The techniques used to detect genetically modified organisms (GMO), including qualitative polymerase chain reaction (PCR), quantitative PCR, enzyme-linked immunosorbent assay (ELISA) and many others, are systematically described and discussed. The application progress of GMO in species-specific detection, endogenous genes, standard substances and restraining factors influencing detection are reviewed. The ongoing problems and development prospects of detection techniques of GMO are also pointed out.

The plant expression vectors pCAMBIA1301PMI and pBIPMI were constructed by substituting the Escherichia coli phosphomannose-isomerase (PMI) gene for the hpt gene of pCAMBIA1301 and gus gene of pBI121, respectively. Epicotyl explants of the Xuegan sweet orange (Citrus sinensis L. Osbeck) were inoculated with Agrobacterium tumefaciens EHA105- pCAMBIA1301PMI and EHA105-pBIPMI and subsequently selected in a medium supplemented with a combination of 25 g/l mannose and 5 g/l sucrose as the carbon source. The transformation efficiency rate was 27.7% when transformed by pCAMBIA1301PMI and 12.7% by pBIPMI. Genetic transformation was confirmed by chlorophenol red assay and polymerase chain reaction (PCR). A new method for obtaining transgenic Xuegan sweet orange plants was developed using the PMI/mannose selection system.

Four bioengineered bacteria having the fasG gene were constructed using two Escherichia coli expression strains (BL21 (DE3) and Rossetta (DE3)) with different tRNA profiles and two E. coli expression vectors (pET28a(+) and pGEX-KG) to examine the influence of rare codons on the expression of the fasG gene. The induction expression result of bioengineered bacteria showed that the expression levels of target proteins in Rossetta (DE3), which contained the tRNAs of the rare codons, were higher than in BL21 (DE3), either using pET-fasG or pKG-fasG.

The expression of the YUCAA1 gene and the amount of endogenous indole acetic acid (IAA) in rice (Oryza sativa subsp. japonica) plants and rice suspension cells infected by Rice stripe virus (RSV) were investigated by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and high-performance liquid chromatography, respectively. The results showed that the expression of the YUCAA1 gene and the amount of endogenous IAA increased at various times (16, 32, 48 and 64 h) after infection of rice suspension cells by RSV. In rice plants infected with RSV, the expression of the YUCAA1 gene and the amount of endogenous IAA increased, in comparison with healthy rice plants, at 4–8 days after infection, and decreased at 12 and 16 days. These results indicated that RSV infection could regulate auxin biosynthesis in rice. Additionally, the expression of the RSV gene CP increased 2.9 times in rice plants after they were treated with a KPSC buffer to deplete the endogenous auxins, and decreased 45% after treatment with 30 μmol/l IAA. All of these results suggest that auxin may play a role in RSV replication in rice plants.

Epidermal growth factor receptor (EGFR) is becoming a perfect target for killing carcinoma cells, especially because of its overexpression on the surface of these cells. Cationic antimicrobial peptides (CAP) have their own special mechanism of membrane-lytic cytotoxicity. In this study, a membrane-lytic immunotoxin (IT), chimeric protein MEGFMEL, was constructed to kill carcinoma cells with EGFR overexpression. This protein is composed of mouse (Mus musculus) epidermal growth factor (MEGF), as the target part, and melittin (MEL), as the cytotoxic part. Using Escherichia coli BL21 and pET30a as expression strain and vector, respectively, 63.45 μg/ml of MEGFMEL (68% purity) was obtained through low-temperature induction of expression and a thawing-freezing purification procedure (without cytolysis). In vitro activity measurement showed that this MEGFMEL significantly induced a lethal effect on A431 carcinoma cells overexpressing EGFR on the surface, with an LD50 value 52.6 μg/ml. The results suggest that the use of CAP as the toxin in the construction of unique membrane-lytic ITs aimed at EGFR is feasible.

The testosterone-inducible regulator (teiR) gene was cloned from Comamonas testosteroni chromosomal DNA, and introduced into plasmids pKtac2 (containing a tac promoter) and pK18 to yield plasmids pKtac2-teiR and pKteiR100. The recombinant plasmids were transformed into competent Escherichia coli HB101 and total protein was extracted to detect the TeiR protein expression level using enzyme-linked immunosorbent assay (ELISA). E. coli transformed by pKtac2-teiR and pKteiR100 produced 6.65 and 5.93 μg/mg of TeiR protein, respectively. Recombinant plasmids were also co-transformed into competent E. coli HB101 with plasmid p6 [containing hsdA gene (3α-HSD/CR, 3α-hydroxysteroid dehydrogenase/carbonyl reductase encoding gene)] to reveal the relationship between 3α-HSD/CR and TeiR by ELISA. The amounts of TeiR protein expressed by E. coli containing pKtac2-teiR and pKteiR100 were 5.94 μg/mg and 5.33 μg/mg, respectively, and these increased up to 6.81 μg/mg and 6.10 μg/mg after inducing with 1 mmol/l isopropyl-β-d-thiogalactoside (IPTG). Interestingly, 3α-HSD/CR protein expression level, after co-transformation with plasmids pKtac2-teiR and p6, was lower than that observed in the co-transformation with pKteiR100 and p6. The first co-transformation induced 1.20 μg/mg 3α-HSD/CR protein and the second 1.71 μg/mg. These values rose to 1.42 and 1.80 μg/mg, respectively, after treatment with 1 mmol/l IPTG. Our results proved that the tac promoter was more efficient than the lacZ promoter and that the teiR gene could act as an activator for hsdA gene expression.

An efficient AHL (N-acyl-homoserine lactone) bioassay strain, JZA1, of Agrobacterium tumefaciens was used to detect the AHL production from Acidovorax avenae subsp. citrulli [the pathogen causing bacterial fruit blotch (BFB) of melons], and the results showed that A. avenae subsp. citrulli produced a 3-O-C8-homoserine (HSL) type signal molecule. Gene aiiA, which could degrade AHL molecules, was transformed into A. avenae subsp. citrulli strain NJF10, creating strain NJF10-aiiA. The AHL production from NJF10-aiiA was significantly reduced compared with wild-type NJF10. Inoculation tests showed that NJF10-aiiA had an obvious reduction of virulence on watermelon fruits. Our finds showed that AHL production by A. avenae subsp. citrulli was related to its pathogenicity. This work might provide a novel way to control BFB by QS (quorum sensing) interference.

A rapid real-time polymerase chain reaction (RT-PCR) for detecting West Nile virus (WNV) was established. Primers were designed according to the sequence of the capsid protein gene of WNV by Primer Premier 5.0. In this way, an inexpensive assay using the intercalating dye SYBR Green I was developed and validated. The amplifying curve showed that this method could successfully amplify 102 copies/μl of the WNV gene, while reference to Japanese encephalitis virus (JEV) and blank control were all negative. Tenfold successive dilutions of positive WNV DNA were used to measure the sensitivity of RT-PCR. The assay system showed high reproducibility with coefficient of variation (CV) <2%. Thus the newly established RT-PCR assay was shown to be a rapid, sensitive and specific test for detecting WNV.

A rapid and sensitive real-time polymerase chain reaction (PCR) assay coupled with SYBR Green I chemistry was developed for the quantitative detection of Turbot reddish body iridovirus (TRBIV) isolated from farmed turbot (Scophthalmus maximus). A 152 bp DNA fragment from the TRBIV major capsid protein (MCP) gene was involved in the real-time PCR (RT-PCR) assay using the Roter Gene 3000 sequence detection system. The PCR mixture contained a fluorescent dye, SYBR Green I, which exhibited fluorescence enhancement when bound to double-stranded (ds) DNA. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. The positive control plasmid, pUCm-T/TRBIV MCP, containing the target sequence, was quantified to make a standard curve for sample detection after serial tenfold dilution. Linear coefficient correlations between the cycle threshold (CT) value and logarithmic positive plasmid concentration were close to one (R2=0.9952) and the detection limit of the assay was 102 copies of positive plasmids. The quantitative detection of virus in different tissues from TRBIV-infected fish showed that the spleen and kidney contained the largest number of viral particles (5.23×106 and 2.18×106 viral genome copies/mg tissue, respectively), while no viral DNA was detected in the muscular tissue. The molecular epidemic investigation of TRBIV showed that many cultured turbots were infected and TRBIV has become epidemic in turbot farms located along the Shandong peninsula. The virus number varied from 1.27×102 to 2.33×106 viral genome copies/mg tissue in spleens of infected turbot. These results suggest that the RT-PCR assay reported here can be used as a rapid, sensitive and quantitative method for TRBIV.

Macrophages from the head kidney (HK) and peripheral blood leucocytes were isolated fromCyprinus carpio by Percoll gradient density centrifugation, cultured in vitro and exposed to different concentrations of the immunomodulator lentinan. To evaluate the immunostimulating effects of lentinan, proliferation of the peripheral blood leucocytes, respiratory burst of macrophages and interleukin-1β (IL-1β) gene mRNA expression of macrophages were investigated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], NBT (nitroblue tetrazolium) reduction, Griess reaction and real-time polymerase chain reaction (PCR). Results showed that proliferation of peripheral blood leucocytes after 24 h incubation by the induction of lentinan at 100 and 1000 μg/ml was markedly stimulated. Lentinan at 1, 100 and 1000 μg/ml could significantly induce superoxide anions in macrophages. Production of nitric oxide by HK macrophages after 96 h incubation by lentinan showed that it had no conspicuous effect on nitrogen burst activity of macrophages. Moreover, it inhibited the nitrogen burst activity at higher doses. The expression of IL-1β in the HK macrophages after 24 h of polysaccharide stimulation showed that lentinan stimulated IL-1β expression in the head kidney in carp. Lentinan can modulate the immune response of C. carpio.

Amniotic fluid stem (AFS) cells isolated from human amniotic fluid were cultured and the factors affecting the primary culture of these cells were investigated. The isolated AFS cells express both embryonic stem cell markers, such as Oct-4, and adult stem cell markers, such as CD29, and possess a high proliferation capability. Moreover, isolated AFS cells were able to differentiate into Nestin-positive and α-actin-positive cells. There were several factors, including the date of gestation, the level of blood contamination and the volume of amniotic fluid, which could significantly affect the attachment time and numbers of AFS cells in primary culture. The results showed that the cell attachment time (4.7±0.6 days) during the second trimester of gestation was significantly different from that during the third trimester of gestation (6±0.5 days) (P<0.05), suggesting that cells collected from the fluid during the third trimester of gestation needed a longer attachment time. Blood contamination could significantly affect the cell attachment time. The attachment time of the brown-coloured fluid group (10.8±0.3 days) was significantly different from the blood-cell-free fluid group (6±0.5 days) and the blood-cell group (6.3±0.6 days) (P<0.05). The volume of amniotic fluid influenced, to some extent, both cell attachment numbers and time. With the increase in amniotic fluid volume, cell attachment numbers significantly increased (P<0.05), and cell attachment time extended, but not significantly (P>0.05). The present studies systematically examined the factors that influence the primary culture of human AFS cells and provided useful data on AFS cell research. Additionally, the isolated AFS cells maintain the capacity for differentiation into other cell types and are able to become seed cells for clinical applications.

This study aimed to explore high-throughput cDNA array monitoring technology and to apply it to the gene expression spectrum analysis of salinity-challenged tobacco plants. A Nicotiana tabacum cDNA library was sequenced and found to consist of 5927 high-quality sequences (GenBank accession nos CV015900-CV021826). By analysing the expressed sequence tags (ESTs), the proportion of N. tabacum genes was identified at the EST level. A cDNA array was constructed based on the tentative unique transcripts (TUTs) derived from EST assembling results. A total of 42 differentially expressed genes were identified, including plasma membrane intrinsic protein 2a, ethylene-responsive proteinase and pre-mRNA splicing factor prp31 gene, suggesting that there was a complicated biological response in N. tabacum under saline stress.