Simplex RT-PCR Research Articles

Development of simplex and quintuplex RT-PCR for simultaneous detection of soybean viruses

Five simplex and a multiplex-RT-PCR (m-RT-PCR) protocols were developed for detection and differentiation of bean pod mottle virus (BPMV), cherry leaf roll virus (CLRV), raspberry ringspot virus (RpRSV), soybean mosaic virus (SMV) and tomato ringspot virus (ToRSV) infecting soybean. The simplex RT-PCR protocols produced virus-specific amplicons of 538 bp for BPMV, 139 bp for CLRV, 298 bp for RpRSV, 403 bp for SMV, and 282 bp for ToRSV, with sensitivity down to 10−4 diluted cDNA. Further, to detect all the five viruses simultaneously in a single tube a quintuplex RT-PCR protocol was optimized with as low as 10−3 diluted cDNA and 0.05 µM primer. To validate the reliability of the simplex RT-PCR protocol, imported soybean samples were tested by ELISA as well as RT-PCR. The results revealed that the developed protocol could detect the viruses in imported soybean, and found to be efficient than ELISA in resolving ambiguity in detection of seed borne viruses. The developed simplex and quintuplex RT-PCR protocol will be quite helpful for the diagnosis of soybean germplasm co-infected with viruses during the quarantine processing for ensuring virus free long term seed conservation in the National Gene Bank as well as for quarantine certification.

Development of a multiplex RT-PCR assay for the detection of soybean mosaic virus, bean common mosaic virus and cucumber mosaic virus in field samples of soybean

Soybean is susceptible to viral diseases which are often present as mixed infections. The individual simplex RT-PCR methods used for the identification of multiple viruses are more tedious and time-consuming than the corresponding multiplex RT-PCR. This study used soybean mosaic virus (SMV), bean common mosaic virus (BCMV) and cucumber mosaic virus (CMV)-infected leaf samples from southern China as the test materials to evaluate a multiplex RT-PCR assay developed for the simultaneous detection of these viruses. The parameters optimised included the annealing temperature, extension time, number of cycles, and primer type and concentration. The specific fragments sizes obtained by the multiplex RT-PCR were 550 bp (SMV), 288 bp (BCMV) and 99 bp (CMV). The assay was tested using infected soybean samples obtained from farmers’ fields in Sichuan Province, China. The multiplex RT-PCR assay had high sensitivity, was rapid and simple, and could be used for the diagnosis of soybean infected with various combinations of these viruses in the field.

Development of a multiplex reverse transcription-PCR assay for simultaneous detection of pepper viruses

The aim of the present study was to establish a fourplex RT-PCR method for examining the infecting peppers to improve the efficiency of pepper virus detection, which includes Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Broad bean wilt virus- 2 (BBWV-2) and Potato virus Y (PVY). According to the genome sequences of TMV, CMV, BBWV-2 and PVY published by GenBank, four specific pairs of primers were designed to verify the specificity and sensitivity of detection system by simplex and multiplex RT-PCR method. In addition, using set multiplex RT-PCR method pepper plant samples of two geographic locations in China were tested and verified by simplex RT-PCR. The anticipated amplicon sizes of four primer pairs targeting TMV, CMV, BBWV-2 and PVY in both simplex and multiplex RT-PCR were 237 bp, 323 bp, 480 bp and 1057 bp, respectively, and established fourplex RT-PCR system was able to check out all sorts of viruses accurately from the pepper leaves of two geographic locations in China, which was 37.21%, 81.4%, 23.26%, and 2.33% respectively, and two or more mixed infections viruses sample accounted for 48.83% of the pepper samples. The research results show a detection technique of multiplex RT-PCR method for major peppers' viruses, which is a rapid, sensitive and reliable method and examines CMV, TMV, PVY and BBWV-2.

Occurrence of Grapevine Pinot gris virus in Poland and description of asymptomatic exhibitions in grapevines

Sixty five grapevines in productive areas from southern regions in Poland were tested for the presence of Grapevine Pinot gris virus (GPGV) by RT-PCR. Twenty two percent of the tested plants were infected by GPGV. GPGV positive grapevines were also tested for the presence of other viruses by multiplex and simplex RT-PCR. Part of the movement and coat protein coding regions and a part of RdRp domain were sequenced. Phylogenetic analyses of the 2 genes revealed that south Polish GPGV isolates grouped with already described asymptomatic isolates. This study provides the first survey of the GPGV occurrence in Poland.

FIRST REPORT OF CUCUMBER MOSAIC VIRUS INFECTING APPLE IN CHINA

A survey for apple viruses was conducted in October of 2014 in Shanxi, Gansu, Sichuan, Liaoning and Hebei provinces of China. A total of 189 leaf samples with mosaic symptoms and five symptomless samples were collected and tested for viruses. Apple mosaic virus (ApMV) (Lakshmi et al., 2011; Robertson, 2012) was not detected by RT-PCR with total RNA and primers ApMV-F (5’-CGTGAGGAAGTTTAGGTTG-3’)/ApMV-R (5’-GCCTCCTAATCGGGGCATCAA-3’). Neither were Tobacco mosaic virus (TMV), Turnip mosaic virus (TuMV), Potato virus Y (PVY) and Potato virus X (PVX) by a multiplex RT-PCR assay previously developed in our laboratory, with the exception of Cucumber mosaic virus (CMV), for which a partial fragment (322 bp) of the coat protein (CP) gene was amplified from 153 symptomatic samples using specific primers CMV-F (5’-GATAAGAAGCTTGTTTCGCG-3’)/CMV-R (5’-GCTCGATGTCGACATGAAGT-3’). To confirm these preliminary results, primer pair CMV-CP-F (5’-A TGGACAAA TCTGAA TCAACC-3’)/ CMV-CP-R (5’-TCAGACTGGGAGCACCCC-3’) were designed to amplify a 657 bp CMV CP amplicon by simplex RT-PCR. The expected product was obtained from each sample identified as positive by multiplex RT-PCR. Five amplicons were randomly selected for cloning and sequencing (GenBank accession Nos. KP307919-KP307922 and KP641344). Sequence alignments showed the highest CMV sequence identity at the nucleotide level (97.3% to 99.2%) of the five isolates with that of a CMV isolate from tobacco in Sichuan province (KJ746016). Phylogenetic analysis indicated the five isolates cluster into CMV subgroup I. Using the CMV-specific monoclonal antibody 3C12 (Yu et al., 2005), the 153 symptomatic samples were positive for CMV in ELISA. No virus was detected in the five symptomless samples. To our knowledge, this is the first report of CMV infecting apple in China.

First report of Citrus bent leaf viroid in the United Arab Emirates.

Citrus species are among the three top fruit trees in the United Arab Emirates (UAE), with a combined total pro- duction of 12230 tons in 2011. Leaf samples were collected from four lemon (Citrus lemon L.) and five sweet orange (C. sinensis (L.) Osbeck) groves located in Al-Ain (south east of Abu Dhabi, UAE). Sampled trees showed weakened growth, but no typical symptoms of viroids and were tested for the presence of viroids by multiplex RT-PCR using two sets of primers specific for Citrus exocortis viroid (CEVd) and Cit- rus bent leaf viroid (CBLVd) (Al-Harthi et al., 2013). Testing disclosed the association of CEVd with 15 of 16 lemons in four groves, and with 18 of 18 sweet oranges in five groves. CBLVd was detected in six of 16 lemons in three groves and in five of 18 sweet oranges in three groves. To sequence PCR products, simplex RT-PCR was carried out with each primer set and two positive samples for CEVd and two for CBLVd. Amplifications produced fragments of the expected size, 371 bp for CEVd and 234 bp for CBLVd. PCR products were pu- rified and sequenced by Macrogen (Korea). CEVd sequences from the UAE were identical to each other and shared 99% nucleotide similarity to CEVd isolate CEVd-XNM-XY (Gen- Bank accession No. DQ431993), while the CBLVd sequence was identical to that of CBLVd isolate 201-1-2 Uy (accession No. AF428053). Sequences of both viroids from the UAE were deposited in GenBank (accession No. pending) This is the first record of CBLVd in the UAE. Presence of citrus viroids in Al-Ain, the most important agricultural area in the UAE, necessitates a larger scale study to characterize their distribution, pathogenicity and economic impact.

A duplex real-time RT-PCR assay for the simultaneous genogroup-specific detection of noroviruses in both clinical and environmental specimens

Norovirus (NoV) is the major etiological agent causing foodborne and waterborne outbreaks worldwide. We developed a novel duplex real-time quantitative RT-PCR assay designed for the simultaneous detection of and discrimination between NoV genogroups GI and GII, by targeting the short junction region between ORF1 and ORF2, with sensitivity and efficiency comparable to those of each simplex RT-PCR assay. This new duplex assay was evaluated against clinical stool (n = 82) and environmental (groundwater or surface water, n = 60) specimens from South Korea, and the results were compared with those of conventional RT-PCR (cRT-PCR) assays. The duplex assay detected more positive samples than did the cRT-PCR for both clinical (74 vs. 71) and, more strikingly, environmental (24 vs. 10) specimens. No cross-reactivity against specimens containing other enteric viruses such as rotavirus, adenovirus, and poliovirus were observed. These results suggest that this newly developed duplex real-time RT-PCR assay can be used for the sensitive and simultaneous genogroup-specific detection of NoV in both clinical and environmental specimens.

Simultaneous detection of stone fruit tree viruses by one-step multiplex RT-PCR

A protocol of multiplex RT-PCR in a one-tube system for the detection of the most common stone fruit trees viruses [e.g., plum pox virus (PPV), prune dwarf virus (PDV), and Prunus necrotic ringspot virus (PNRSV)], including the internal control of NADH dehydrogenase subunit 5 ( nad5) gene are described here. The method specificity was tested on more than 80 different samples with various isolates and strains of the viruses. It showed that the targeted viruses produced the expected amplicons, whereas all other related viruses produced only the nad5 internal control amplicon. The method sensitivity was evaluated by comparing it with Simplex RT-PCR with the same primers; no significant differences in detection limits were recorded. Furthermore, the competitiveness of the primers in the assay was tested by serial RNA dilutions of samples with mixed and single infections. The least competitive was the internal control nad5 gene primer pair; therefore, there is a reduced risk of false negatives as all the other primers tend to be more efficient in the given primer cocktail than in the primers for internal control.

Simultaneous detection and quantitation of Chikungunya, Dengue and West Nile viruses by multiplex RT-PCR assays and Dengue virus typing using High Resolution Melting

Chikungunya (CHIKV), Dengue (DENV) and West Nile (WNV) viruses are arthropod-borne viruses that are able to emerge or re-emerge in many regions due to climatic changes and increase in travel. Since these viruses produce similar clinical signs it is important for physicians and epidemiologists to differentiate them rapidly. A molecular method was developed for their detection and quantitation in plasma samples and a DENV typing technique were developed. The method consisted in performing two multiplex real-time one-step RT-PCR assays, to detect and quantify the three viruses. Both assays were conducted in a single run, from a single RNA extract containing a unique coextracted and coamplified composite internal control. The quantitation results were close to the best detection thresholds obtained with simplex RT-PCR techniques. The differentiation of DENV types was performed using a High Resolution Melting technique. The assays enable the early diagnosis of the three arboviruses during viremia, including cases of coinfection. The method is rapid, specific and highly sensitive with a potential for clinical diagnosis and epidemiological surveillance. A DENV positive sample can be typed conveniently using the High Resolution Melting technique using the same apparatus.

Epidemiological aspects of astrovirus and coronavirus in poults in the South Eastern Region of Brazil

A survey of Turkey Coronavirus (TCoV) and Astrovirus (TAstV-2) prevalence was carried out from February to December during 2006 year in semiarid region of Brazil, from a turkey producer area, localized in South Eastern of Brazil. To asses the risk factor related to clinical material, climatic condition and type of RT-PCR applied, cloacal swabs (CS), faeces, sera, bursa of Fabricius (BF), thymus (TH) and spleen (SP) and ileum-caeca region were collected from 30-day-old poults suffering of enteritis episode characterized as poult enteritis mortality syndrome (PEMS). The PEMS clinical features were characterized by watery to foamy faeces, light brown-yellow in colour and low mortality rate. Meteorological data (rainfall and relative humidity) observed during along the study presented monthly average temperature ranging from 39.3 and 31.2ºC, precipitation in rainy season from 40 to 270.3 mm/month, and no rain during dry season. Simplex RT-PCR gave odds ratio (OR) values suggesting that ileum-caeca region is at higher chance (OR=1.9; p=0.9741) to have both viral RNA than faeces (OR=1.5; p=0.7319). However, multiplex RT-PCR showed 3.98 (p=0.89982) more chance to give positive results in faeces than CS at dry season. The major risk factors seem to be low rate of humidity and high temperatures at winter, probably responsible for spread, easily, the TCoV and TAstv-2 among the flocks. The positive results of both virus suggested that they can play an important role in enteric disorders, associated to low humidity and high temperatures frequently found in tropical countries.

Simultaneous detection of enteropathogenic viruses in buffalos faeces using multiplex reverse transcription-polymerase chain reaction (mRT-PCR)

A multiplex reverse transcription- polymerase chain reaction (mRT-PCR) assay that detects Bovine Viral Diarrhoea Virus, Bovine Coronavirus, and Group A Rotaviruses in infected cell-culture fluids and clinical faecal samples is described. One hundred twenty faecal samples from buffalo calves with acute gastroenteritis were tested. The mRT-PCR was validated against simplex RT-PCR with published primers for Pestivirus, Coronavirus and Rotavirus. The multiplex RT-PCR was equally sensitive and specific in detecting viral infections compared with simplex RT-PCR. The mRT-PCR readily identified viruses by discriminating the size of their amplified gene products. This mRT-PCR may be a sensitive and rapid assay for surveillance of buffalo enteric viruses in field specimens. This novel multiplex RT-PCR is an attractive technique for the rapid, specific, and cost-effective laboratory diagnosis of acute gastroenteritis.

Rapid identification of potato virus Y strains by one-step triplex RT-PCR

A one-step triplex RT-PCR method was characterised that allows rapid, strain-specific detection of potato virus Y (PVY) occurring on potato: PVY N, PVY O, PVY NTN (recombinant isolates), PVY NWi and PVY C. Three specific primer pairs were designed on aligned PVY sequences available from genomic data banks. The specificity of the selected primers was first examined by simplex RT-PCR with a large number of PVY reference isolates. Two fragments of 0.44 and 1.11 kb were amplified for PVY N and non-recombinant PVY NTN isolates, two fragments of 0.53 and 0.66 kb for PVY O isolates, a single fragment of 0.44 kb for recombinant PVY NTN isolates, a 0.66 kb fragment for PVY C isolates and a 0.53 kb fragment for PVY NWi isolate. The primers were then combined in a one-step triplex RT-PCR reaction, optimised stepwise and validated with the reference isolates. The great similarity between the genomes of PVY N and non-recombinant PVY NTN prevented their differentiation using this method. No fragments were amplified with samples infected by non-related potato viruses, as well as with samples from healthy tobacco and potato plants. The one-step triplex RT-PCR described here fastens specific detection of PVY strains that are otherwise only distinguishable by combined serological and biological assays.

Expression and significance of tumor-related genes in HCC

To investigate the expression and clinical significance of DEK, cyclin D1, insulin-like growth factor II (IGF-II), glypican 3 (GPC3), ribosomal phosphoprotein 0 (rpP0) mRNA in hepatocellular carcinoma (HCC) and its paraneoplastic tissues. The expression of mRNAs of DEK, cyclin D1, IGF-II, GPC3 and rpP0 mRNA was detected in HCC and its paraneoplastic tissues by multiplex RT-PCR. By the simplex RT-PCR, the overexpression of mRNAs of DEK, cyclin D1, IGF-II, GPC3, rpP0 mRNA in HCC and its paraneoplastic tissues was 78.1%, 87.5%, 87.5%, 75.0%, 81.3% and 15.6%, 40.6%, 37.5%, 21.9%, 31.3% respectively (P<0.05). By the multiplex RT-PCR, at least one of the mRNAs was detected in all HCC samples and in 75.0% of paraneoplastic samples (P>0.05). However, all these five mRNAs were found in 68.8% of HCC samples, but only in 9.4% of paraneoplastic tissues (P<0.05). The positive expression of mRNAs of DEK, cyclin D1, IGF-II, GPC3, rpP0 in well- and poorly-differentiated HCC was 89.0%, 66.7%, 66.7%, 66.7%, 77.8% and 73.9%, 95.7%, 95.7%, 95.7%, 82.6%, respectively (P>0.05). The expression of these genes in HCCs with alpha-feto protein (AFP) negative and positive was 90.0%, 80.0%, 90.0%, 90.0%, 90.0% and 72.7%, 86.3%, 77.3%, 90.9%, 68.2% respectively (P>0.05). The expression of DEK, cyclin D1, IGF-II, GPC3, rpP0 mRNA in HCC is much higher in HCC than in its paraneoplastic tissues. Multiplex RT-PCR assay is an effective, sensitive, accurate, and cost-effective diagnostic method of HCC.