一种肺炎链球菌常见血清型多重PCR分型方案的评估

doi:10.3969/j.issn.1673-8640.2015.06.014

摘要/Abstract

摘要： 目的

评估一种肺炎链球菌常见血清型多重聚合酶链反应(PCR)分型方案的分型能力。

方法

以传统荚膜肿胀试验为参考,采用多重PCR分型方案对50株肺炎链球菌进行血清分型。

结果

50株肺炎链球菌经传统荚膜肿胀试验分为19F型9株、 19A型9株、 23F型9株、 6A/B型9株、 14型9株和15B/C型5株;而多重PCR分型方案结果为 19F型8株、 19A型10株、 23F型9株、 6A/B型9株、 14型10株和15B/C型4株,2种方法符合率为96%,Kappa值为0.952。

结论

多重PCR分型技术与常规方法一致性好,具有简便、快速、成本相对较低等优点,能用于监测临床肺炎链球菌流行血清型。

关键词: 多重聚合酶链反应, 血清型, 肺炎链球菌

Abstract: Objective

To evaluate the performance of multiplex polymerase chain reaction (PCR) for detecting the common serotypes of one kind of Streptococcus pneumoniae.

Methods

A total of 50 isolates of Streptococcus pneumoniae were analyzed by multiplex PCR with traditional quellung reaction as reference method for detecting the common serotypes of Streptococcus pneumoniae.

Results

The 50 isolates were typed by traditional quellung reaction with 9 isolates being 19F, 19A, 23F, 6A/B and 14, respectively, and 5 isolates being 15B/C. However, according to the results of multiplex PCR, 8 of 50 isolates were serotype 19F, and there also were 19A (10 isolates), 23F (9 isolates), 6A/B (9 isolates), 14 (10 isolates) and 15B/C (4 isolates), which showed coincidence rate 96%. The value of Kappa was 0.952.

Conclusions

Multiplex PCR has good consistency with traditional method, and it has the advantages of easy, rapid performance and low costing, which could provide the reference for monitoring the serotypes of Streptococcus pneumoniae in clinic.

Key words: Multiplex polymerase chain reaction, Serotype, Streptococcus pneumoniae

中图分类号:

Q503

潘芬, 赵声远, 秦惠宏, 祝俊英, 肖淑珍, 张泓. 一种肺炎链球菌常见血清型多重PCR分型方案的评估[J]. 检验医学, 2015, 30(6): 599-602.

PAN Fen, ZHAO Shengyuan, QIN Huihong, ZHU Junying, XIAO Shuzhen, ZHANG Hong. Evaluation on multiplex PCR for detecting the common serotypes of one kind of Streptococcus pneumoniae[J]. Laboratory Medicine, 2015, 30(6): 599-602.

图/表 3

表1 肺炎链球菌棋盘法分型方案

组	P	Q	R	S	T	非疫苗覆盖的血清群/型
A	1^*	1 $8 18 F 、 18 A 、 18 B 、 18 C) *$	4^*	5^*	2^*
B	1 $9 19 F 、 19 A 、 19 B 、 19 C) *$	$6 6 A 、 6 B 、 6 C) *$	3^*	8^*
C	$7 7 F 、 7 A 、 7 B 、 7 C) *$				20^*	24_{(24F、24A、24B)},31,40
D			$9 9 A 、 9 L 、 9 N 、 9 V) *$		1 $1 11 F 、 11 A 、 11 B 、 11 C 、 11 D) *$	16_(16F、16A),36,37
E			1 $2 12 F 、 12 A 、 12 B) *$	1 $0 10 F 、 10 A 、 10 B 、 10 C) *$	3 $3 33 F 、 33 A 、 33 B 、 33 C 、 33 D) *$	21,39
F				1 $7 17 F 、 17 A) *$	2 $2 22 F 、 22 A) *$	27,32_(32F、32A),41_(41F、41A)
G						29,34,35_{(35F、35A、35B、35C)},42,47_(47F、47A)
H	14^*	2 $3 23 F 、 23 A 、 23 B) *$		1 $5 15 F 、 15 A 、 15 B 、 15 C) *$		13,28_(28F、28A)
I						25_(25F、25A),38,43,44,45,46,48

表1 肺炎链球菌棋盘法分型方案

组	P	Q	R	S	T	非疫苗覆盖的血清群/型
A	1^*	1 $8 18 F 、 18 A 、 18 B 、 18 C) *$	4^*	5^*	2^*
B	1 $9 19 F 、 19 A 、 19 B 、 19 C) *$	$6 6 A 、 6 B 、 6 C) *$	3^*	8^*
C	$7 7 F 、 7 A 、 7 B 、 7 C) *$				20^*	24_{(24F、24A、24B)},31,40
D			$9 9 A 、 9 L 、 9 N 、 9 V) *$		1 $1 11 F 、 11 A 、 11 B 、 11 C 、 11 D) *$	16_(16F、16A),36,37
E			1 $2 12 F 、 12 A 、 12 B) *$	1 $0 10 F 、 10 A 、 10 B 、 10 C) *$	3 $3 33 F 、 33 A 、 33 B 、 33 C 、 33 D) *$	21,39
F				1 $7 17 F 、 17 A) *$	2 $2 22 F 、 22 A) *$	27,32_(32F、32A),41_(41F、41A)
G						29,34,35_{(35F、35A、35B、35C)},42,47_(47F、47A)
H	14^*	2 $3 23 F 、 23 A 、 23 B) *$		1 $5 15 F 、 15 A 、 15 B 、 15 C) *$		13,28_(28F、28A)
I						25_(25F、25A),38,43,44,45,46,48

表2 肺炎链球菌8个血清型及cpsA引物序列

血清型	GenBank编号	引物序列	产物大小(bp)
19F	CR931678	F: GTTAAGATTGCTGATCGATTAATTGATATCC	304
		R: GTAATATGTCTTTAGGGCGTTTATGGCGATAG
19A	CR931675	F: GAGAGATTCATAATCTTGCACTTAGCCA	566
		R: CATAATAGCTACAAATGACTCATCGCC
23F	CR931685	F: GTAACAGTTGCTGTAGAGGGAATTGGCTTTTC	384
		R: CACAACACCTAACACTCGATGGCTATATGATTC
6A/B	CR931639	F: AATTTGTATTTTATTCATGCCTATATCTGG	250
		R: TTAGCGGAGATAATTTAAAATGATGACTA
14	CR931653	F: CTTGGCGCAGGTGTCAGAATTCCCTCTAC
		R: GCCAAAATACTGACAAAGCTAGAATATAGCC
15B/C	CR931664	F: TTGGAATTTTTTAATTAGTGGCTTACCTA
		R: CATCCGCTTATTAATTGAAGTAATCTGAACC
cpsA	AF057294	F: GCAGTACAGCAGTTTGTTGGACTGACC
		R: GAATATTTTCATTATCAGTCCCAGTC

图1 6种常见血清型的电泳图

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