Hi Anh Tai,

There are several things that you can try. Since you only indicate that you try different primers, I will just share some other tricks or experiences. 

1. cDNA synthesis: There could be something wrong with you viral RNA in the beginning. For example, RNA concentration too low or inappropriate handling which introduces RNase and degrades your samples during RNA extraction. According to my experience, RNA concentration from Nanodrop after viral RNA exaction does not represent your true viral RNA concentration because carrier RNA is added to protect your viral RNA and most of the readout is contributed by carrier RNA. What you can do is lyse more virus and pull them into one column and elute in a smaller volume. Low RNA template could be troublesome as reverse transcriptase has exonuclease activity. It will degrade your RNA template immediately after synthesizing one copy of cDNA. RNase contamination is not a technical issue, it can be solved by just exercise extra precaution during extraction. 

2. PCR of cDNA: Your cDNA synthesis might be successful, however, your PCR condition might not work. Do a gradient PCR, test different annealing temperature. If you didn't get any band from your previous experiment, I would suggest go for lower annealing temperature such as 53 degree. If you have unspecific bands, but you can't find your expected band, try to increase your annealing temperature. Put as much of cDNA as possible in your PCR. Personal experience is when I put more cDNA that band just pop up. 

3. Other small things to consider, make sure your gene specific primer is a reverse primer. Sometimes people might make a small mistake by putting in forward primer. Another thing you can do is to separate your gene into two fragments, cDNA synthesis has a higher success rate for smaller pieces. The disadvantage is you have to assembly this two fragments together for your downstream work which might take some time. However, it can be done easily by using nested PCR. 

Hope this help. 

If you want more help, please send the Rt and PCR protocols you used, proportions are also important.

Beside what Donald recommended to you,

I am thinking also of Mg++ concentration (assuming that your enzyme is Mg dependent) - if this is too low, no PCR product is obtained. Sometimes increasing Mg concentration with just 0.5 helps.

Also the extension time should be longer for a 3kb product

Thank you, Donald and Doinita. your advices would be verry useful for my experiment. I am a new person accessing to molecular cloning. the problem would be derived from my weak experience as well as the steps I conducted the research. 

I used the possitive control and the band was well appeared. I also tried to amplify the Beta-actin gene from insect together with the virus gene, but no band of beta-actin gene as well. So, the problem would come from the cDNA synthesis. I am now try to quickly detect the virus with a smaller DNA segment to make sure that my samples contain the virus and then I will carefully fix the cDNA synthesis step. My lab mates, they have already successfully amplified the same size but on other virus. But I tried their protocol I am still fail. 

Hi Anh Tai, 

Glad that you provide more details. 

1. The first thing to clarify is the positive control for PCR that you are talking about is it using the primer pairs for your target gene? If it is, then that means your PCR condition and primers work. If you are just using another 3kb gene, the positive control you got is only telling you that your PCR condition such as your enzyme, dNTPs or buffer are working fine, it still can't tell you that is annealing temperature a problem. 

2. "I also tried to amplify the Beta-actin gene from insect together with the virus gene, but no band of beta-actin gene as well." I think beta-actin gene belongs to your host cell while virus gene belongs to the virus. When I extract viral RNA, I don't lyse the host cells and viruses together, I will just take the viral supernatant and extract the RNA. 

3. Yes, I think your approach is right, using a small fragment to detect your virus. If there is nothing in the beginning, then no virus no viral RNA. Alternatively, you can observe is there any CPE occurring when you grow your virus provide that your virus can produce CPE.