A Novel Nested Polymerase Chain Reaction (n-PCR) Assay for Identifying Sorghum nitidum

This work developed a novel nested polymerase chain reaction (n-PCR) assay to identify Sorghum nitidum ( S. nitidum ). It has been designed a set of specific n-PCR inner primers Snit5/Snit2 and outer primers Nout1/Nout2 based on a conserved nucleotide sequence of adh1-like gene of S. nitidum . Fourteen samples of sorghum were used to investigate the specificity of the primers and the n-PCR assay. The result showed that 9 samples of S. nitidum displayed a positive strong, specific amplified band at ~873 bp in gel spectra, while other relatives, including Sorghum halepense , Sorghum almum , Sorghum bicolor , Sorghum propinum and Sorghum sudanse exhibited negative amplifications. This assay was able to specifically identify S. nitidum fast and effectively, which could be applied widely in field inspection, agriculture production and plant protection.


Introduction
Sorghum nitidum is a related species of the hard weed Sorghum halepense (Guo et al., 1996), which originated from west of India and then spread widely into southeast Asia, Indonesia and Australia. Sorghum Moench could be classified into five groups, namely Stiposorghum, Parasorghum, Eusorghum, Heterosorghum and Chaetosorghum (Celarier, 1959;DeWet, 1978), while S. nitidum was recognized to be one of the most widely spreading species in the group of Parasorghum (Snowden, 1955). Like S. halepense and S. almum, S. nitidum is a perennial weed and the chromosome is as big as S. halepense and S. biocolor. Sun et al. (1994) believed that the S. nitidum should be cataloged into the Eusorghum group, while DeWet et al. (1978) thought S. nitidum should be classified into the Parasorghum group. The correct relationship of S. nitidum should be further studied. It is very difficult to distinguish the seed of different sorghum species morphologically from each other (Guo et al., 1996). Because seeds of S. nitidum among farm products may be deformed and abraded during storage, loading and unloading, in addition, the morphology of the sorghum seed usually impacted by the environment, climate, condition of maturation. The method of cellular diagnostics would also be very tedious and time -consuming, which would not be very suitable for field detection (Harlan and De Wet, 1974).
ADH (Ethanol Dehydrogenase, EC 1.1.1.1), coded by the gene adh, which was widely distributed over various organs and tissues throughout the plant, catalyzes the conversion between the ethanol and acetaldehyde. Sivaraman et al. (1984) was the first to study the DNA polymorphism of the adh gene of Arabidopsis. The polymorphism of the adh gene could help discover the genetic relationship between different species and provide biological dates for identifying some specific species.
Nested PCR (n-PCR) originated from the typical PCR technique, which has been widely used in basic biological researches and clinical diagnostics (Khan et al., 2004;Mayer and Palmer, 1996;Tanzer, 2000). The basic principle of n-PCR based on two pairs of specific primers designed to amplify one gene fragment by two round thermo-cycling. N-PCR technique could decrease the pseudo-positive amplification and improve the sensitivity of the assay. In this work, we designed a set of specific n-PCR primers based on a conserved nucleotide gene fragment of adh1-like gene of S. nitidum and developed a fast assay to identify the S. nitidum with high specificity and effectivity.

N-PCR primers design for identifying S. nitidum
The discrimination of the nucleotide sequence of the adh 1-like gene between the S. nitidum and other sorghum species were analyzed by the sorfware Cluster X. The typical PCR primers were designed based on a conserved gene fragment of S. nitidum (Tab. 2). The outer primers of n-PCR for the S. nitidum were shown as Tab. 3, while the inner n-PCR primers were as the same as the primer of typical PCR (Tab. 4).

Typical PCR method
The typical PCR for identifying S. nitidum was performed in a volume of 20 µL system including ddH 2 O 12.8 µl, PCR buffer 2 µl, Mg 2+ 1.2 µl, dNTP (2.5 mmol/L) 0.5 µl, rTaq DNA polymerase (5 U/µl) 0.2 µl, Primer Snit5 (20 µmol/L) 1 µl, Primer Snit2 (20 µmol/L) 1µl and template DNA 1 µl. The PCR was carried out in a thermocycler, commenced with 3 min at 94 o C, followed by the 35 cycles of 30 s at 94 o C, 30 s at 50 o C and 80 s at 72 o C, finally ended with 6 min at 72 o C. The amplified product was analyzed by the agarose gel electrophoresis and the result was visualized and recorded by the gel documentation system.

Nested PCR method
The n-PCR was performed in a volume system of 20 µl, which include ddH 2 O 12.8 µl, PCR buffer 2 µl, Mg 2+ 1.2 µl, dNTP (2.5 mmol/L) 0.5 µl, rTaq DNA polymerase (5 U/µl) 0.2 µl, Primer Nout1 (20 µmol/L) 1 µl, Primer Nout4 (20 µmol/L) 1 µl and template DNA 1 µl. The amplification was carried out with the commence of 3 min pre-denaturing at 94 o C, followed by the 20 cycles of 30 s denaturing at 94 o C, 30 s annealing at 50 o C and elongation 80 s at 72 o C. The second amplification system was as the same as the first round PCR, other than the primers and the template DNA, while the number of thermo-cycles was adjusted to 20 cyclers in the second round. The amplified product was separated by the agarose gel electrophoresis and analyzed by gel documentation system.

Results and discussion
Establishment of typical-PCR assay for identifying sorghum nitidum Primers of typical PCR Snit5/Snit2 were used to amplify the genomic DNA extracted from a single seed of S. nitidum. The result was shown in Fig. 1, from which, a strong, specific band at ~873 bp appeared, according well with our predicting.

The application of the typical-PCR
The specificity of the typical PCR assay was verified using 2 samples of S. nitidum and 1 sample of S. halepense.
Extraction of genomic DNA form a seed of sorghum Total Genomic DNA was extracted from a single seed of sorghum using an improved method developed in our laboratory (Chen et al., 2009). After verified by the aga- The result was as shown in Fig. 2, from which, we observed that a specific amplified band occurred to the sample of S. nitidum while there was no positive signal to the S. halepense, indicating the high specificity of the assay.

Establishment of nested PCR assay for identifying S. nitidum
We made use of primers Nout1/Nout4 and Snit5/ Snit2 to amplify the genomic DNA extracted from a single seed of S. nitidum. The result was as shown in Fig. 3, from which, we could observe a strong specific band at ~873 bp, which was well accorded with our predicting.

The application of n-PCR
We used the n-PCR assay established above to detect 14 sorghum samples. The result was as following (Fig. 4). Samples 1-9 displayed strong specific amplified band at ~873 bp while other samples exhibited negative amplification on the electrophoretic gel. As a matter of fact, these 9 samples were confirmed to be S. nitidum while others were S. halepense, S. almum, S. bicolor, S. propinum and S. sudanse. The reproducibility of the n-PCR assay was con-firmed to be higher than typical PCR after many tests and meet the requirement of clinical applications.
The specificity of the PCR primers is very critical to the PCR assay. In this study, we compared the nucleotide sequence of the adh1-like gene between the S. nitidum and other 28 sorghum species using the software Cluster X. The analysis showed the great discrimination of the adh1like gene of the S. nitidum with other sorghum species. Although the high-similarity of the nucleotide sequence of adh1 gene with the S. leiocladum, there is a great diversity within the gene fragment of 1035-1068 bp. Therefore, we designed a backward primer in the gene region of 1035-1068 bp, while the forward primer was designed in the low-similarity gene fragment, which would like to guarantee the high specificity of the primers for identifying the S. nitidum. After all, the high-specificity of the primers was the technical foundation of the nested-PCR assay.
The reason of the low-efficiency of the typical PCR when compared with the nested PCR is as follows: 1) the space/advanced structure of the adh1-like gene may be very complex; 2) the number of the loci of adh1-like gene is small throughout the genomic gene of the sorghum, which reduce the target number and the amplification efficiency. In order to proof the first suggestion, we designed series of primers targeting at the nucleotide fragment throughout ~2000 bp nucleotide acids near the adh1-like gene. However, all of the primers showed low-efficient amplification, which tells us that the first reason seems not like exit. In order to verify the second reason, we blast the adh1-like loci in the database of S. bicolor and observed that there is only one locus in the genome of the sorghum. Therefore, typical PCR would possibly displayed low-efficiency; especially the DNA concentration is low in the amplification system. Wen WG et al. (2008) applied the  4. The specificity of the n-PCR for identifying the S. nitidum; M, DL2000 (TAKARA); 1~9, Snit1~Snit9; 10, Shal1; 11, Salm1; 12, Sbic1; 13, Spro1; 14, Ssud1, 0, Blank semi-nested PCR assay to detect the genetically modified rice in the food and observed the high sensitivity of the assay when compared with the typical PCR. Nested PCR was much more specific and sensitive than typical PCR and the stability of the n-PCR could be very good in some cases. We herein established a novel n-PCR assay for fast and reproducible detection of the S. nitidum.

Conclusions
A set of specific n-PCR inner primers Snit5/Snit2 and outer primers Nout1/Nout2 based on a conserved nucleotide sequence of adh1-like gene of S. nitidum was designed. A novel nested polymerase chain reaction (n-PCR) assay to identify Sorghum nitidum (S. nitidum) was developed. This assay was able to specifically identify S. nitidum fast and effectively, which could be applied widely in field inspection, agriculture production and plant protection.