US 12,448,658 B2
Primer and probe sets for pathogen detection of infection in transplant patient, kit and use thereof
Miao He, Chengdu (CN); and Shan Liang, Chengdu (CN)
Assigned to Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu (CN)
Filed by Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu (CN)
Filed on Jul. 14, 2022, as Appl. No. 17/812,496.
Claims priority of application No. 202110794646.1 (CN), filed on Jul. 14, 2021.
Prior Publication US 2023/0071792 A1, Mar. 9, 2023
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/689 (2018.01); C12Q 1/6895 (2018.01); C12Q 1/70 (2006.01); C12Q 1/6818 (2018.01); C12Q 1/6851 (2018.01)
CPC C12Q 1/701 (2013.01) [C12Q 1/689 (2013.01); C12Q 1/6895 (2013.01); C12Q 1/705 (2013.01); C12Q 1/6818 (2013.01); C12Q 1/6851 (2013.01); C12Q 2600/16 (2013.01)] 8 Claims
 
1. Primer and probe sets for pathogen detection of infection in a transplant patient, comprising the following 23 primer and probe sets, with each probe having a reporter group at a 5′ end and a quencher group at a 3′ end:
a 1st primer and probe set for detecting an adenovirus type B, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-TCAGGGATAACTTTGTAGG-3′ (SEQ ID NO: 1),
the downstream primer: 5′-GACAGTTCAGTATTTCTGTC-3′ (SEQ ID NO: 2), and
the probe: 5′-TCAACCACTGCATTCAGTTGTGATG-3′ (SEQ ID NO: 3);
a 2nd primer and probe set for detecting an adenovirus type C, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-CAGTGGTCTTACATGCAC-3′ (SEQ ID NO: 4),
the downstream primer: 5′-TGGGGTTTCTAAACTTGTTA-3′ (SEQ ID NO: 5), and the probe: 5′-ACGCCTCGGAGTACCTGAGC-3′ (SEQ ID NO: 6);
a 3rd primer and probe set for detecting a varicella-zoster virus (VZV), wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-GGTGTCTCCCTAATCTTG-3′ (SEQ ID NO: 7),
the downstream primer: 5′-CTCTCTTTCTGTGTGTCC-3′ (SEQ ID NO: 8), and
the probe: 5′-AGGCTTCTGCTCTCGACTGG-3′ (SEQ ID NO: 9);
a 4th primer and probe set for detecting an Epstein-Barr virus (EB virus), wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-CTCCCTTTACAACCTCAG-3′ (SEQ ID NO: 10),
the downstream primer: 5′-GACGACTCAATGGTGTAA-3′ (SEQ ID NO: 11), and
the probe: 5′-CCTTGCTGTTCCACAATGTCGT-3′ (SEQ ID NO: 12);
a 5th primer and probe set for detecting a human herpes simplex virus type 1, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-GGATGGCGAACATCTTAA-3′ (SEQ ID NO: 13),
the downstream primer: 5′-GTTGGGTTTGTCCTTCTC-3′ (SEQ ID NO: 14), and
the probe: 5′-ATGGGACCCGTGACACCCTA-3′ (SEQ ID NO: 15);
a 6th primer and probe set for detecting a cytomegalovirus (CMV), wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-CGTGACGTGCATAAAGAG-3′ (SEQ ID NO: 16),
the downstream primer: 5′-GGTGATAGGTGACCAGTA-3′ (SEQ ID NO: 17), and
the probe: 5′-CTCGCAGAAGGACTCCAGGT-3′ (SEQ ID NO: 18);
a 7th primer and probe set for detecting a human herpes virus type 6, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-GTGTGGTAATGGACTAAG-3′ (SEQ ID NO: 19),
the downstream primer: 5′-CGACCATCAAAATATAAAGAG-3′ (SEQ ID NO: 20), and
the probe: 5′-AGCTGCTACAATACACACGTAAGTAC-3′ (SEQ ID NO: 21);
an 8th primer and probe set for detecting Staphylococcus aureus, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-GCGAATGAGCGTTTATTTAG-3′ (SEQ ID NO: 22),
the downstream primer: 5′-GACCTTTAGATAAAGCTGTAATG-3′ (SEQ ID NO: 23), and
the probe: 5′-CGCCATAACTCATACCAGATTGTCCTA-3′ (SEQ ID NO: 24);
a 9th primer and probe set for detecting Streptococcus pneumoniae, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-CAGCTACCAACGACAGTC-3′ (SEQ ID NO: 25),
the downstream primer: 5′-CGCAAGAAGAGTGGGATTA-3′ (SEQ ID NO: 26), and
the probe: 5′-TCTCTAACAAGGTCTCATCCACTACGA-3′ (SEQ ID NO: 27);
a 10th primer and probe set for detecting Staphylococcus epidermidis, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-CGGTGCTTATACTGGAGA-3′ (SEQ ID NO: 28),
the downstream primer: 5′-TCGTCAGTTTCGTGGAAA-3′ (SEQ ID NO: 29), and
the probe: 5′-ACGACGCTCTGAGTGACCAAT-3′ (SEQ ID NO: 30);
an 11th primer and probe set for detecting Pseudomonas aeruginosa, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-TCACCACCTTCTACTTCG-3′ (SEQ ID NO: 31),
the downstream primer: 5′-CAGAGCCATGTTGTACTC-3′ (SEQ ID NO: 32), and
the probe: 5′-ACGACAGCTCCGACCTGAAG-3′ (SEQ ID NO: 33);
a 12th primer and probe set for detecting Acinetobacter baumannii, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-TCCAGTTTATCAAGATTTAGC-3′ (SEQ ID NO: 34),
the downstream primer: 5′-CTTGGGTACCGATATCTG-3′ (SEQ ID NO: 35), and
the probe: 5′-CATAACCAACACGCTTCACTTCCTTAG-3′ (SEQ ID NO: 36);
a 13th primer and probe set for detecting Escherichia coli, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-CCGCAAGAAAACTATCCC-3′ (SEQ ID NO: 37),
the downstream primer: 5′-TAGCGACTGATATTGAACTG-3′ (SEQ ID NO: 38), and
the probe: 5′-CCGCCTTACTGCCGCCTGTT-3′ (SEQ ID NO: 39);
a 14th primer and probe set for detecting Enterococcus faecalis, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-TGCCATCATTTAGTTGGG-3′ (SEQ ID NO: 40),
the downstream primer: 5′-GAAGCTTTTAAGAGATTTGCATG-3′ (SEQ ID NO: 41), and
the probe: 5′-CGGTCTAGCGACTCGTTGTACTTC-3′ (SEQ ID NO: 42);
a 15th primer and probe set for detecting a Mycobacterium tuberculosis complex, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-GTCGAGTACGCCTTCTTG-3′ (SEQ ID NO: 43),
the downstream primer: 5′-CACCTATGTGTCGACCTG-3′ (SEQ ID NO: 44), and
the probe: 5′-CCTTTGTCACCGACGCCTACG-3′ (SEQ ID NO: 45);
a 16th primer and probe set for detecting Enterococcus faecium, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-CATCCAAGATATTCTGCC-3′ (SEQ ID NO: 46),
the downstream primer: 5′-CGGATTTTGTTCAATACAAG-3′ (SEQ ID NO: 47), and
the probe: 5′-CACCATCTACATCGTCAGCAATAATCA-3′ (SEQ ID NO: 48);
a 17th primer and probe set for detecting Clostridium difficile, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-GCGTAGATATTAGGAGGAA-3′ (SEQ ID NO: 49),
the downstream primer: 5′-GGAGTACTTAATGCGTTAG-3′ (SEQ ID NO: 50), and
the probe: 5′-TCTCTGGACTGTAACTGACGCTG-3′ (SEQ ID NO: 51);
an 18th primer and probe set for detecting Stenotrophomonas maltophilia, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-TCCTCCAAGGCTAAATAC-3′ (SEQ ID NO: 52),
the downstream primer: 5′-CGGTTTCAGGTTCTATTTC-3′ (SEQ ID NO: 53), and
the probe: 5′-TGACCGACCGATAGTGAACCAG-3′ (SEQ ID NO: 54);
a 19th primer and probe set for detecting Haemophilus influenzae, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-CGTTCAAATCTTAGACGC-3′ (SEQ ID NO: 55),
the downstream primer: 5′-CGATGTTGTATTCTGGTG-3′ (SEQ ID NO: 56), and
the probe: 5′-CGTTCATCAGTGTTACCTTCTACTAAT-3′ (SEQ ID NO: 57);
a 20th primer and probe set for detecting Klebsiella pneumoniae, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-CCCGTGAACCTTTACTATA-3′ (SEQ ID NO: 58),
the downstream primer: 5′-AAGGTGGTATTTCAAGG-3′ (SEQ ID NO: 59), and
the probe: 5′-TGACACTGAACATTGAGCCTTGAT-3′ (SEQ ID NO: 60);
a 21st primer and probe set for detecting Cryptococcus neoformans, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-CTGGAAGCTCATTTACCTA-3′ (SEQ ID NO: 61),
the downstream primer: 5′-GGCCATGGAATTGGAATA-3′ (SEQ ID NO: 62), and
the probe: 5′-ATTCACCGTAAGCCATCAATCAGG-3′ (SEQ ID NO: 63);
a 22nd primer and probe set for detecting Candida albicans, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-GAGGTCTAAACTTACAACC-3′ (SEQ ID NO: 64),
the downstream primer: 5′-CGTTGTTGAAAGTTTTGAC-3′ (SEQ ID NO: 65), and
the probe: 5′-TCAACTTGTCACACCAGATTATTACTT-3′ (SEQ ID NO: 66); and
a 23rd primer and probe set for detecting Aspergillus fumigatus, wherein sequences of an upstream primer, a downstream primer, and a probe are as follows:
the upstream primer: 5′-TCCCTCTTATCCATTTTCC-3′ (SEQ ID NO: 67),
the downstream primer: 5′-GTTCCAAAGCCGAAAGAATA-3′ (SEQ ID NO: 68), and
the probe: 5′-CCTCATCACTGCAACTCTAATCCTCG-3′ (SEQ ID NO: 69).