Nested RT-PCR method and primer combination for detecting potato mop-top virus

Abstract

The invention discloses a nested RT-PCR method and a primer combination for detecting potato mop-top virus. The method comprises the following steps: firstly, conducting reverse transcription by virtue of a random primer by taking RNA as a template, so as to obtain cDNA; secondly, taking the cDNA as a template; and thirdly, conducting the second turn of PCR by taking the fragment 1 of the first PCR amplified product as a template, wherein by virtue of two turns of the PCR reactions, the specificity and the sensitivity of detecting and identifying the virus are improved. Therefore, the nested RT-PCR method, when used for detecting the PMTV, is high in virus detection sensitivity and strong in specificity.

Description

technical field

[0001] The invention belongs to the technical field of crop virus detection, and in particular relates to a nested RT-PCR method for detecting potato broom top virus and a primer combination thereof. Background technique

[0002] Potatoes, also known as potatoes, artichokes, etc., are common food and vegetable crops in people's daily life. As food crops, they are the fourth largest food crops in the world after wheat, corn and rice. As far as our country is concerned, the cultivated area and total output of potatoes rank among the top in the world, but its per unit yield and quality are far behind other advanced countries that produce potatoes. The harm of potato virus disease is one of the main reasons for this situation. Potato virus disease is seriously harmful in my country. It is not only affected by one infection, but also can be transmitted through seed potatoes, causing re-infection. The degree of damage is serious from generation to generation, resu...

Examples

Embodiment 1

[0041] Example 1 Nested RT-PCR detects the progeny tuber PMTV infected with PMTV potatoes

[0042] 1. Primer design

[0043] Due to the need to detect potato broom top virus with low virus expression content in plants, a nested RT-PCR detection method with high detection sensitivity, strong specificity, simple detection steps, accuracy and economy was established. According to the coat protein sequence of PMTV in Genbank, use Primer5.0 to design universal primer pairs PMTV-N1 and PMTV-N2. The target fragment size of PMTV-N1 amplification is 460bp, and PMTV-N2 is the amplification product bound to PMTV-N1. Internally, the second PCR amplification fragment is shorter than the first amplification, and the size of the amplified target fragment is 263bp. The primer sequences are shown in Table 1. The size of the fragments amplified by the two pairs of primers is quite different. The target fragment amplified by the second pair of primers is smaller than the amplified product of t...

Embodiment 2

[0059] Example 2 Nested RT-PCR detects tobacco plants infected with PMTV

[0060] Tobacco plants were inoculated with PMTV-infected potato juice by rubbing, and mosaic symptoms appeared on the leaves of tobacco plants after friction inoculation with PMTV. The leaves of tobacco plants with mosaic symptoms were taken, and RNA was extracted, and RT-PCR and nested RT-PCR were used respectively. Methods To detect tobacco infection with PMTV, the detection results are as follows: figure 2 Shown, utilize the method detection of RT-PCR once, the positive rate of tobacco PMTV is 50% (see figure 2 -A), and the target band is not clear and difficult to distinguish, but the positive rate of tobacco PMTV reaches 100% by nested RT-PCR method (see figure 2 -B), the target band is very clear and there is no non-specific band, which shows that the detection sensitivity of nested RT-PCR is significantly higher than that of one-time PCR, and the specificity is also stronger than that of one-...

technical field

[0001] The invention belongs to the technical field of crop virus detection, and in particular relates to a nested RT-PCR method for detecting potato broom top virus and a primer combination thereof. Background technique

[0002] Potatoes, also known as potatoes, artichokes, etc., are common food and vegetable crops in people's daily life. As food crops, they are the fourth largest food crops in the world after wheat, corn and rice. As far as our country is concerned, the cultivated area and total output of potatoes rank among the top in the world, but its per unit yield and quality are far behind other advanced countries that produce potatoes. The harm of potato virus disease is one of the main reasons for this situation. Potato virus disease is seriously harmful in my country. It is not only affected by one infection, but also can be transmitted through seed potatoes, causing re-infection. The degree of damage is serious from generation to generation, resu...

Examples

Embodiment 1

[0041] Example 1 Nested RT-PCR detects the progeny tuber PMTV infected with PMTV potatoes

[0042] 1. Primer design

[0043] Due to the need to detect potato broom top virus with low virus expression content in plants, a nested RT-PCR detection method with high detection sensitivity, strong specificity, simple detection steps, accuracy and economy was established. According to the coat protein sequence of PMTV in Genbank, use Primer5.0 to design universal primer pairs PMTV-N1 and PMTV-N2. The target fragment size of PMTV-N1 amplification is 460bp, and PMTV-N2 is the amplification product bound to PMTV-N1. Internally, the second PCR amplification fragment is shorter than the first amplification, and the size of the amplified target fragment is 263bp. The primer sequences are shown in Table 1. The size of the fragments amplified by the two pairs of primers is quite different. The target fragment amplified by the second pair of primers is smaller than the amplified product of t...

Embodiment 2

[0059] Example 2 Nested RT-PCR detects tobacco plants infected with PMTV

[0060] Tobacco plants were inoculated with PMTV-infected potato juice by rubbing, and mosaic symptoms appeared on the leaves of tobacco plants after friction inoculation with PMTV. The leaves of tobacco plants with mosaic symptoms were taken, and RNA was extracted, and RT-PCR and nested RT-PCR were used respectively. Methods To detect tobacco infection with PMTV, the detection results are as follows: figure 2 Shown, utilize the method detection of RT-PCR once, the positive rate of tobacco PMTV is 50% (see figure 2 -A), and the target band is not clear and difficult to distinguish, but the positive rate of tobacco PMTV reaches 100% by nested RT-PCR method (see figure 2 -B), the target band is very clear and there is no non-specific band, which shows that the detection sensitivity of nested RT-PCR is significantly higher than that of one-time PCR, and the specificity is also stronger than that of one-...

Images