The nested RT-PCR has been developed to detect all Lyssaviruses, including all 16 ICTV approved and two novel Lyssavirus species. The validation of this method has been proved by using over 9000 animal brain specimens in 10 years of clinical rabies diagnosis and surveillance in China. The nested RT-PCR not only can produce credible result on fresh brain tissue samples, but also on degraded samples.
The inundation this method can be used to assay biological fluid specimens such as cerebrospinal fluid, saliva, and urine, thereby this method can be applied into ultimatum diagnoses. In order to minimize PCR carry-over contamination, it is important to prepare negative and positive controls It's really important to take steps. For example, for each RNA extraction needs preparation, PCR set up, and gel electrophoresis should be operated in physically separate areas.
It is also recommended to use spiral tapes in RNA extraction and reverse transcription in order to prevent rabies contamination to the peptide chain. Proper handling such as preparing your agents on ice, changing gloves regularly, and use of ribonuclease free materials will prevent the introduction of ribonuclease and eliminate degradation of RNA. To begin this procedure, extract the RNA from rabies suspected tissue, skin biopsies, saliva, cerebrospinal fluid or RABV infected cell culture as outlined in the text protocol.
When ready to proceed, removed the needed reagents from the freezer and keep them on ice. Make sure to thaw and vortex each reagent before use. For reverse transcription of the viral RNA to cDNA prepare 12 micro liters of reaction mix for each sample on ice and a 1.5 milliliter centrifuge tube as outlined in Table 1 of the text protocol in a cleanroom.
To account for pipetting variations, prepare a volume of master mix at least one reaction size greater than required, and then divide the reaction mix into 0.2 milliliter PCR tubes. In a PCR workstation in a template room, add eight microliters of either the sample or one of the controls to the reaction mix. The positive control is RNA extracted from the cell culture infected with fixed-RABV strain CVS11, while the negative control contains Rnase-free double distilled water.
Mix the contents of each tube by first vortexing and then by briefly centrifuging. After this, load the reaction tubes into a thermocycler. Set up and run the cDNA synthesis program as outlined in the text protocol.
First, retrieve the necessary reagents and keep them on ice in a cleanroom until ready to use. When ready, thaw and vortex the reagents. Next, prepare 48 microliters of first round PCR mix for each sample in a 1.5 milliliter centrifuge tube as outlined in Table 2 of the text protocol and divide the reaction mix into 0.2 milliliter PCR tubes.
In a PCR workstation in a template room, add a two microliter sample of the cDNA into the first round PCR mix. Include two microliters of CVS11 cDNA as a positive control, and two microliters of double distilled water as a negative control. Then, transfer the sealed tubes into a PCR thermocycler and cycle using the parameters listed in Table 3 of the text protocol.
To begin, prepare 48 microliters of second round PCR mix for each sample in a 1.5 milliliter centrifuge tube as outlined in Table 4 of the text protocol and divide the reaction mix into 0.2 milliliter PCR tubes. Add two microliters of the first round PCR product into the second round PCR mix. Include double distilled water as a negative control for this round of PCR.
Then, perform PCR thermocycling using the parameters listed in Table 3 of the text protocol. First, add 1.5 grams of agarose to 100 milliliters of Tris-acetate EDTA and heat it in a microwave oven to dissolve it thoroughly. Next, add ethidium bromide at a final concentration of 0.01%Pour the gel into the mold and leave it to solidify at ambient temperature for at least 30 minutes.
Prepare the loading samples by mixing five microliters of each PCR product with one microliter of 6x loading buffer. Separately load the samples and suitable DNA marker into the wells and run the gel for approximately 30 to 45 minutes at 120 volts until the dye line is approximately 75 to 80%down the gel. When the run is complete, turn off the power and disconnect the electrodes from the power source.
Then, carefully remove the gel from the gel box. Use a UV gel documenting device to visualize and photograph the DNA fragments. Characterize the nested reverse-transcription PCR as outlined in the text protocol.
The results of a representative nested RT-PCR to detect 18 Lyssavirus species is shown here. The PCR positive controls show the expected band at 845 base pairs in the first round amplification and at 371 base pairs for the second. No band is seen for the negative controls.
All 18 Lyssaviruses are seen to produce the same two bands as the positive control, indicating that the nested RT-PCR detected all 18 Lyssaviruses. While 16 of the plasmids have efficient amplification in two rounds of PCR, two of them, Aravan virus and Ikoma virus, are amplified only in either the first or second round, respectively. The sensitivity of the method varies in the detection of different Lyssavirus plasmids, from values ranging from 2.24 to 2, 240 molecules per microliter.
These differences can be attributed to the mismatches between the primers and templates due to viral sequence diversity. A comparison of 9, 624 brain tissues from clinical specimens that are tested by nested PCR versus those tested with FAT shows that nested RT-PCR as a sensitivity of 100%and a specificity of 99.97%The accordance between the two methods is 99.07%Ten other rabies laboratories were invited to conduct the test to further validate the nested RT-PCR. All ten laboratories obtained nested RT-PCR results that are 100%in accordance with the FAT, with no false negatives or false positives, indicating that the nested RT-PCR has a high specificity and reproducibility.
RT-PCR is now recommended by OIE for using rabies technologies. And our nested RT-PCR has been proved as the national standard of rabies technologies in China in 2018.
