US2012309643A1PendingUtilityA1
Method and/or apparatus of oligonucleotide design and/or nucleic acid detection
Assignee: WONG CHRISTOPHER WING CHEONGPriority: Aug 12, 2005Filed: Jul 13, 2012Published: Dec 6, 2012
Est. expiryAug 12, 2025(expired)· nominal 20-yr term from priority
G16B 25/20G16B 25/00C12Q 1/6806C12Q 1/6813C12Q 1/6811G16B 99/00
56
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Claims
Abstract
A method of designing at least one oligonucleotide for nucleic acid detection including: (I) identifying and/of selecting region(s) of at least one target nucleic acid to be amplified, the region(s) having an efficiency of amplification (AE) higher than the average AE; and (II) designing at least one oligonucleotide capable of hybridizing to the selected region(s). Also, a method of detecting at least one target nucleic acid including: (i) providing at least one biological sample; (ii) amplifying nucleic acid(s) in the biological sample; (iii) providing at least one oligonucleotide capable of hybridizing to at least one target nucleic acid, if present in the biological sample; and (iv) contacting the oligonucleotide(s) with the amplified nucleic acids and detecting the oligonucleotide(s) hybridized to the target nucleic acid(s). The method is useful for detecting the presence of at least one pathogen, such as a virus, in a human biological sample.
Claims
exact text as granted — not AI-modified1 . A method of detecting at least one target nucleic acid comprising the steps of:
(i) providing at least one biological sample; (ii) amplifying nucleic acid(s) comprised in the biological sample; (iii) providing at least one oligonucleotide capable of hybridizing to at least one target nucleic acid, if present in the biological sample; and (iv) contacting the oligonucleotide(s) with the amplified nucleic acids and/or detecting the oligonucleotide(s) hybridized to the target nucleic acid(s).
2 . The method according to claim 1 , wherein the target nucleic acid to be detected is nucleic acid exogenous to the nucleic acid of the biological sample.
3 . The method according to claim 1 , wherein in the detection step (iv), the mean of the signal intensities of the probes which hybridize to v a is statistically higher than the mean of the probes ∉v a , thereby indicating the presence of v a in the biological sample.
4 . The method according to claim 1 , wherein in the detection step (iv), the mean of the signal intensities of the probes which hybridize to v a is statistically higher than the mean of the probes ∉v a , and the method further comprises the step of computing the relative difference of the proportion of probes ∉v a having high signal intensities to the proportion of the probes used in the detection method having high signal intensities, the density distribution of the signal intensities of probes v a being more positively skewed than that of probes ∉v a , thereby indicating the presence of v a in the biological sample.
5 . The method according to claim 1 , wherein in the detection step (iv), the presence of at least one target nucleic acid in a biological sample is given by a value of Weighted Kullback-Leibler divergence score of 1.0.
6 . The method according to claim 1 , wherein the detection step (iv) comprises evaluating the signal intensity of probe(s) in each signature probe set (SPS) for the target nucleic acid(s) v a by calculating the distribution of Weighted Kullback-Leibler (WKL) divergence scores:
WKL
(
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a
|
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=
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a
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where Q a (j) is the cumulative distribution function of the signal intensities of the probes in P a found in bin b j ; Q ā (j) is the cumulative distribution function of the signal intensities of the probes in P a found in bin b j ; P a is the set of probes of a virus v a and P a =P−P a .
7 . The method according to claim 6 , wherein each signature probe set (SPS) which represents the absence of target nucleic acid(s) v a has a Weighted Kullback-Leibler (WKL) divergence score of WKL<5, and wherein each signature probe set (SPS) which represents the presence of at least one target nucleic acid v a has a Weighted Kullback-Leibler (WKL) divergence score of WKL>5.
8 . The method according to claim 6 , further comprising performing Anderson-Darling test on the distribution of WKL score(s), wherein a result of P>0.05 thereby indicates the absence of target nucleic acid(s) v a and wherein a result of P<0.05 thereby indicates the presence of target nucleic acid(s) V a .
9 . An apparatus configured to perform a method of detecting at least one target nucleic acid comprising the steps of:
(i) providing at least one biological sample; (ii) amplifying nucleic acid(s) comprised in the biological sample; (iii) providing at least one oligonucleotide capable of hybridizing to at least one target nucleic acid, if present in the biological sample; and (iv) contacting the oligonucleotide(s) with the amplified nucleic acids and/or detecting the oligonucleotide(s) hybridized to the target nucleic acid(s); wherein the detection step comprises evaluating the signal intensity of probe(s) in each signature probe set (SPS) for the target nucleic acid(s) by calculating the distribution of Weighted Kullback-Leibler (WKL) divergence scores:
WKL
(
P
a
|
P
a
_
)
=
∑
j
=
0
k
-
1
Q
a
(
j
)
log
(
Q
a
(
j
)
Q
a
_
(
j
)
)
Q
a
_
(
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)
[
1
-
Q
a
_
(
j
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]
where Q a (j) is the cumulative distribution function of the signal intensities of the probes in P a found in bin b j ; Q ā (i) is the cumulative distribution function of the signal intensities of the probes in P a found in bin b j ; and where P a is the set of probes of a virus v a and P a =P−P a .
10 . The apparatus according to claim 9 , wherein the target nucleic acid to be detected is at least one nucleic acid exogenous to the nucleic acid of the biological sample.
11 . The apparatus according to claim 9 , wherein the presence of a target nucleic acid in a biological sample is given by a value of Weighted Kullback-Leibler divergence of ≧1.0.
12 . The apparatus according to claim 9 , wherein each signature probe set (SPS) which represents the absence of target nucleic acid(s) has a Weighted Kullback-Leibler (WKL) divergence score of WKL<5, and wherein each signature probe set (SPS) which represents the presence of at least one target nucleic acid has and/or a Weighted Kullback-Leibler (WKL) divergence score of WKL>5.
13 . The apparatus according to claim 9 , further comprising performing an Anderson-Darling test on the distribution of WKL score(s), wherein a result of P>0.05 thereby indicates the absence of target nucleic acid(s) and wherein a result of P<0.05 thereby indicates the presence of target nucleic acid(s).
14 . A non-transitory electronic storage medium comprising a software with instructions to cause a computing processing unit to perform the method according to claim 1 .
15 . A non-transitory electronic storage medium comprising a software with instructions to cause a computing processing unit to determine the WKL divergence score according to claim 6 and/or perform the Anderson Darling test according to claim 8 .
16 . The method according to claim 8 , wherein P<0.05 indicates the distribution of WKL scores is not normal and P>0.05 indicates the distribution of WKL scores is normal.
17 . The method according to claim 16 , wherein if the distribution of WKL scores is not normal, the target nucleic acid molecule with the highest WKL score is identified as present in the biological sample.
18 . The method according to claim 17 , further comprising removing the highest WKL score from the WKL scores, and repeating the Anderson-Darling test on the remaining WKL scores to determine if the distribution of the remaining WKL scores is normal.
19 . The method according to claim 18 , wherein if the distribution of the remaining WKL scores is not normal, the target nucleic acid molecule with the next highest WKL score is also identified as present.
20 . The method according to claim 19 , wherein the target nucleic acid molecule with the next highest WKL score is indicative of a co-infecting pathogen.
21 . The method according to claim 19 , comprising repeating the steps of removing the next highest WKL score and repeating the Anderson-Darling test until the distribution of the WKL scores becomes normal, thereby detecting the presence of any other target nucleic acid molecules and/or co-infecting pathogens.Cited by (0)
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