Methods and devices for nasopharyngeal carcinoma screening
Methods and devices for nasopharyngeal carcinoma screening are disclosed. The method comprises providing a nasopharyngeal sample from a subject, isolating DNA from the sample, amplifying and detecting at least one EBV target sequence from the DNA using real-time PCR, wherein a real-time PCR cycle threshold number of less than or equal to 31.5 is indicative of the subject having nasopharyngeal carcinoma or a risk of developing nasopharyngeal carcinoma. The device for obtaining a brush biopsy sample comprises a longitudinal shaft having a first end and a second end, wherein at least two brush heads extend from the second end of the shaft.
1. A method of detecting nasopharyngeal carcinoma or a risk of developing nasopharyngeal carcinoma in a test subject comprising:
a. providing a nasopharyngeal sample obtained transorally from the fossae of Rosenmueller of the subject using a brush biopsy,
b. isolating DNA from the sample,
c. amplifying and detecting at least one EBV target sequence from the DNA using real-time PCR,
wherein a real-time PCR cycle threshold number is verified based on batch specific, plate specific, sample specific, and quantitated EBV DNA controls, and
wherein a real-time PCR cycle threshold number of less than or equal to 31.5 is indicative of the test subject having nasopharyngeal carcinoma or a risk of developing nasopharyngeal carcinoma.
2. The method of claim 1 , wherein a real-time PCR cycle threshold number of 28 to 31.5 is indicative of the test subject having a risk of developing nasopharyngeal carcinoma.
3. The method of claim 1 , wherein a real-time PCR cycle threshold number of less than 28 is indicative of the test subject having nasopharyngeal carcinoma.
4. The method of claim 1 further comprising determining an Epstein-Barr Virus Detection Level (EDL) based on the real-time PCR cycle threshold number, wherein an EDL of greater than or equal to 2.57 is indicative of the test subject having nasopharyngeal carcinoma.
5. The method of claim 1 , wherein the nasopharyngeal sample comprises epithelial cells.
6. The method of claim 1 , wherein the at least one EBV target sequence is amplified from 40-60 ng, optionally about 50 ng of DNA.
7. The method of claim 1 , wherein the at least one EBV target sequence is in the EBNA1 gene.
8. The method of claim 1 , wherein the at least one EBV target sequence is amplified using primers corresponding to SEQ ID NO: 1 and 2.
9. The method of claim 6 , wherein the at least one EBV target sequence is detected using a probe corresponding to SEQ ID NO: 4, wherein the probe is labeled with a reporter fluorophore at the 5′-end and a quencher fluorophore at the 3′-end.