Detection of the root-lesion nematode, Pratylenchus penetrans (Cobb), in a nematode community using real-time PCR

Quantitative detection of Pratylenchus penetrans from soil by using soil compaction and real-time PCR

“…The compacted soils were removed from the cylinder and mixed well with TE buffer at a ratio of 1:1 (w/w) by using a homogenizer (AUTO CELL MASTER CM-200, AS ONE Corporation, Tokyo, Japan) at 15,000 rpm for 10 min. Then, DNA was extracted from 0.5 g of the mixed soil by the method of Sato et al (2007) and finally suspended in 100 µl of TE buffer. The extracted DNA was used as a template for real-time PCR after 10-fold dilution (Griffiths et al, 2000).…”

“…Real-time PCR was performed by using a Step One Real-Time PCR System (Life Technologies Japan, Tokyo, Japan) in a final volume of 10 µl containing 5 µl of Fast SYBR Green Master Mix (Life Technologies Japan, Tokyo, Japan), 5 mM of each primer (NEGf: 5 , -ATT CCG TCC GTG GTT GCT ATG -3 , , NEGr: 5 , -GCC GAG TGA TCC ACC GAT AAG -3 , , Sato et al, 2007), and 2 µl of template DNA under the manufacturer's recommended conditions (95 for 20 sec, (95 for 3 sec and 62 for 30 sec) 40 cycles). As controls, non-compacted soils were mixed well with TE buffer and DNA was extracted by the same method as described above.…”

“…Recently, DNA analysis has been used to identify and quantify soil nematodes (Madani et al, 2005;MacMillan et al, 2006;Sato et al, 2007;Toyota et al, 2008) and to assess nematode community structure (Sato et al, 2006;Okada and Oba, 2008;Donn et al, 2008). For these studies, nematodes must first be extracted from soil by the Baermann methods using funnels and trays, which are time-consuming and yield a recovery rate of around 50% (Ingham, 1994).…”

Development of a direct quantitative detection method for Meloidogyne incognita and M. hapla in andosol and analysis of relationship between the initial population of Meloidogyne spp. and yield of eggplant in an andosol

“…The rapid development of DNA technology has been a breakthrough for overcoming the weak points of traditional identification methods (Atkins et al, 2005;Berry et al, 2007). Since Madani et al (2005) reported real-time PCR specific primers for quantitative purposes of the potato cyst nematode Globodera pallida and the sugarbeet cyst nematode Heterodera schachtii, many specific primers have been designed for different plant-parasitic nematodes such as Bursaphelenchus xylophilus (Leal et al, 2007), Pratylenchus penetrans (Sato et al, 2007), M. incognita and G. rostochiensis (Toyota et al, 2008), M. javanica, P. zeae and Xiphinema elongatum (Berry et al, 2008), H. glycines (Goto et al, 2009) and P. thornei (Yan et al, 2012). However, no specific primers have been developed for M. hapla until now.…”

Suppressed-Priming PCR, a Novel Concept of DNA Quantification Based on PCR Kinetics

“…Among these methods, real-time PCR is preferably applied to estimate accurately the abundance of the corresponding species, e.g., a nematode, 1) based on the kinetics of PCR. However, it is still hard to quantify many species in a mixture simultaneously.…”