DNA Marker;1.SPFMV/SPCSV;2.SPFMV/SPVG;3.SPFMV/SPVC;4.SPCSV/SPVG;5.SPVG/SPVC;6.SPCSV/SPVC。

Fig.3 Detection of the viruses by RT-PCR using 3 pairs of primers M.DL2000 DNA Marker;1.SPFMV/SPCSV/SPVG;2.SPFMV/SPCSV/SPVC;3.SPFMV/SPVG/SPVC;4.SPCSV/SPVG/SPVC。

Fig.4 Detection by RT-PCR using 4 pairs of primers M.DL2000 DNA Marker;1-2.SPFMV/SPCSV/ SPVG/SPVC.The same as Fig.5.

Fig.5 Reaction system of multiplex RT-PCR optimized

Tab.2 The best reaction system for cDNA synthesis (10 μL)

名称 Heading	体积/μL Bulk
gDNA Eraser	1.0
模板Template	2.0
SPFMV-R	0.5
SPCSV-R	0.5
SPVG-R	0.5
SPVC-R	1.0
RNase Free H₂O	4.5

Tab.3 The best combination of PCR reactions

名称 Heading	体积/μL Bulk
10×PCR Buffer	2.50
rTaq	0.25
dNTPs	2.50
F/R(10 μmol/L)	1.00
模板Template	2.50
ddH₂O	9.25

Tab.4 Optimal reaction conditions for multiplex PCR

温度/℃ Temperature	时间 Time	循环数 Number of cycles
94 3 min $9430 s 5830 s 721 m i n$ 35 72 10 min 12 ∞

Tab.4 Optimal reaction conditions for multiplex PCR

温度/℃ Temperature	时间 Time	循环数 Number of cycles
94 3 min $9430 s 5830 s 721 m i n$ 35 72 10 min 12 ∞

Fig.6 Detection sensitivity of multiplex RT-PCR M.DL2000 DNA Marker; 1-7.10⁰~10^-6.

Fig.7 Specific detection of multiplex RT-PCR of sample 1 and 2 M.DL2000 DNA Marker;1.Positive control of sample 1; 2.Multiple detection of sample 1;3.Positive control of sample 2;4.Multiple detection of sample 2.

Fig.8 Specific detection of multiplex RT-PCR of sample 3 M.DL2000 DNA Marker;1.SPFMV-F/SPFMV-R;2.SPCSV-F/SPCSV-R;3.SPVG-F/SPVG-R;4.SPVC-F/SPVC-R;5.SPBV-F/SPBV-R;6.SPLV-F/SPLV-R;7.Multiplex RT-PCR system.

Fig.9 Challenge test A M. DL2000 DNA Marker;1-4.Detection of sample 1 with SPFMV,SPCSV,SPVG,SPVC single pair primer respectively;5.Multiple RT-PCR detection of sample 1;6-9.Detection of sample 2 with SPFMV,SPCSV,SPVG,SPVC,single pair primer respectively;10.Multiple RT-PCR detection of sample 2.

Fig.10 Challenge test B M.DL2000 DNA Marker;1-4.SPFMV,SPCSV,SPVG,SPVC single pair primer detection of sample 3 respectively;5.Multiple RT-PCR detection of sample 3;6-9.SPFMV,SPVG,SPCSV,SPVC single pair primer detection of sample 4 respectively;10.Multiple RT-PCR detection of sample 4.

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